Thermodynamic Analysis of Interaction of eIF4E Protein with mRNA 5

Thermodynamic Analysis of Interaction of eIF4E Protein
with mRNA 5' Cap Structure and 4E-BP1 or eIF4G
Protein Fragments
Anna NiedŸwiecka
Praca doktorska
pod kierunkiem dr. hab. Ryszarda Stolarskiego, prof. UW
Zak³ad Biofizyki, Instytut Fizyki Doœwiadczalnej, Wydzia³ Fizyki
Uniwersytet Warszawski
Warszawa 2003

Recenzenci: prof. dr hab. Kazimierz Wierzchowski
dr hab. Maciej Geller
II

Sk‡adam serdeczne podziŒkowania
dr. hab. Edwardowi Dar¿ynkiewiczowi, prof. UW, ktry swoim
wysi‡kiem organizatorskim umo¿liwi‡ mi prowadzenie badaæ,
oraz za za wsparcie w rozmaitych sytuacjach,
dr. hab. Ryszardowi Stolarskiemu, prof. UW, za wszechstronn„
pomoc, cierpliwo i ¿yczliwe nastawienie do rozwijaj„cej siŒ
tematyki pracy,
prof. dr. Stephenowi K. Burley’owi z Uniwersytetu Rockefellera
w Nowym Jorku za inspiracjŒ,
dr hab. Agnieszce Bzowskiej, dr Beacie Wielgus-Kutrowskiej,
mgr Joannie fluberek, dr. hab. Janowi Antosiewiczowi, prof.
UW, p. dr hab. Ewie Kulikowskiej, p. Dorocie Haber oraz innym
pracownikom Zak‡adu Biofizyki za cenne dyskusje i
zapewnienie mi‡ej atmosfery,
dr. hab. Januszowi StŒpiæskiemu, dr Marzenie Jankowskiej-
Anyszce i mgr Lidii Chlebickiej za syntezy chemiczne,
biosyntezŒ bia‡ka oraz wiele po¿ytecznych wskazwek,
mgr. Jzefowi Greupnerowi i mgr Urszuli Wonikowskiej-Bezak
oraz lek. med. Tomaszowi Zawadzkiemu za skierowanie moich
zainteresowaæ ku fizyce i biologii molekularnej,
moim Rodzicom za wszelk„ pomoc.
Praca by‡a finansowana przez Komitet Badaæ Naukowych (3 PO4A 071 22 - grant
promotorski, 6 PO4A 055 17, 6 P04A 034 09), Human Frontier Science Program
(RG0303/1998-M) i II. Amerykaæsko-Polski Fundusz im. Marii Sk‡odowskiej-Curie
(MEN/NSF-98-337, MEN/NSF-96-257).
III

Table of Contents
ABBREVIATIONS ................................................................................................................VIII
PREFACE................................................................................................................................XI
1. INTRODUCTION ............................................................................................................. 1
1.1. Brief Description of Biological Aspects................................................................................ 1
1.1.1. Central Dogma of Molecular Biology............................................................................. 1
1.1.2. Protein Functions.............................................................................................................. 2
1.1.3. Translation Initiation........................................................................................................ 2
1.1.4. Eukaryotic Initiation Factor 4E ....................................................................................... 5
1.1.5. Translational Control ....................................................................................................... 9
1.1.5.1. Regulation of eIF4F Complex Formation ................................................................ 10
1.1.6. Clinical Aspects of the Cellular eIF4E Activity Control ............................................. 11
1.1.6.1. Relation of eIF4E Activity to Immunology and Malignancy.................................. 11
1.1.6.2. Relation of 4E-BP1 and eIF4G Activities to Human Health and Disease.............. 11
1.1.6.2.1. Phosphorylation of 4E-BP1................................................................................. 11
1.1.6.2.2. Dephosphorylation of 4E-BP1 and Cleavage of eIF4G..................................... 12
1.1.6.3. Anticancer Gene Therapy and Pharmacotherapy..................................................... 12
1.2. Some Remarks about Thermodynamic Approach to Molecular Interactions in
Solution................................................................................................................................................. 13
1.2.1. Thermodynamic and Apparent Association Constant.................................................. 13
1.2.2. Gibbs Free Energy.......................................................................................................... 14
1.2.3. Thermodynamic Parameters of Macromolecular Interactions in Solution.................. 15
1.2.4. Noncovalent Interactions in Aqueous Solutions........................................................... 17
1.2.5. Parsing of Gibbs Free Energy........................................................................................ 18
1.2.6. Thermodynamics of Coupled Processes ....................................................................... 18
2. AIM OF THE WORK...................................................................................................... 20
3. MATERIALS AND METHODS ..................................................................................... 22
3.1. Chemical and Biochemical Syntheses................................................................................. 22
3.1.1. Cap Analogues ............................................................................................................... 22
3.1.2. Peptides........................................................................................................................... 22
3.1.3. Protein Biosynthesis....................................................................................................... 24
3.2. Preparation of Protein and Peptide Samples to Spectroscopic Measurements ........... 24
3.3. Absorption and Emission Spectroscopy............................................................................. 25
3.3.1. Fluorescence Quenching................................................................................................ 26
3.3.1.1. Static Quenching........................................................................................................ 26
3.3.1.2. Dynamic Quenching.................................................................................................. 27
3.3.1.3. Resonance Energy Transfer ...................................................................................... 27
3.3.1.3.1. Resonance Energy Transfer in eIF4E ................................................................. 28
3.3.2. Tryptophan Fluorescence in Proteins............................................................................ 28
3.3.3. Quenching of eIF4E Fluorescence by Cap Analogues................................................. 29
V

3.4. Experimental Conditions of Spectroscopic Measurements.............................................30
3.4.1. Titration Assay................................................................................................................31
3.4.2. Fluorescence Data Corrections ......................................................................................31
3.4.2.1. Inner Filter Effect......................................................................................................31
3.4.2.2. Dilution.......................................................................................................................32
3.4.2.3. Fluorescence Drift in Time........................................................................................33
3.5. Fluorescence Data Analysis..................................................................................................33
3.5.1. Simultaneous Determination of Association Constants and Protein Activity.............33
3.5.2. The Gibbs Free Energy of Binding................................................................................35
3.5.3. Osmotic Stress and Electrostatic Interactions...............................................................35
3.5.3.1. Davies-Stockes-Robinson Electrostatic Screening Approach .................................35
3.5.3.2. Wyman Linkage Analysis .........................................................................................36
3.5.4. Protonation Equilibria ....................................................................................................37
3.5.5. Thermodynamics ............................................................................................................38
3.5.5.1. Coupling Between Binding and Conformational Transition of Ligand..................38
3.5.5.2. Enthalpy-Entropy Compensation in Congener Series – General Model 199 .............39
3.6. Isothermal Titration Calorimetry.......................................................................................40
3.6.1. Calorimetric Measurements for m 7 GpppG....................................................................41
3.6.1.1. Modified "Single Injection" Experiment ..................................................................41
3.6.2. Calorimetric Data Treatment .........................................................................................42
3.6.2.1. Buffer Ionization Heats..............................................................................................42
3.7. NMR Spectroscopy................................................................................................................42
3.7.1. NMR Spectra Recording................................................................................................43
3.7.2. NMR Data Analysis .......................................................................................................44
3.7.2.1. Concentration Dependence........................................................................................44
3.7.2.2. Temperature Dependence..........................................................................................44
3.8. Dynamic Light Scattering ....................................................................................................45
3.9. Statistical Analysis.................................................................................................................46
3.10. Additional Information − Crystallography...................................................................47
4. RESULTS AND DISCUSSION..................................................................................... 48
4.1. Methodological Aspects of Studies of Non-Enzymatic Protein.......................................48
4.1.1. Solution of Spectroscopic Difficulties...........................................................................49
4.1.2. Activity of Non-Enzymatic Protein...............................................................................51
4.1.3. Incorrectness of Data Linearization in Case of Strong Interactions ............................58
4.2. Molecular Mechanism of Recognition of mRNA 5' Cap Structure by eIF4E Cap-
Binding Protein ...................................................................................................................................60
4.2.1. Affinity of Cap Analogues for eIF4E............................................................................60
4.2.1.1. Equilibrium Binding Constants.................................................................................60
4.2.1.2. Binding Affinity vs Inhibitory Properties of Cap Analogues ..................................62
4.2.2. Parsing the Free Energy of eIF4E Binding to mRNA 5' Cap.......................................64
4.2.2.1. Energetic Cost of 7-Substituent Alteration...............................................................66
4.2.2.2. General Separation of the Binding Free Energy.......................................................68
4.2.2.3. Relation of Stacking - Hydrogen Bonding Cooperativity to Other Cap-Binding
Molecules ....................................................................................................................................69
4.2.2.4. Energetic Cost of Methylation of the N(2)-Amino Group.......................................70
VI

4.2.2.5. Energetic Cost of the Second Nucleoside Addition and Modification ................... 70
4.2.2.6. Contributions of Phosphate Groups and Trapped Water Molecules....................... 72
4.2.3. Interactions in Context of Environment........................................................................ 73
4.2.3.1. Interaction of eIF4E-Cap in the Presence of Electrolyte ......................................... 74
4.2.3.1.1. Davies-Stockes-Robinson Electrostatic Screening Approach........................... 74
4.2.3.1.1.1. Electrostatic Effects ...................................................................................... 74
4.2.3.1.1.2. Two-Step Mechanism of the Cap-eIF4E Association ............................. 77
4.2.3.1.1.3. Relation to the "Clamping Cycle" ............................................................ 78
4.2.3.1.1.4. Osmotic Stress............................................................................................... 78
4.2.3.1.2. Wyman Linkage Analysis ................................................................................... 79
4.2.3.1.2.1. Electrolyte Effect........................................................................................... 79
4.2.3.1.2.2. Osmotic Stress............................................................................................... 80
4.2.3.1.3. Comparison of the Two Approaches .................................................................. 80
4.2.3.1.4. Reasons for KCl-Dependence of Internal Rearrangement Rate Constants....... 80
4.2.3.1.5. Discussion of Hydration Effects ......................................................................... 81
4.2.3.1.6. Conformational Change of eIF4E upon Cap Binding........................................ 82
4.2.3.1.6.1. Deaggregation of eIF4E Induced by Cap Binding ...................................... 82
4.2.3.2. Ionic Equilibria in eIF4E-Cap Complex................................................................... 83
4.2.4. Conclusions .................................................................................................................... 85
4.3. Molecular Interactions within Ternary Complexes Involving eIF4E, a Cap Analogue,
and an eIF4E-Binding Motif from eIF4G or 4E-BP1 Proteins.................................................... 86
4.3.1. eIF4E Fluorescence Quenching Pattern upon Peptide Binding................................... 87
4.3.2. Cooperativity in Ternary Peptide-eIF4E-Cap Complexes ........................................... 90
4.3.2.1. Cooperativity of Cap and eIF4G Peptides Binding.................................................. 91
4.3.2.2. Cooperativity of Cap and 4E-BP1 Peptides Binding............................................... 93
4.3.3. Regulation of 4E-BP1 Activity by Phosphorylation .................................................... 94
4.3.4. Concluding Remarks...................................................................................................... 96
4.4. Thermodynamics of mRNA 5' Cap Binding by eIF4E .................................................... 97
4.4.1. Van't Hoff Analysis of Binding of eIF4E to Cap Analogues....................................... 97
4.4.2. Direct Calorimetric Measurements of Binding Enthalpy........................................... 106
4.4.3. Protonation Equilibrium of m 7 GpppG ........................................................................ 108
4.4.4. Conformational Equilibrium of Cap Analogues......................................................... 110
4.4.5. Could the Positive Values of ΔCp° Result from Protein Deaggregation ? ................ 113
4.4.6. Enthalpy-Entropy Compensation for Individual Cap Analogues .............................. 113
4.4.7. Biological Implications of Non-Linear van't Hoff Thermodynamics........................ 116
4.4.8. Isothermal Enthalpy-Entropy Compensation in Congener Series ............................. 117
4.4.8.1. Molecular Interpretation of Isothermal Enthalpy-Entropy Compensation ........... 119
4.4.9. Inquiries about Origins of the Unusual Positive Heat Capacity Change................... 121
4.4.9.1. Changes of Solvent Accessible Surfaces Upon Binding ....................................... 122
4.4.9.2. Electrostatic Contributions to Heat Capacity Changes.......................................... 123
4.4.9.3. Tryptophan Stacking with Cationic 7-Methylguanosine Moiety .......................... 124
4.4.9.4. Coupling of Other Intermolecular Equilibria to eIF4E – Cap Binding................. 127
4.4.10. Linear Correlation between ΔCp° and ΔG°................................................................. 128
4.4.11. Discussion with Other Authors.................................................................................... 130
4.4.12. Conclusions .................................................................................................................. 131
5. SUMMARY................................................................................................................... 133
6. REFERENCES............................................................................................................. 135
VII

Abbreviations
used throughout the text:
4E-BP 4E binding protein;
A absorbance;
Å angström, 10 -10 m;
bz 7 GTP 7-benzylguanosine 5’-triphosphate;
CPK colours scheme developed by Corey, Pauling and Koltun: carbon - light grey;
oxygen – red; hydrogen – white; nitrogen - light blue; phosphorus –
orange.
cx complex;
DLS Dynamic Light Scattering;
DNA deoxyribonucleic acid;
DTT dithiothreitol;
EDTA disodium ethylenediaminetetraacetate;
eIF eukaryotic initiation factor;
ESI MS Electrospray Ionization Mass Spectrometry;
et 7 GTP 7-ethylguanosine 5’-triphosphate;
F observed fluorescence;
F0
calculated initial fluorescence;
GDP guanosine 5’-diphosphate;
GMP guanosine 5’-monophosphate;
GpppG P 1 -guanosine-5’ P 3 -guanosine-5’ triphosphate;
GTP guanosine 5’-triphosphate;
Hepes N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid];
HPLC High Performance Liquid Chromatography;
I ionic strength;
ITC Isothermal Titration Calorimetry;
k Boltzmann constant, 1.38 ⋅ 10 -23 J⋅K -1 ;
K observed association constant for stacking, determined for individual
protons from NMR signals;
Kas
Kas
observed association constant;
(micro)
Kas
observed microscopic association constant;
pH-ind
calculated, hypothetical association constant for binding of two
species in the ionic states optimal for binding;
Kt
thermodynamic association constant;
kDa kilodalton, 1000 atomic mass units;
1K self-stacking/unstacking equlibrium constant;
L ligand;
m2 2,7 GTP N 2 ,7-dimethylguanosine 5’-triphosphate;
m3 2,2,7 GTP N 2 ,N 2 ,7-trimethylguanosine 5’-triphosphate;
m 7 G 7-methylguanosine;
m 7 GDP 7-methylguanosine 5’-diphosphate;
m 7 GMP 7-methylguanosine 5’-monophosphate;
m 7 Gpppm 2’O G P 1 -7-methylguanosine-5’ P 3 -2'-O-methylguanosine-5’ triphosphate;
m 7 GpppA P 1 -7-methylguanosine-5’ P 3 -adenosine-5’ triphosphate;
m 7 GpppC P 1 -7-methylguanosine-5’ P 3 -cytosine-5’ triphosphate;
VIII

m 7 GpppG P 1 -7-methylguanosine-5’ P 3 -guanosine-5’ triphosphate;
m 7 Gpppm 7 G P 1 -7-methylguanosine-5’ P 3 -7-methylguanosine-5’ triphosphate;
m 7 GppppG P 1 -7-methylguanosine-5’ P 3 -guanosine-5’ tetraphosphate;
m 7 Gppppm 7 G P 1 -7-methylguanosine-5’ P 3 -7-methylguanosine-5’ tetraphosphate;
m 7 GTP 7-methylguanosine 5’-triphosphate;
M molar concentration (1 M = 1 mol⋅dm -3 );
MALDI-TOF MS Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass
Spectrometry;
mRNA messenger ribonucleic acid;
NMR Nuclear Magnetic Resonance;
NMWL nominal molecular weigh limit;
P probability that distribution of fitting residuals is random;
p, PO3 - ; phosphate group;
p-Cl-bz 7 GTP 7-(p-chlorobenzyl)-guanosine 5’-triphosphate;
Pact
active protein;
PDB Protein Data Bank (http://rcsb.icm.edu.pl/);
pH -log10 of H + activity approximated by concentration;
pHopt
pKa
optimal pH value;
-log10 of H + acidic dissociation constant;
pKL
-log10 from the effective acidic dissociation constant of a ligand;
pKP
-log10 from the effective acidic dissociation constant of a protein
residue;
P(ν1,ν2) probability that improvement of the fit with the smaller number of
degrees of freedom (ν2) in comparison with that with the greater
number (ν2) is random;
R universal gas constant, 8.3143 J⋅mol -1 ⋅K -1 ;
r 2
linear correlation coefficient;
R 2
goodness of fit;
RNA ribonucleic acid;
SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis;
t temperature in °C;
T absolute temperature in K;
TH
temperature at which ΔH° = 0;
TS
temperature at which ΔS° = 0;
[x] molar concentration of x expressed in M (1 M = 1 mol⋅dm -3 );
° denotes standard state; the hypothetical state of solute at the standard
concentration (1 mol⋅dm -3 ) and at arbitrarily chosen temperature of
293 K (20 °C), and exhibiting infinitely dilute solution behaviour; ∗
ε molar extinction coefficient;
δ chemical shift;
ΔCp° standard molar heat capacity change at constant pressure;
ΔCp°sst
contribution to standard molar heat capacity change, resulting from
thermodynamic coupling with self-stacking;
Δδ change of chemical shift (Δδ = δ(mixture) − δ(free));
ΔG° standard molar Gibbs free energy change (observed);
∗ according to IUPAC Compendium of Chemical Terminology, 2 nd Edition (1997)
IX

ΔG°el
contribution of electrolyte effect to standard molar Gibbs free energy
of complex stabilization;
ΔG° pH-ind
standard molar Gibbs free energy of binding of two species in the
ionic states that are optimal for binding;
ΔG°L
standard molar Gibbs free energy change of partial protonation of a
ligand at a given pH;
ΔG°P
standard molar Gibbs free energy change of partial deprotonation of a
protein residue at a given pH;
ΔH° standard molar enthalpy change;
ΔH°0
intrinsic standard molar enthalpy change;
ΔH°cal
calorimetric standard molar enthalpy change;
ΔH°ion
standard molar enthalpy of buffer ionization at given pH;
ΔH°sst
contribution to standard molar enthalpy change, resulting from
thermodynamic coupling with self-stacking;
ΔH°vH
standard molar van't Hoff enthalpy change;
1ΔH° standard molar enthalpy change of self-stacking;
ΔN number of exchanged water molecules;
ΔS° standard molar entropy change;
ΔS°0
intrinsic standard molar entropy change;
ΔS°el
contribution of electrolyte effect to standard molar entropy change;
ΔS°sst
contribution to standard molar entropy change, resulting from
thermodynamic coupling with selfsatcking;
1ΔS° standard molar entropy change of self-stacking;
λem
emission wavelength;
excitation wavelength.
λex
X

Preface
The thesis concerns the rules of intermolecular recognition of mRNA 5' terminus by
eukaryotic translation initiation eIF4E factor and thermodynamic origins of the complex
stability. Molecular spectroscopy and isothermal calorimetry were used to address the
mechanistic bases of the process of association. Conclusions resulting from the
experimental data were discussed in context of biological processes in the living cell and of
general thermodynamic features of protein-ligand interactions.
The work is composed of the following chapters:
1. INTRODUCTION which contains a concise and far from exhaustive survey of the
present knowledge about significance of the studied objects in biology and medicine,
and points to some selected thermodynamic aspects of macromolecular interactions,
that are fundamental but not always explicitly accented in literature;
2. AIM OF THE WORK defines the context and the particular goals of the thesis;
3. MATERIALS AND METHODS – provide necessary information about chemicals,
syntheses, applied equipment, experimental protocols, and procedures of data
analysing;
4. RESULT AND DISCUSSION are merged so that the results can be immediately
interpreted in light of other phenomena or literature data, without perpetual referring to
different chapters. The chapter addresses four main problems:
4.1. Methodological aspects of studies of non-enzymatic proteins;
4.2. Molecular mechanism of recognition of mRNA 5' cap structure by eIF4E;
4.3. Molecular interactions within ternary complexes that consist on eIF4E, a cap
analogue, and additionally involve an eIF4E-binding motif from either the
initiation factor 4G (eIF4G) or the translation inhibitor (4E-BP1). The former is
represented by two isoforms (eIF4GI and eIF4GII), and the latter is studied as the
unphosphorylated, monophosphorylated, and diphosphorylated species.
4.4. Thermodynamics of mRNA 5' cap binding to eIF4E and its possible consequences
for biological activity.
5. SUMMARY recollects the main conclusions drawn from the studies.
The results presented in this work were partially published, 1-7 some of them are in
press (Chapter 4.4.9.3.) or in preparation for submission (data from 4.1. and 4.4.).
XI

1. Introduction
1.1. Brief Description of Biological Aspects
Life of organisms depends on delicate balance of many biochemical processes. Such
balance needs sensitive and efficient regulatory mechanisms to coordinate growth,
development and reproduction. The most fundamental mechanisms are related to gene
expression.
1.1.1. Central Dogma of Molecular Biology
The Central Dogma was first proposed by F. Crick in 1958, and states that genetic
information flow is unidirectional, from DNA to protein via an RNA intermediate. 8 In
order to be expressed, genetic information encoded in DNA has first to be transcribed into
a complementary copy of RNA. There are some exceptions from this rule, i. e. reverse
transcriptases of retroviruses synthesize DNA from an RNA template, and RNA viruses
such as poliovirus or influenza virus apply RNA replication. 9
DNA replication
DNA
Reverse transcription
Transcription
(RNA synthesis)
Scheme 1-1. Central Dogma of Molecular Biology.
RNA transcripts are either used directly as transfer RNAs (tRNAs), ribosomal RNAs
(rRNAs), and small nuclear RNAs (snRNAs) or, as messenger RNAs (mRNAs), are
translated into proteins. The mRNA transcripts are cotranscriptionally processed by
capping (linkage of 7-methylguanosine by a 5' - 5' triphosphate bridge to the first
transcribed nucleoside), splicing (intron excision), and polyadenylation of the 3'
terminus. 10 The mature mRNA is transported into the cytoplasm (except for mitochondrial
or chloroplast protein mRNAs). In eukaryotes, transcripts contain one sequence encoding a
1
mRNA PROTEIN
RNA
replication
Translation initiation
Translation
(protein synthesis)

polypeptide, but some sequences can yield more than one polypeptide as a consequence of
alternative transcriptional starting sites and/or alternative splicing. Translation occurs on
ribosomes. The ribosome is made up of two nonidentical subunits, the large (60S) and
small one (40S). Each of them contains one or more rRNA molecules and 20-50 different
ribosomal proteins. Several ribosomes may simultaneously translate the same mRNA
molecule. Proteins are synthesized starting with their N termini, corresponding to
translation of the mRNA in the 5' to 3' direction. Translation may be divided into three
stages: initiation, elongation, and termination. Some proteins can fold to their native
conformation during elongation of the peptide chain, i. e. before the synthesis is
completed, or it is only after that, often with help of other proteins, molecular chaperones.
Proteins can undergo many post-translational covalent modifications, e. g. proteolysis,
protein splicing, phosphorylation, and disulphide bond formation, yielding finally the
biologically active form. 11
1.1.2. Protein Functions
Proteins participate in almost every biological process. Exemplary functions, which
proteins are responsible for, are as follows: structure building, metabolism, transport
throughout body, storage of ions, membrane transport, muscle contraction, osmotic
regulation, immunological defence, cell recognition, generation and transduction of
nervous signals, and control functions. 9 Control factors are even by orders of magnitude
less abundant than structural proteins or enzymes, but they perform superior functions in
the living cell, regulating abundancies and activities of the remaining proteins. Protein
control factors act as hormones and fulfil control on gene expression, cell growth,
proliferation, and differentiation.
One of the eukaryotic regulatory proteins is eIF4E, initiation translation factor 4E,
the key protein in translational control of biosynthesis. 12
1.1.3. Translation Initiation
Translation initiation is a complex (Scheme 1-2), rate-limiting step of gene
expression. 13 During initiation, the correct starting site on mRNA is identified and binding
of the ribosome occurs. All mRNAs and snRNAs that are transcribed in the nucleus
possess a 5'-terminal "cap", m 7 GpppN, in which 7-methylguanosine is linked by a 5’-5’-
triphosphate bridge to the first nucleoside 14 (Scheme 1-3). The cap structure participates in
2

Scheme 1-2. Eukaryotic translation initiation pathway. Initiation factors (eIF) are shown as colourcoded
labelled circles, and appear as rectangulars when first implicated in the pathway. 60S, 40S -
large and small ribosomal subunits, respectively; Met – initiator methionyl-tRNA; m7G AUG -
mRNA with the 7-methylguanosine cap and the starting codon (AUG). eIF4E is responsible for
recognition of the mRNA cap and mediates joining of the remaining eIFs, which finally leads to
ribosome recruitment and positioning. Scheme adopted from Hershey and Merrick. 15
3

the splicing of mRNA precursors, 16,17 is necessary for RNA nuclear export 18-20 and for
optimal mRNA translation, 14,21 and affects mRNA stability. 19 After nuclear export to the
cytosol, the cap of snRNAs is further methylated at the amino group of the guanosine
moiety, forming m GpppN
7 , 2 , 2
22
This trimethylated structure is responsible for import of
3
the snRN-protein spliceosomal complexes (snRNPs) back into the nucleus, 23,24 where
snRNPs take part in pre-mRNA splicing. 25 A variety of mRNA cap structures can be
present in the cytoplasm, and most of them are additionally methylated at 2'O of the second
ribose moiety: m 7 Gpppm2 6,2'O A, m 7 Gpppm 2'O G, m 7 Gpppm 2'O A, m 7 Gpppm 2'O C, m 7 GpppA,
and m 7 GpppG. 26 The original mRNA cap can be replaced by the trimethylguanosine cap
structure as a result of trans-splicing which occurs in primitive animals, 27,28 e. g. in the C.
elegans nematode about 70% of the mRNA population contains GpppN
4
m 7 , 2 , 2
3
Regular translation initiation is cap-dependent, though there are also two alternative
ways to recruit the 40S ribosome subunit to the mRNA, mediated by an internal ribosome
entry site (IRES) or by the poly(A) tail. 30 The cap function in translation initiation is
mediated by the eukaryotic initiation factor 4E (eIF4E), 12 which is the smallest subunit of
the heterotrimeric eIF4F initiation complex. 31 eIF-4F appears to be also required for the
cap-independent translation of naturally uncapped mRNAs. 32 In higher eukaryotes eIF4F
consists of eIF4E, eIF4A, an RNA-dependent ATPase and RNA helicase, and eIF4G, a
multifunctional, adaptor protein. 30,31,33 Two isoforms of eIF4G, namely eIF4GI (171
kDa) 34 and eIF4GII (176 kDa) 35 are present in mammals. These scaffolding molecules act
as a molecular bridge between eIF4E and eIF4A. 33,35 The eIF4Gs interact with the
ribosome-bound multiprotein factor eIF3, to recruit the 40S ribosomal subunit. Additional
interaction of the eIF4Gs with the poly(A)-binding protein (PABP) allows mRNA
circularization 30,31 and ribosome recycling. 36
* H
N
H 2
O
N 6
1
2
3
N
CH 3
N
7
+
9
N
8
H
H
H
H
OH
HO
O
CH 2
H
O O O
O P O P O P O
O - O - O -
Scheme 1-3. Chemical structure of eukaryotic mRNA 5' terminus called "cap". Proton that partially
dissociates at physiological pH is marked with asterisk.
CH 2
H
H
O
O
H
Base
H
. 29
OH O-H or -CH3 mRNA chain

1.1.4. Eukaryotic Initiation Factor 4E
The eukaryotic initiation factor 4E (eIF4E) is a 25 kDa protein (217 amino acids)
which plays a pivotal role in translational control. eIF4E is a cytoplasmic protein, and acts
as a subunit of the eIF4F initiation complex. However, a fraction of eIF4E localizes also in
the nucleus 37 and participates in nucleocytoplasmic transport of specific, growth regulatory
mRNA. 38 Since eIF4E colocalizes in nuclear speckles with splicing factors, it is also
supposed to be involved in splicing and 3' mRNA processing. 39 All these additional
functions of eIF4E appear to be dependent on its intrinsic ability to recognize the mRNA 5'
cap. Thus, this single biochemical activity of eIF4E is versatile enough to play roles in
divergent processes in different cellular compartments. The shuttling protein, eIF4E-
transporter (4E-T), that mediates the nuclear import of eIF4E via the α/β importin pathway
by a piggy-back mechanism was discovered recently. 40
eIF4E is the least abundant protein initiation factor in the cell (0.8 ⋅ 10 6 molecules
per 1 HeLa cell, i. e. only 0.021 % of cell protein), 41 and its accessibility for formation of
the initiation eIF4F heterocomplex is supposed to regulate the overall efficiency of
ribosome recruitment. 31,42 Expression of eIF4E is regulated at the transcription level. 43 On
the other hand, eIF4E is a very instable protein; its instability index calculated on the basis
of the sequence 44 is 52.17 for human full length eIF4E and 47.41 for murine protein
(residues 28-217) (Table 1-1). This biochemical property may account for the low
abundance of eIF4E in the cell. Interaction between eIF4E and eIF4G upon eIF4F complex
formation is inhibited by 4E-binding proteins (mammalian 4E-BP1, 4E-BP2, 4E-BP3, 31
and yeast p20 45 ).
Table 1-1. Comparison of protein stabilities resulting from frequency of occurrence of particular
dipeptides in the amino acid sequences. 44
Protein (Organism) Instability Index
eIF4E (residues 1-217; human) 52.17
eIF4E (residues 28-217; mouse) 47.41
stability limit 40
BPTI (bovine) 32.60
S100 (human) 25.22
PNP (E. coli) 23.13
lysozyme (mouse) 17.76
5

Figure 1-1. Sequence alignments of eIF4E from mammalia. The relative accessibility of each
protein residue was extracted from DSSP 46 and PHD 47 files and shown as colour boxes: white –
buried; cyan – intermediate accessible; blue – accessible; blue with red borders – exposed into
solvent; red – unstructured lysine side chains; gap – unstructured main chain. The secondary
structural elements were assigned from the X-ray structures of human eIF4E in complex with
m 7 GpppA (1IPB.pdb 48 , top) and murine eIF4E with m 7 GpppG (1L8B 1 , bottom); α - alpha helices;
η - 310 helices; β - beta strands; TT - strict beta turns.
Table 1-2. Characteristics of eIF4E sequence conservation among organisms
Group of Organisms (Protein Isoform) Identity (%) Similarity (%)
Mammalia 98 99
Vertebrates 88 95
Animals a
19 49
Plants (eIF4E-1) 55 69
Plants (eIF4E-2) 52 69
Plants b
26 53
Fungi c
28 50
All eukaryotes (eIF4E-1) 10 35
All eukaryotes d
5 23
Human vs Yeast 29 44
a including only eIF4E-1 from Caenorhabditis elegans
b including both eIF4E-1 and eIF4E-2 isoforms
b including only eIF4E-1 from Schizosaccharomyces pombe
c including all isoforms of eIF4E (33 sequences)
6

Figure 1-2 (in the previous page). Sequence alignments of eIF4E (or eIF4E-1 if more isoforms
exist) from various organisms: human 49 , mouse 50 , rabbit 51 , Xenopus laevis 52 , Aplysia californica 53 ,
Drosophila melanogaster 54 , Caenorhabditis elegans 55 , maize 56 , rice 57 , wheat 58 , Arabidopsis
thaliana 59 , Candida glabrata 60 , Saccharomyces cerevisiae 61 , Schizosaccharomyces pombe 62 . The
sequences are separated in groups corresponding to the animals (1), plants (2) and fungi kingdoms
(3). The alignments were done by the ClustalW program 63 and are colour coded according to the
following rules: red box, white character - strict identity; red character - similarity in a group (>
70% of residues are similar according to physico-chemical properties); blue frame - similarity
across groups; orange box - differences between conserved groups (> 50% of residues are
conserved within a group but not conserved from one group to the other). Risler matrix was used
for similarity and difference score calculations 64 . A consensus line indicates as follows: uppercase
– identity; lowercase - consensus level > 0.5; ! is I or V; $ is L or M; % is F or Y; # is N, D, Q or E.
eIF4E activity is also regulated by phosphorylation at Ser209 in response to
treatment of cells with growth factors, hormones, and mitogens. 65-67 Phosphorylation
seems to positively correlate with protein synthesis, cell growth and proliferation, 66,68,69 but
the explanation how it could happen at the molecular level remains unclear. 70-74
eIF4E is phylogenetically highly conserved (Table 1-2, Fig. 1-1, 1-2) and unusually
rich in tryptophans. Each of its eight tryptophans is absolutely conserved among all known
sequences, from fungi to human. Three-dimensional structures of murine eIF4E bound to
7-methylGDP 75 and 7-methylGpppG, 1 and recently also of human eIF4E in complex with
7-methylGTP and 7-methylGpppA 48 were solved by X-ray crystallography. The structure
of their yeast homologue bound to 7-methylGDP was established by solution NMR
spectroscopy. 76 Each protein consists of one α/β domain, and is shaped like a cupped hand,
with the cap analogue located in a narrow cap-binding slot on the concave surface of the
protein (Fig. 1-3). Recognition of the 7-methylguanine moiety by the human and murine
proteins is mediated by base sandwich-stacking between Trp56 and Trp102, formation of
Watson-Crick-like hydrogen bonds with a side chain carboxylate of a conserved Glu103
and a backbone NH of Trp102, and a van der Waals contact of the N(7)-methyl group with
Trp166. The phosphate chain forms salt bridges and direct or water-mediated hydrogen
bonds with the NH of Trp102 and Trp166 indole rings, and side chains of Arg112, Lys162,
Arg157, and Lys 206. As regards the second nucleoside, adenosine is stabilized by a direct
hydrogen bond between N 6 -amino group and the backbone carbonyl of Thr205. The
second guanosine in invisible in the crystal structure since it is not capable of forming this
hydrogen bond. At the same time, the protein loop containing Thr205 is disordered.
8

Figure 1-3. Global fold of eIF4E bound to a fragment of cap, 7-methylguanosine 5’-triphosphate
(shown as sticks in CPK colours). 1 Secondary structure elements are colour coded: yellow - β
strands, magenta - α helices, white - loops and turns. Amino acid belonging to the cap-binding site
are shown as balls and sticks: blue – Lys206, Arg112, Lys162, Arg157 (from the left); purple –
absolutely conserved tryptophans 102, 166, 56 (from the left), red – glutamic acid 103.
1.1.5. Translational Control
To the middle nineties of the XX-th century transcription was thought to be the main
subject to a multitude of controls. 9 However, the cell applies also a lot of regulatory
mechanism at the level of translation. Translational control provides the cell with benefits
more important than the energetic cost paid for the reaching that step of gene expression. 77
The most conspicuous advantage of translational control is immediacy. Direct and rapid
intervention gives the cell no time to adjust coupled biochemical reactions and the protein
synthesis is immediately stopped. On the other hand, most translational controls consist in
reversible modifications of translation factors, e. g. phosphorylation. The reversibility is
fast and economical in energetic terms, especially for energy-deprived cells. The changes
in transcription rates are considerably greater in magnitude than the changes in translation
9

ates. Thus, while transcription is responsible for the coarse control, translation provides a
means for fine regulation. Moreover, it enables a spatial control of protein synthesis, which
plays a key role in effecting synaptic plasticity in the hippocampus. 78 The local protein
synthesis at a stimulated synapse leads to the synapse-specific growth. This, in turn, is
necessary to long-term potentiation that underlies processes of learning and memory
encoding. 79,80 Translation gives a possibility to control of expression of a single gene or of
whole classes of mRNAs. For instance, host-cell shutoff induced by heat shock 81 or by
picornaviruses, 82 as well as retroviral gene expression (such as human immunodeficiency
virus type 1, HIV-1 83 ) are controlled at the translational level. Translational controls are
common for regulation of gene expression during development (e. g. in oocytes), and in
other systems lacking transcriptional control. Finally, translational control has proven to be
an important factor in human cancer. 84-87
1.1.5.1. Regulation of eIF4F Complex Formation
The accessibility of eIF4E in mammals is regulated by interactions with small
translational repressors, eIF4E-binding proteins, 4E-BP1, 4E-BP2 and 4E-BP3, which
prevent productive interactions between eIF4E and eIF4G. 88,89 They also inhibit
phosphorylation of eIF4E at Ser 209. 68
Sequence analysis of the 4E-BPs and eIF4Gs suggests that these otherwise unrelated
protein families have converged on the same eIF4E binding strategy. 45,89 The eIF4E
binding site shared between the 4E-BPs and the eIF4Gs is BXXYDRXFLΦ, where B is a
conserved basic residue, X is variable, Φ is a conserved hydrophobic residue, and invariant
residues are shown in boldface. 34,35,67,90 Crystal structures of two ternary complexes of
eIF4E with 7-methyl-GDP and peptides encompassing the eIF4E binding sites, derived
from eIF4GII or 4E-BP1, revealed that both peptides recognize a phylogenetically
invariant, partially hydrophobic, partially acidic surface of the convex dorsum of eIF4E in
the vicinity of Trp73, and share a common mode of interaction. 91
Some of the hypophosphorylated forms of 4E-BPs bind to eIF4E and these
interactions are highly regulated in cells. 82 Hyperphosphorylation abrogates the interaction
with eIF4E, which enables recruitment of eIF4G, allowing eIF4F complex formation and
translation initiation to proceed. 67,92 The phosphorylation mechanism has been extensively
investigated for 4E-BP1. The protein possesses at least six phosphorylation sites: Thr37,
Thr46, Ser65, Thr70, Ser83 and Ser112 (numbering for the human protein). The ordered,
10

hierarchical model of phosphate addition to endogenous 4E-BP1, in which phosphorylation
of Thr37 and Thr46 is followed by phosphorylation of Thr70 and finally of Ser65, 3,92 is
generally accepted, although some points remain controversial. 93-95 At issue is the
influence of the single phosphorylation of Ser65 or combined Ser65/Thr70
phosphorylation on the 4E-BP1 binding to eIF4E. 96
1.1.6. Clinical Aspects of the Cellular eIF4E Activity Control
1.1.6.1. Relation of eIF4E Activity to Immunology and Malignancy
Since initiation is a rate-limiting step for translation, it is not surprising that eIF4E
activity is frequently related to disorder of cellular proliferation, growth and
differentiation. 87 The eIF4E gene is a proto-oncogene. Overexpression of eIF4E is caused
by gene amplification. 85 When present in excess of the normal physiological amount,
eIF4E can stimulate global protein synthesis. 97 Elevated levels of eIF4E were found in
many cancers, and were shown to stimulate division and cause malignant transformation of
both normal and immortal cells. 87 Immunological detection of eIF4E was even reported as
a useful diagnostic tool to identify malignant cells for removal during surgery of head and
neck cancer. 98 Affinity of eIF4E for mRNA 5' cap is strongly reduced by the nuclear
promyelocytic leukaemia protein (PML) which mediates suppression of oncogenic
transformation and of growth. 99
On the other hand, enhanced eIF4E activity and its phosphorylation at Ser209 are
engaged in signalling pathways necessary for human peripheral blood T lymphocytes to
progress from the resting state to proliferation and to attain immune competency. 66
1.1.6.2. Relation of 4E-BP1 and eIF4G Activities to Human Health and Disease
1.1.6.2.1. Phosphorylation of 4E-BP1
4E-BP1 is also known as PHAS-I, phosphorylated heat- and acid-stable protein
regulated by insulin, since insulin causes 4E-BP1 to become hyperphosphorylated and
dissociate from eIF-4E, thus stimulating the overall rate of translation and promoting cell
growth. 67
Activation of human blood T lymphocytes needs the release of eIF4E by
hyperphosphorylation of 4E-BP1 to enable synthesis of proteins that are important for
proliferation. 66
Dissociation of 4E-BP1 from eIF4E is involved in tumour cell survival. In particular,
11

the α6β4 integrin stimulates the phosphorylation and inactivation of 4E-BP1, thus
preventing inhibition of eIF4E, and finally enhancing translation of vascular endothelial
growth factor (VEGF) in human breast carcinoma cells. 100
1.1.6.2.2. Dephosphorylation of 4E-BP1 and Cleavage of eIF4G
In contrast, heat shock 81 and infection of cells with picornaviruses, such as poliovirus
and encephalomyocarditis virus, 82 render 4E-BP1 dephosphorylated. Picornaviruses use a
cap-independent mechanism for the translation of their RNA, and specific inhibition of
cap-dependent translation by activation of 4E-BP1 is thought to be the major cause of a
shutoff of host protein synthesis in encephalomyocarditis virus-infected cells. Poliovirus
acts doubly, and its second strategy involves also translational control at the initiation
level, since the infection results in the cleavage of the eIF4GII protein. 101 The eIF4GII
protein is also a target of the rhino and foot-and-mouth-disease viral proteases, 102 while the
HIV-1 viral protease cleaves the eIF4GI protein. 83
Rapid cleavage of eIF4GI by caspase-3 occurs also during induced apoptosis. 103,104
1.1.6.3. Anticancer Gene Therapy and Pharmacotherapy
4E-BP1 belongs to a class of multiphosphorylated proteins that are dephosphorylated
rapidly after rapamycin treatment. 105 Rapamycin represents a novel family of agents of
antibiotic, antifungal, and, which is the most important, immunosuppressory and
antitumour activity. 106 Rapamycin acts successfully as a cytostatic or induces apoptosis in
malignant cells through inhibition of a function of mTOR (mammalian target of
rapamycin) which is a huge, highly conserved protein (~280 kDa). mTOR is a central
controller of cell growth and proliferation, influencing an unusually abundant and diverse
set of reactions. 107 It acts both as a protein-kinase and protein-phosphatase regulator, and
among others it modulates 4E-BP1 phosphorylation. The mTOR − 4E-BP1 signalling
pathway is important not only for malignancy but also for proliferation in T lymphocytes. 66
The Food and Drug Administration has approved rapamycin in 1999, and the European
Commission in 2000. However, recent data indicate that genetic mutations or
compensatory changes in tumour cells render resistance to rapamycin. 108,109
The important role for translation initiation in the regulation of the cell cycle makes
that other components of the initiation machinery already begin to be considered attractive
targets for therapeutic intervention. Attention is focused at the activity of 4E-BP1, as a
12

crucial mTOR-controlled downstream effector molecule, and at eIF4E. The most recent
publications (2002) report that cellular activity of eIF4E is already now exploited to
selectively kill breast cancer cells during clinical gene therapy. 110 However, there are still
no chemotherapeutic inhibitors of the eIF4E activity. Thorough biophysical studies of
functional complexes involving eIF4E can make up for this delay and provide a specific
inhibitory agent based on the rationally modified cap structure.
1.2. Some Remarks about Thermodynamic Approach to
Molecular Interactions in Solution
Mechanistic insights into molecular bases of biological activity do not flow
naturally from the description of the crystal structure. The stationary structure does not
reveal all exchange processes that need completing so that the final state, detectable by
crystallography, can come into being. In general, proteins and ligands structures are not
rigid. In particular, non-enzymatic regulatory protein factors are often very flexible and
experience large and even global conformational changes upon binding to their targets, in
contrast to most of enzymes. Such features are characteristic for e. g. transcription factors
that interact with DNA. 111 In addition to conformational transitions, formation of the
functional macromolecular complexes is accompanied by changes of interactions with the
environment, first of all within the first-layer solvation shell. Hence, the static structural
view needs to be supplemented by thermodynamic description in order to render the
biological functioning on the molecular level comprehensible.
Binding studies of specific complexes involving proteins, nucleic acids and small
ligands have proven to be useful to investigate the stabilization energy and the influence of
solvent on the complex formation, e. g. electrolytic effect, 112-116 water exchange between
the macromolecules and the bulk solvent, 117-121 and linked protonation equilibria. 122-124
1.2.1. Thermodynamic and Apparent Association Constant
A thermodynamic equilibrium constant (Kt) which is a function of temperature (T)
and pressure (p) only, is expressed in terms of equilibrium molar activities (ai): 125
∏ α
νi
t = a i
i=
1
K , Eq. 1-1
where νi are stoichiometric coefficients of the reaction and summation ranges by all
species taking part in the reaction. A logarithmic form of this relationship:
13

log( K
t
∑ i
i 1
α
=
) = ν ⋅ log( a )
Eq. 1-2
i
makes it possible to analyse easily the dependence of Kt on the presence of individual
participants of the reaction: macromolecules, cations, anions, protons, and water.
An experimentally observed equilibrium association constant (Kas) is defined for
the process converting chosen reactants (e. g. protein (P) and ligand (L)) to products (e. g.
complex (cx)) at a specified set of solution conditions, i. e. when activities of remaining
reactants (e. g. ions, protons, water molecules) are fixed, e. g.: 118
K
as
[ cx]
= Eq. 1-3
[ P]
⋅[
L]
0
0
This apparent constant is formulated in terms of concentrations of only a protein, a ligand
and their complex, instead of thermodynamic activities of all interacting species that could
putatively participate in the reaction. Kas contains involved, hidden information of both the
true thermodynamic association constant (Kt) and the changes of the activity coefficients
as a function of the solution conditions. Therefore, the observed equilibrium binding
constant depends on the environmental variables, i.e. pH, ionic strength and osmolality of
the solution. 118,119,126 Hence, Kas is a relative quantity. To probe the molecular and
thermodynamic origins of stability and specificity of eIF4E interactions with cap in
solution the binding studies need to be enriched by measurements at different experimental
conditions. This is a starting point to analyse possible intermolecular processes that can
accompany binding of cap analogues by eIF4E: protonation or ion and water exchange.
1.2.2. Gibbs Free Energy
Chemical potential (μi) of interacting species at given conditions consists on the
standard potential (μi°, index "°" refers to the pseudostandard state at concentrations of 1
mol/dm 3 , i.e. unit molarity 127 ) and a contribution (μi m ) which depends on the presence of
other solution components (xi): 125
o
m
μ = μ T,
p)
+ μ ( T,
p,
x ,..., x )
Eq. 1-4
i i ( i 1 α−1
The activity of species is defined as:
⎛ m
⎞
⎜
μi
( T,
p,
x1,...,
x α−1
)
a
≡
⎟
i ( T,
p,
x1,...,
x α−1
) exp
, Eq. 1-5
⎜
⎟
⎝
RT
⎠
m
so: i ≡ RT ln a i
μ . Eq. 1-6
14

Definition of the molar Gibbs free energy change (ΔG):
∑ α
i
i=
1
ΔG
≡ μ ν
Eq. 1-7
at equilibrium, where:
m
Δ ∑ i
i 1
α
=
i
G = ΔG°
+ μ νi
= 0
leads to the relationship for the standard molar Gibbs free energy change (ΔG°):
o
t
15
Eq. 1-8
Δ G = −RT
ln K . Eq. 1-9
Stability of a noncovalent complex is determined by the difference in noncovalent
interactions involving both the macromolecular recognition surfaces and solvent
components in the complex and in the uncomplexed state. Endeavour to interpret the
apparent Gibbs free energy of binding (ΔG°app) defined by the apparent association
constant (Kas) and not by the thermodynamic association constant must take into account
the fact of the participation of water, electrolyte ions and other solution components in the
interactions of a protein with a ligand, as well as the other macromolecular equilibria.
For shortening, the apparent Gibbs free energy change (ΔG°app) will be simply
referred to here as "the Gibbs free energy change" but one should bear in mind that this
"ΔG°" will be related to the observed and not to the thermodynamic association constant.
1.2.3. Thermodynamic Parameters of Macromolecular Interactions in
Solution
The Gibbs free energy change in an isothermal-isobaric process is related to changes
in standard molar enthalpy (ΔH°) and entropy (ΔS°): 128
ΔG ° = ΔH°
− TΔS°
. Eq. 1-10
At constant but different temperatures the process is described by different pairs of
the ΔH° and ΔS° values, since they can vary with temperature according to the standard
molar heat capacity change at constant pressure ( Δ ) and to two critical temperatures that
are characteristic for the analysed process:
o o
H p H
Δ = ΔC
( T − T ) ,
o
C p
o
Δ H = 0 at TH; Eq. 1-11
o o ⎛ T ⎞
ΔS
= ΔC
p ln
⎜
⎟ , ΔS° = 0 at TS; Eq. 1-12
⎝ TS
⎠

⎛ ∂H°
⎞
C p = ⎜ ⎟
⎝ ∂T
⎠ p
Δ o
The parameters of interest can be determined from the van't Hoff equation:
ln K
as
o p
ΔC
⎡TH
⎛ TS
⎞ ⎤
= ⎢ − ln⎜
⎟ −1⎥
R ⎣ T ⎝ T ⎠ ⎦
16
Eq. 1-13
Eq. 1-14
The isobaric heat capacity in solution is related to the fluctuations in internal energy
(E) of the system ( X denotes an ensemble average of a quantity X):
B
( ) 2
E E
1
C p ≈ C v = −
Eq. 1-15
2
k T
In most cases, the thermodynamic properties of the system expressed by ΔCp° are
dominated by changes in water structure induced by macromolecular association. The
increased energy fluctuations are related to lack of possibility of formation of hydrogen
bonds by water molecules forced to contact with nonpolar (nonpolarizable) or aromatic
(polarizable) solvent accessible groups of macromolecules (so called "hydrophobic
effect"). The nature of aromatic rings shows that the terms "hydrophobic" and "nonpolar,
apolar" must not be used equivalently, since the aromatic systems can carry induced charge
distribution, being then partly polar, yet still hydrophobic objects. 129 Consequently, their
contribution to the heat capacity changes, related to changes in the solvent accessible
surface area (SASA), differs from that of aliphatic groups 130 (Table 1-3). The fluctuations
of water energy are silenced when hydrogen bonds are maximally satisfied, i. e. when
polar (uncharged) or charged molecular surface is water exposed. The second main origin
of energy fluctuation in the system is related to macromolecular internal soft vibrations. 131
Table 1-3. Increments to the standard molar heat capacity resulting from exposure of 1 Å of
aliphatic, aromatic, and polar molecular surface to water (ΔCp°/SASA); data from different groups
of authors.
ΔCp° transfer /SASA (J⋅mol -1 ⋅K -1 ⋅Å -2 ) Authors (date) ref
Surface: aliphatic aromatic polar
+1.88 ± 0.08 a
−1.09 ± 0.13 Murphy et al. (1991, 1992) 132,133
+1.34 ± 0.17 a
−0.59 ± 0.17 Spolar et al. (1992) 134
+2.14 +1.55 −1.27 Makhatadze & Privalov (1995) 130
a average values for aliphatic and aromatic surfaces

1.2.4. Noncovalent Interactions in Aqueous Solutions
"Energy of hydrogen bonds ranges from −12.5 to −38 kJ/mol" is the habitually
repeated statement in the context of protein stability and protein-ligand interactions, both
in many biochemical textbooks 135-137 as well as during academic lectures. However, this is
true only if the "energy" means the internal energy (ΔE°) calculated usually in vacuum,
and not the enthalpy (ΔH°) and Gibbs free energy (ΔG°). Binding between
macromolecules in aqueous solution is an exchange, entropy-related process involving
both water and solutes, since it needs disruption of pre-existing interactions of individual
groups of each macromolecule with water and ions, and replacement of these interactions
by contacts between complementary groups on each macromolecule. Hence, the stability
of noncovalent complexes is totally different than in vacuum, and is highly dependent on
the details of the solution environment. The real Gibbs free energy of the hydrogen bond
formed in the water accessible region of the macromolecule dissolved in aqueous solution
is only from −3 to −8 kJ/mol, 138-143 which may appear surprising but is a well documented
rule. This is due mainly to entropic effects related to the presence of surrounding polar
water molecules that compete for the hydrogen bond formation with the residues of the
macromolecule. The strong decrease in free energy of a single amide hydrogen bond
formation in water in comparison with that in apolar solvent (CCl4), from –35.31 ± 0.46 to
−1.42 ± 1.2 kJ/mol, has been also demonstrated theoretically. 144 Similar entropic effects
due to the presence of counterions should be taken into account when analysing the Gibbs
free energy of salt bridges (Coulombic interactions with some contribution of hydrogen
bonding) that usually link unlikely ionised residues.
The second factor decreasing ΔG° of hydrogen bonds and salt bridges in
macromolecules is the entropic penalty for ordering, e. g. conformational entropy of
protein side chains packing. Consequently, for macromolecules and their complexes
interacting in buffered aqueous solutions (dielectric constant ε ~ 80, ionic strength I ~ 100-
150 mM), the free energy of stabilization of a hydrogen bond is the same as that of a salt
bridge, van der Waals interaction (induction and dispersion forces), and a hydrophobic
contact. 145
Stacking of aromatic rings (van der Waals and hydrophobic interactions) can attain
free energy from 0 to −42 kJ/mol, depending on interatomic distances, but the energy is
usually close to that of hydrogen bonds in proteins, 4 - 12.5 kJ/mol. 128,146,147 A nontypical
17

ut biologically important example of stacking is cation - π stacking which consists in
interaction between a charge and an induced quadrupole moment of charge distribution at
the aromatic ring. 129,148,149
Each type of noncovalent bonds has a typical range of interatomic distances. The
optimal configuration of hydrogen bonds and salt bridges is linear, with the atoms/ions
distant by 2.6 – 3.1 Å. The optimal distance for the van der Waals interaction is usually by
0.3 - 0.5 Å greater than the sum of the van der Waals atomic radii, and this is ~ 4 Å for the
C – C pair.
1.2.5. Parsing of Gibbs Free Energy
At issue is widely utilized parsing of ΔG° into contributions from individual,
specific, non-covalent interactions, 120,137,139,150,151 i. e. hydrogen bonding, salt bridges, van
der Waals contacts etc., from stating it to be meaningful 152 to proving its unreliability. 153
The argument against parsing is based on the fact that even if the energy of the system can
be approximated as linear combination of terms, it is not possible to write such general
expression for the total free energy. The ΔG° value can be approximately factorized only
when the resulting interactions are not correlated, e. g. operate on different coordinates.
The free energy parsing gives reliable physical insight into macromolecular processes as
long as the experimentally or theoretically obtained data are interpreted with care. 137,152,154
1.2.6. Thermodynamics of Coupled Processes
Thermodynamically coupled processes that accompany binding of a ligand to a
protein influence the thermodynamic parameters of binding to be determined. 122 In
particular, the existence of many of protein conformational microstates can significantly
shift the binding equilibrium in comparison with that for the binding to the single state.
Additionally, conformational rearrangement of the ligand as well as protonation, ionic and
hydration equilibria within the interacting molecules can modulate the resultant
thermodynamic features of the complex formation.
A0
1K, 1ΔH°
(+B) K0, ΔH°0 K1, ΔH°1 (+B)
BA0
18
A1
A1B

For instance, if the molecule (A) exists in equilibrium of only two states (A0, A1) that
is described by the constant (1K) and the transition enthalpy (1ΔH°), and can bind the
second molecule (B) with different affinities and binding enthalpies of K0, K1 and ΔH°0,
ΔH°1, respectively, then the observed parameters are as follows: 122
( ΔH°
+ ΔH°
− ΔH°
)
1ΔH°⋅1
K γ⋅1
K 1
1 0
Δ H°
= ΔH°
0 − +
; Eq. 1-16
1+
K
1 + γ⋅
K
1
( 1+
K)
1
19
( 1ΔH°
+ ΔH°
1 − ΔH°
0 )
T(
1+
γ⋅
K)
1ΔH°⋅1
K
γ⋅1
K
Δ S°
= ΔS°
0 − R ln(
1+
1K)
− + R ln(
1+
γ⋅1
K)
+
; Eq. 1-17
T
p
p 0
2 ( 1ΔH°
) ⋅1
2
RT ( 1+
K)
1
2
1
( )
( ) 2
1ΔH°
+ ΔH°
1 − ΔH°
0
2
RT 1+
γ⋅
K
K γ⋅1
K
Δ C°
= ΔC°
−
+
; Eq. 1.18
where γ = K1/K0 (K1 < K0, γ [0,1]), and terms indexed "0" denote intrinsic parameters
for binding of B to A in the A0 state. This case is called "nonmandatory coupling" since
both A states can participate in the binding to some extent. In contrast, "mandatory
coupling" occurs when only one state of A is capable of binding B, then K1 = 0 and γ = 0.
In this case, the equations simplify significantly.
Due to the two-state transition of A, the resultant binding free energy is always
shifted toward less negative values:
⎛ 1+
⎞
⎜ 1K
ΔG°
= ΔG°
0 + RT ln
⎟ . Eq. 1-19
⎝1
+ γ⋅1
K ⎠
It is interesting that macromolecular equilibria that have no intrinsic heat capacity
changes resulting from surface-related phenomena and conformational stiffening (silencing
of internal soft modes) can yield a non-zero heat capacity change due only to the presence
of coupled processes, e. g. due to ligand-induced shift in the equilibrium of protein states.
The heat capacity change resulting from mandatory coupling is always negative, while the
nonmandatory coupling can yield both negative and positive contribution, depending on
the particular values of equilibrium constants and enthalpy changes.
Hence, the overall heat capacity change of association that is accompanied by
coupled processes can be written as a sum of the following terms:
ΔC
o p
= ΔC
+ ΔC
o transfer
p aliphatic
o transfer
+ ΔCp
aromatic
o
p mandatory _ coupling
+ Δ
+ ΔC
C
o transfer
p polar
o
p nonmandatory
_ coupling
1
+ ΔC
2
o
pint
ernal _ soft _ vibrations
1
+
Eq. 1-20

2. Aim of The Work
While the structure determinations provided important information on the mode of
cap binding, the consistent energetic description of the cap-eIF4E interaction was still
lacking. In order to build a biophysical basis of the cap-dependent translation initiation and
explain some discrepancies among structural, biochemical and biological
observations, 75,155-157 measurements of molecular interactions in solution were necessary.
The eIF4E protein possesses eight tryptophans which are conserved both in number
and location, 21 and binding of cap results in quenching of the intrinsic protein
fluorescence. 5,6,156,158-164 However, previous fluorescence measurements of the interaction
between cap analogues and eIF4E purified by m 7 G-affinity chromatography yielded
puzzling data. The reported association constants, 5,156,161-163 in the range of 10 5 -10 6 M -1 , did
not reflect the differing inhibitory potency of cap analogues observed in vitro, 157 and
contrasted with the structural data. 75,91 The crystal structure suggested that the binding
constant for the eIF4E-m 7 GDP complex should be a few orders of magnitude higher, since
there was a clearly visible hole in the middle of the six-membered ring of 7-
methylguanosine in the electron density omit map. Such a high resolution within the
protein binding centre is usually observed by crystallographers for the complexes that have
binding constants much higher than 10 6 . 165 These divergences were a prompt to perform
systematic measurements to reveal the true affinity of cap analogues for eIF4E.
The work was focused on the process of association of recombinant untagged murine
eIF4E (residues 28-217) and full length human eIF4E with a large series of mono- and
dinucleotide cap analogues, in order to attain several particular goals leading together to a
complete decription of the molecular mechanism of specific recognition of the mRNA 5'
structure by eIF4E. The particular goals were as follows:
1. determination of association constants that would reflect the structure-activity
relationship;
2. parsing of the free energy of the binding into contributions of individual structural
elements in the cap-binding centre,
3. elucidation of the nature of the stacking-hydrogen bonding cooperativity,
4. searching for conformational changes and water exchange between the complex and
bulk solution,
20

5. revealing the role for ionic equilibria and electrostatic interactions in cap-eIF4E
recognition,
6. analysis of the specific binding between eIF4E and mRNA 5' cap in terms of exact
quantitative thermodynamic parameters determined independently from the van't
Hoff equation and isothermal titration calorimetry (ITC).
Such quantitative data are of primary importance for rational design of new cap-
analogues of potential therapeutic activity since the high eIF4E cellular level is relevant to
malignancy and apoptosis 13 .
The fluorescence affinity measurements have been extended to include ternary
complexes which consisted of eIF4E, a cap-analogue, and synthetic peptides corresponding
to the eIF4E recognition motifs from the mammalian proteins eIF4GI, eIF4GII, 4E-BP1,
and 4E-BP1 peptides monophosphorylated at Ser65 and diphosphorylated at Ser65/Thr70.
The particular questions asked in the work were related to two issues:
1. what is the influence of phosphorylation at Ser65 and Thr70 on regulation of 4E-BP1
binding to eIF4E,
2. how to reconcile a lot of contradictory biological and biochemical experimental data
reported hitherto, regarding the putative cooperation between two eIF4E binding
sites. 166-168
21

3. Materials and Methods
3.1. Chemical and Biochemical Syntheses
3.1.1. Cap Analogues
Syntheses and purification of cap analogues (Scheme 3-1) as sodium salts were
performed by Dr. Dr. Edward Dar¿ynkiewicz, Janusz Stêpiñski and Marzena Jankowska-
Anyszka as described previously 169-173 . Their concentrations were obtained from weighed
amounts (± 5 %) and checked spectrophotometrically. 157 The cap analogues in solution at
elevated pH (> 8) undergo opening of the five-membered ring of 7-substituted guanine,
followed by hydrolysis of the glycosidic bond. 174 As checked by NMR, 175 the cap was very
stable at pH 7.2, and stable enough to perform fast experiments at higher pH.
3.1.2. Peptides
Peptides corresponding to residues 569-580 of mammalian eIF4GI:
KKRYDREFLLGF, 34 621-637 of mammalian eIF4GII: KKQYDREFLLDFQFMPA, 35 and
51-67 of mammalian 4E-BP1: RIIYDRKFLMECRNSPV 67 were synthesized by Dr. Joseph
Marcotrigiano (The Rockefeller University, N.Y., U.S.A.) by Boc protocols for solid phase
peptide synthesis and cleaved using a standard HF procedure. 176 Syntheses of a
mammalian phosphopeptide P-Ser65 51-67 4E-BP1 and a diphosphopeptide P-
Ser65/Thr70 51-75 4E-BP1 RIIYDRKFLMECRNSpPVTKTpPPKDL 67 were performed
by Dr. Aleksandra Wys³ouch-Cieszyñska (IBB PAS, Warszawa) on a Wang resin
(Novabiochem) using Fmoc protocols for solid phase phosphopeptide synthesis. 1 Peptides
were purified to homogeneity by semi-preparative HPLC (> 95 % purity), and
characterized by MALDI-TOF or ESI mass spectrometry (569-580 eIF4GI peptide,
predicted mass 1697.0 Da, measured mass 1698.0 Da; 621-637 eIF4GII peptide, predicted
mass 2177.8 Da, measured mass 2178.0 Da; 51-67 4E-BP1 peptide, predicted mass 2141.8
measured mass 2141; phosphopeptide, P-Ser65 51-67 4E-BP1, predicted mass 2220.1,
measured mass 2221.0; diphosphopeptide, P-Ser65/Thr70 51-75 4E-BP1, predicted mass
3180.55, measured mass 3181.0).
Concentrations of the five investigated peptides were determined by acid gas-phase
hydrolysis and three independent repeats of amino acid analysis, by Dr. Pawe³ Mak
(Jagiellonian University, Kraków).
22

Scheme 3-1. Structures of the 16 methylated cap-analogues. Φ denotes the phenyl ring. Protons
which partially dissociate at pH 7.2, are marked with asterisk (pKa N(1)-H ~7.24-7.54, depending on
R2, R3, and n 177 ; pKa phosph ~6.1-6.5, depending on n 178 ).
m 7 GTP: R1 = CH3, R2 = R3 = H, n = 3;
m 7 GDP: R1 = CH3, R2 = R3 = H, n = 2;
m 7 GMP: R1 = CH3, R2 = R3 = H, n = 1;
m 7 G: R1 = CH3, R2 = R3 = H, n = 0;
et 7 GTP: R1 = CH2-CH3, R2 = R3 = H, n = 3;
bz 7 GTP R1 = CH2-Φ, R2 = R3 = H, n = 3;
p-Cl-bz 7 GTP: R1 = CH2-Φ-Cl, R2 = R3 = H, n = 3;
m 7 , 2
2 GTP : R1 = CH3, R2 = CH3, R3 = H, n = 3;
m 7 , 2 , 2
R2
R2
* H
N
R3
*
H
N
R3
O
N 6
1
2
3
N
N
O
N
R1
N
7
+
9
N
8
H
H
H
H
R1
N
+
N
H
OH
HO
3 GTP : R1 = CH3, R2 = R3 = CH3, n = 3;
O
H
H H
OH
HO
O
m 7 Gpppm 2’O G: R1 = CH3, R2 = R3 = H, n = 3, R4 = OCH3, B = G
m 7 GpppG: R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = G
m 7 GpppA: R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = A
m 7 GpppC R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = C
m 7 Gpppm 7 G: R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = m 7 G
m 7 GppppG: R1 = CH3, R2 = R3 = H, n = 4, R4 = OH, B = G
m 7 Gppppm 7 G: R1 = CH3, R2 = R3 = H, n = 4, R4 = OH, B = m 7 G
23
CH 2
H
CH 2
H
O
O (P O) n
O -
O
O (P O) n CH 2
O -
H
H *
H
OH
O
H
B
H
R4

A dodecapeptide DGIEPMWEDEKN was kindly provided by Dr. Laslo Balaspiri
(A. Szent-Gyorgyi Medical University, Szeged, Hungary).
All other chemicals were analytical grade, purchased from Sigma-Aldrich, Merck,
Carl Roth (Germany) or Fluka (U.S.A.).
3.1.3. Protein Biosynthesis
Human eukaryotic initiation factor eIF4E (residues 1-217) and murine eIF4E
(residues 28-217 and residues 33-217) was expressed by Mrs. Lidia Chlebicka with a help
of Dr. Micha³ Dadlez (IBB PAS, Warszawa) in Escherichia coli (strain
BL21(DE3)pLys). 179,180 The bacterial cells were transformed by a pET11d plasmid,
containing the cloned coding region for eIF4E, and the T4 promoter. Induction of T7
polymerase in liquid culture of bacteria on Luria-Bertani broth, with ampicillin and
chloramphenicol, was initiated by addition of isopropyl-β-D-galactopyranoside. Bacterial
pellets were lysed by sonication.
The human protein was initially isolated from the soluble fraction by means of
affinity chromatography 181 and purified on an ion exchange MonoQ column. Next, the
human and the murine proteins were purified from inclusion bodies pellets and folded by
one step dialysis from 6 M guanidine hydrochloride, followed by ion-exchange
chromatography on a MonoQ (human) or MonoS (murine) column, thus avoiding contact
with cap analogues at any stage of purification. The protein purity, checked by a routine
SDS PAGE, was > 95 %.
3.2. Preparation of Protein and Peptide Samples to
Spectroscopic Measurements
The protein solutions were buffer exchanged if necessary with use of Ultrafree-15 ml
filters with Biomax 5 kDa NMWL membrane (Millipore Co., U.S.A.). Immediately before
the spectroscopic measurements, the protein sample was filtered through Millipore
Ultrafree-0.5 ml Biomax 100 kDa NMWL. Total concentration of eIF4E was determined
from absorbance ( 280 53900 = ε cm-1M -1 ).
Solutions of 4E-BP1 peptides were strictly controlled for lack of disulphide dimer
formation. Dimers occurred in the 4E-BP1 solution because of lack of any additives in the
stock water solutions, which was necessary for accurate determination of the peptide
24

concentrations by amino acid analysis. Peptide dimers were broken if incubated at very
high concentration of the reducing agent (13 mM DTT) at pH 7.5 for two weeks. This
secured satisfactory monomerization, as rigorously checked on analytical HPLC (Waters
Inc.) and ESI mass spectroscopy (Q-Tof 2 spectrometer, Micromass Inc.). Then, the
affinity measurements could be performed properly with use of more dilute peptide
solution at 1 mM DTT. The problem concerned the phosphorylated 4E-BP1 peptides to
lesser extent, since the presence of the negatively charged phosphate groups at Ser65 in the
vicinity of Cys62 can partially prevent the dimerization.
3.3. Absorption and Emission Spectroscopy
Emission of light from a fluorophore occurs from electronically excited states. 182
Return to the ground electronic state (S0) from excited singlet states (Sn) is attained by a
rapid (after 10 -10 - 10 -8 s), spin-allowed emission of a photon (fluorescence), while
transition from excited tripled states (phosphorescence) is forbidden and the lifetimes are
longer (10 -3 – 10 1 s). Phosphorescence is not visible in aqueous solutions at temperatures
applied during this work and will not be considered.
Light absorption in UV-Vis wave region occurs typically from the lowest vibrational
level (of S0) to some higher vibrational level of S1 or S2 in a time of about 10 -15 s. In
solutions, molecules rapidly relax to the lowest vibrational level of S1 via internal
conversion in a time ≤ 10 -12 s. Fluorescence return to S0 occurs to one of its vibrational
levels. Then the molecule quickly reaches thermal equilibrium (10 -12 s). The rapid
irradiative decay to the lowest vibrational level of S1 results in the Stokes' shift between the
absorption and emission spectra. The shift is further enhanced by possible decay to higher
vibrational levels of S0, reorientation of fluorophore in respect to solvent molecules,
excited-state reactions, complex formation, and energy transfer.
When incident light (intensity I0) is absorbed by a diluted chromophore solution
(concentration [ch], extinction coefficient ε) on the optical length (l), then the absorbance
(A) and the intensity of transmitted light (I1) can be reliably expressed by the linear Beer-
Lambert law: 183
light:
I0
A = log = ε ⋅[
ch]
⋅ l . Eq. 3-1
I
1
The rate of emission of fluorescence (F) is related to the intensity of the absorbed
25

F = (I0 − I1) ⋅ q, Eq. 3-2
where q is the quantum yield of fluorescence. For weakly absorbing solutions for which
the optical density is very small, the equation can be simplified to:
F = 2.3⋅I0⋅ε⋅[ch]⋅l⋅q. Eq. 3-3
Fluorescence intensities are proportional to the fluorophore concentration over a very
limited range of optical densities, usually 2 orders of magnitude lower than those which
satisfy the Beer-Lambert law for absorption. If the conditions ensuring linearity are
satisfied, the observed fluorescence changes can be directly interpreted in terms of
changing concentrations of the species present in a studied sample. Otherwise suitable
corrections for the inner filter effect are necessary 183 (see below, 3.4.2.1., p. 31).
3.3.1. Fluorescence Quenching
Fluorescence intensity can be decreased by: 182
1. dynamic quenching:
a) irradiative return do S0 by a diffusive collisions of the excited fluorophore with
other molecules,
b) resonance energy transfer (RET) of the excited-state energy from an excited donor
to an acceptor without the appearance of a photon, as a result of long-range
dipole-dipole interactions,
2. static quenching by formation of nonfluorescent complexes of fluorophore with a
quencher in the fluorophore ground state,
3. inner filter effect, i. e. attenuation of the excitation beam and/or reabsorption of the
emitted beam by a layer of an optically dense solution.
3.3.1.1. Static Quenching
Static quenching results from formation of a dark complex that returns to the ground
state immediately after absorption without emission of a photon. A fraction of the
fluorophores is removed from observation and, contrary to dynamic quenching, the
lifetime of the uncomplexed fraction is unchanged. The quenching constant is the
association constant of the dark complex: 182
[ cx]
K s = , Eq. 3-4
[ f ] ⋅[
qu]
0
0
26

where [cx], [f]0 and [qu]0 are the equilibrium concentrations of the complex, free
fluorophore and free quencher, respectively. The fluorescence changes analogically as
upon the dynamic quenching (below):
F0
= 1+
K s[
qu]
0 , Eq. 3-5
F
but the equilibrium concentration the free quencher must be analysed here.
3.3.1.2. Dynamic Quenching
Dynamic quenching by a quencher at a total concentration [qu] is a rate process
which depopulates the excited state, leading to shortening of fluorescence lifetimes (τ0).
The resultant lifetime in the presence of the quencher (τ) is shorter by a decay rate (kq)
which is characteristic to the given quencher and environment:
τ0
= 1+ k qτ0
[ qu]
. Eq. 3-6
τ
An equivalent decrease in fluorescence intensity is:
0 F
F
τ
=
τ
0
. Eq. 3-7
The Stern-Volmer quenching constant is defined as:
KSV = kq ⋅ τ0, Eq. 3-8
so that the Stern-Volmer equation is expressed as:
F0
= 1+
K SV[
qu]
. Eq. 3-9
F
3.3.1.3. Resonance Energy Transfer
The rate of RET (kT) is related to a factor describing the relative space orientation of
the transition dipoles of the donor and acceptor (κ 2 ), to the quantum yield of the donor (qd),
to the lifetime of the donor in the absence of the acceptor (τd), to the reciprocal of sixth
power of their distance (r), and to the integral of spectral overlap between the donor
emission and the acceptor absorption (J(λ)):
k
T
q
~
τ
d
d
⋅ κ
⋅ r
2
6
J(
λ)
. Eq. 3-10
27

The distance at which the transfer rate is equal to the decay rate of the donor in the
absence of acceptor (by the radiative and nonradiative ways) is called the Förster distance
(R0), usually ~ 30 – 60 Å.
The orientation factor (κ 2 ) is given by:
2
κ = cos θ − 3cos
θ ⋅ cos θ , Eq. 3-11
T
d
a
where θT is the angle between the donor and acceptor dipoles, θd and θa are the angles
between these dipoles and the vector joining them. The κ 2 value can range from 0 for the
perpendicular orientation of the dipoles to 4 for the collinear, parallel orientation.
3.3.1.3.1. Resonance Energy Transfer in eIF4E
The eIF4E molecule contains 6 tyrosines and 8 tryptophans. 50 The diameter of the
molecule is ~ 40 Å, i. e. in the typical range of the Förster distances. Hence, the tyrosine
(donor) fluorescence can be transferred to tryptophans (acceptor), 182 and the shape of the
emission spectrum excited at 280 nm is dominated by the final tryptophan fluorescence
(Fig. 4-1, p. 49). It has been checked by comparison of eIF4E emission spectra registered
for different excitation wavelengths (λex) that the contribution of the tyrosine emission in
the resultant fluorescence intensity at λex = 280 nm is not more than 10 %. Putative
conformational changes can modulate both tyrosine and tryptophan emission but
eventually the spectral information about the changes is provided by the tryptophan
emission.
One issue is the interaction of eIF4E with the tyrosine containing peptides 4E-BP1,
4E-BP1 P-Ser65, 4E-BP1 P-Ser65/Thr70, eIF4GI, and eIF4GII. The peptide tyrosine is
distant by only 10 Å from Trp73 of eIF4E in the bound state. 91 However, the mutual
orientation of the two aromatic rings is almost perpendicular (Fig. 4-22, 23, p. 86-87). The
tyrosine is stabilized in this plane by a close phenylalanine ring and an –OH hydrogen
bond, and the tryptophan is tightly bound by a network of intra- and intermolecular
interactions. These constraints suggest that in spite of proximity of the donor and acceptor
the RET is most probably inefficient.
3.3.2. Tryptophan Fluorescence in Proteins
Tryptophan fluorescence varies widely in quantum yield and in the wavelength of
maximum intensity among proteins and solvents. 182 The 280-nm absorption band of Trp is
28

the result of transition to two excited states, called 1 La and 1 Lb, of considerably different
properties. 184 The 1 La state has a large dipole moment, and thus the high sensitivity to
environment. Transition from 1 La usually yields more fluorescence but 1 Lb can be
sometimes also the emitting state and then they are hardly distinguishable in proteins. The
transitions have different structures, intensities and relative spectral positions, but the
details are blurred by inhomogeneous broadening in proteins due mainly to the variation of
the relative orientation of the local electric field felt by tryptophans. This solvent-solute
interaction accounts for the large Stokes' shift in water and much less inside the protein
hydrophobic core. The large red shift is related to the cooperative effect of many water
molecules, ~ 100 cm -1 /1 water molecule within 6 Å. 184 Additionally, protein tryptophans
can be dynamically quenched by neighbouring charged amino acids. 182 Hence, the
spectroscopic properties (quantum yield, accurate shape of the spectrum e.t.c.) of eIF4E
were not the main focus of the work to avoid too far-reaching, uncertain conclusions.
Instead, the fluorescence intensity was used to monitor quantitatively the changes
experienced by eIF4E.
3.3.3. Quenching of eIF4E Fluorescence by Cap Analogues
The intrinsic protein fluorescence quenching observed upon formation of the
complex of eIF4E with a cap analogue can result from two factors: direct quenching of the
tryptophans 102, 56 and 166 in the cap-binding site, and changes of local environment of
other, more distant fluorescent amino acids as a result of possible conformational changes
of the protein upon the binding. The former is thought to be static quenching, since:
1. The Förster resonance energy transfer in unlikely due to several reasons:
a) Spectral overlapping of the cap analogue emission and the protein absorption
spectra, and reversely (Fig. 4-1, p. 49), the protein emission and the cap analogue
absorption spectra, is negligible.
b) Neither any batochromically shifted emission band nor fluorescence
enhancement of cap analogues were observed. The emission of the free cap in the
presence of the protein obtained by fitting of the full equation (Eq. 3-26, p. 34)
was the same as the emission in the absence of the protein;
c) Brownian dynamic calculations involving electrostatic effects together with the
stopped-flow binding studies 4 suggested that quenching can occur only when the
ligand is apart from its final position known from the crystal structure 75 not more
29

than a few angströms. It would probably still diminish this distance if polarization
effects were taken into account in the calculations. 185
2. Collisional quenching of the protein tryptophans seems to be unlikely at such low
concentration of the protein, 50 nM – 1 μM, and of the cap analogue, not exceeding
5 μM for the most specific analogue and 150 μM for the least specific one.
However, the unambiguous assessment could be only provided by time-resolved
measurements.
Fluorescence changes of tyrosines, and the tryptophans which do not have direct
contact with cap are related to dynamic quenching by surrounding charged amino acids, 182
changes of mutual distances and orientations in Trp-Tyr pairs, and changes of local
polar/hydrophobic neighbourhood. The quenching has a dynamic character but reflects the
changes that the protein undergoes upon cap binding and thus depends not on the total
quencher concentration but on the equilibrium concentration. Hence, it is described by the
same equation as static quenching.
3.4. Experimental Conditions of Spectroscopic Measurements
Absorption and fluorescence spectra were recorded on Lambda 20 UV/VIS and LS-
50B instruments (Perkin-Elmer Co., Norwalk, CT., USA), in a quartz semi-micro cuvette
(Hellma, Germany) with optical lengths 4 mm and 10 mm for absorption and emission,
respectively. The titration experiments were performed in a standard buffer: 50mM
Hepes/KOH pH 7.20, 100mM KCl, 1mM dithiothreitol (DTT) and 0.5mM disodium
ethylenediaminetetraacetate (EDTA), except for the experiments at variable pH and ionic
strength. pH (± 0.01 unit) was measured independently at each temperature and ionic
strength (Beckman Φ300 pH-meter, Germany). Solutions were filtered through 0.22 μm
pore size. Protein samples were softly degassed prior to titration. The cuvette was
thermostated and the temperature was controlled with a thermocouple inside it (± 0.2 °C).
For the eIF4E-cap association the excitation wavelength of 280nm (slit 2.5nm, auto
cut-off filter), and the emission wavelength of 335, 336 or 337nm (slit 2.5 to 4nm, 290 nm
cut-off filter) were applied, with an automatic correction for the photomultiplier sensitivity.
For the eIF4E-peptide binding studies, the excitation wavelength of 290nm and the
emission wavelength of 350 or 355 nm were used. These conditions ensured observation of
the protein tryptophan emission, only. The fluorescence intensity was monitored by the
30

egistration of the whole spectrum (310 nm to 400 nm) and during continuous, time-
synchronized titration at a single wavelength, with the integration time of 30 seconds and
the gap of 30 seconds for adding the ligand, with slow but sufficient magnetic stirring to
ensure mixing and keeping the temperature constant in the whole volume of the cuvette.
During the gap, the UV xenon flash lamp was switched off to avoid photobleaching the
photosensitive protein sample. The cuvette has not been touched during the whole titration
experiment to ensure constant geometry for the optical measurements.
3.4.1. Titration Assay
Titrations were performed for eIF4E at several concentrations (50 nM to 1 μM), in
steady-state conditions provided by preincubation of eIF4E in the buffer of the appropriate
pH and ionic strength for the given experiment. 1 μl aliquots of increasing concentrations
(1 μM to 5 mM) of a ligand were injected manually to 1400 μl of eIF4E solution. Each
titration consisted of 28 to 45 data points with a suitable number within the range, at which
the total ligand concentration ([L]) was close to the concentration of active eIF4E ([Pact])
(Fig. 4-7, p. 57). The curvature of the fitted function (Eq. 3-26, p. 34) in this range,
∂
2
F
∂[
L]
2
[L] = [Pact
]
~
K
P
as
act
, Eq. 3-12
mostly influences the accuracy of fitting of [Pact] and Kas. For determination of the active
protein fraction in the samples studied by stopped-flow fluorimetry and ITC, the cap
analogue of the highest association constant for eIF4E was used (7-methylGTP), since this
Kas ensures the optimal quotient of Kas/[Pact].
3.4.2. Fluorescence Data Corrections
3.4.2.1. Inner Filter Effect
The fluorescence intensities were corrected for the inner filter effect 183 (Fig. 3-1).
The cuvette had two thicker walls and thus the optical pathlength for absorption was
significantly shorter than that for emission (4 mm × 10 mm), which minimized both the
inner filter effect and a recapture of the excitation beam from a little mirror behind the
thick cuvette wall. A following polynomial dependence of the fluorescence intensity (F) on
the absorbance (A):
F(
A)
2
3
4
= 1−
2.
348⋅
A + 7.
56 ⋅ A − 20.
2 ⋅ A + 21.
5 ⋅ A
Eq. 3-13
F(
0)
31

proved to be better than the correction proposed for the typical 10 mm × 10 mm cuvette 186
(P < 0.0001). The experimental data (Fobs) were divided by the above factor (F(A)/F(0))
determined individually for each titration, yielding the data-set corrected for absorption
(FcorrA):
F(
A)
= F
Eq. 3-14
F(
0)
FcorrA obs
The inner filter effect was negligible for the specific cap analogues but could change Kas
values ~ 2-fold for the weakly interacting and strongly absorbing cap analogues (Fig. 3-1).
F(A)/F(0)
Residuals
1.0
0.9
0.8
0.7
0.6
0.5
0.02
0
-0.02
Figure 3-1. (left) Inner filter effect measured for tryptophan solution in the 4 mm × 10 mm cuvette,
titrated by GMP in 50mM Hepes/KOH buffer, pH 7.20, 100mM KCl. A comparison of a
polinomial function (solid line, black residuals) with the analytical function proposed for the 10
mm × 10 mm cuvette 186 (broken line, open residuals). The maximal GMP concentration is < 100
μM, which rules out a possibility of dynamic quenching of tryptophan. (right) Fluorescence
titration of eIF4E with GDP. Data uncorrected () and corrected () for the inner filter effect.
3.4.2.2. Dilution
0.0 0.1 0.2 0.3 0.4
Absorbance
0.0 0.1 0.2 0.3 0.4
The final dilution was always ≤ 3.2%. Suitable data correction was applied for these
titrations for which the final dilution was ≥ 2%. FcorrV is the corrected fluorescence, Fobs is
the measured fluorescence, V is the actual sample volume at each point of the titration, and
V0 is the initial sample volume:
V
FcorrV = Fobs
⋅ . Eq. 3-15
V
0
Fluorescence (a. u.)
32
500
400
300
200
100
0 20 40 60 80 100 120
GDP (μM)

3.4.2.3. Fluorescence Drift in Time
In some cases at higher temperatures (~ 40 °C) the protein fluorescence decreased
somewhat in time due most probably to partial thermal denaturation of eIF4E. A correction
was needed to cancel numerically the fluorescence changes that did not result from ligand
binding. Thanks to synchronization of each injection during titration, a correction for a
fluorescence decrease in the time of the titration experiment could be applied. Monitoring
of the fluorescence signal was continued after completing the titration by the next 10 - 20
minutes in the same way as during the titration. The exponential curve was fitted to the
time-dependent data at the constant ligand concentration:
F ( t)
= a ⋅ exp( −b
⋅ t)
+ c . Eq. 3-16
The titration data were then corrected (FcorrT) by the exponential function:
FcorrT obs
= F ⋅ exp( b ⋅ t)
. Eq. 3-17
The latter two corrections had only slight influence on the resultant Kas values
(within 0.3 standard deviation) but improved the goodness of fit (R 2 and P value, see 3.9.,
p. 46)
3.5. Fluorescence Data Analysis
3.5.1. Simultaneous Determination of Association Constants and Protein
Activity
As a result of binding of a ligand at a total ligand concentration of [L] to an active
protein at a total concentration of [Pact], there are three species in the solution: the free
ligand at the equilibrium concentration of [L]0, the free active protein at the equilibrium
concentration of [Pact]0, and their complex at the equilibrium concentration of [cx]:
[L] = [L]0 + [cx], Eq. 3-18
[Pact] = [Pact]0 + [cx]. Eq. 3-19
Equilibrium among the concentrations of free species and the complex is ruled by the
association constant Kas that is defined in the same manner as the static quenching constant
(Eq. 3-4):
[ cx]
K as = . Eq. 3-20
[ L]
[ P ]
0
act
0
Solution of the three above equations yields a square equation with the positive root for
[cx]:
33

[ cx]
( K ([ L]
−[
P ]) + 1)
2
[ L]
+ [ Pact
] 1−
as
act + 4Kas
⋅[
Pact
]
= +
Eq. 3-21
2
2Kas
The total protein concentration [P] is the sum of the active fraction [Pact] and the
inactive [Pinact] fraction that does interact with the ligand:
[P] = [Pact] + [Pinact]. Eq. 3-22
The initial fluorescence intensity (F(0)) of the pure protein is equal:
F(0) = [Pact]⋅φPact_free + [Pinact]⋅φPinact. Eq. 3-23
The observed fluorescence intensity (F) after addition of the fluorescent ligand of the
fluorescence efficiency φlig-free is equal:
F = [Pact]0⋅φPact_free + [cx]⋅φcx + [L]0⋅φlig_free + [Pinact]⋅φPinact. Eq. 3-24
No assumptions regarding the fluorescence efficiency of the inactive protein are necessary.
Simple substitution of F(0) and [cx], and definition of the difference between the
fluorescence efficiencies of the apo-protein and the complex as:
Δφ = φ − − φ
Eq. 3-25
Pact
free
cx
yield the full equation which describes the fluorescence intensity as a function of the total
ligand concentration within the course of titration:
= − ⋅ Δφ
+ φ + ⋅ φ , Eq. 3-26
F F(
0)
[ cx]
( lig −free)
[ L]
lig −free
This theoretical curve was fitted to the experimental data points. The parameters to be
extracted from the fit were as follows: Kas, [Pact], Δφ, φlig-free, F(0). The latter two
parameters were independently verified experimentally. The accordance between the non-
linearly fitted φlig-free value and the value determined from linear regression to the
fluorescence data for the free ligand at increasing concentrations in the absence of the
protein was 4 %. For the experiments at the most elevated temperature, the parameter [Pact]
had a meaning of an average value in the course of the titration.
eliminated.
Total quenching upon binding was calculated as:
Q = F(
0)
− F(
∞)
= [ Pact
] ⋅ Δφ
. Eq. 3-27
For the peptides, φlig-free was fixed as zero, since the direct tyrosine fluorescence was
The final Kas were calculated as a weighted average of 3 to 12 independent titration
series, except for m 7 GTP, for which more than 30 titration experiments were performed, in
order to make statistical analysis of the method and as control for each protein batch. The
34

Eadie-Hofstee representation 187,188 was not used, for its basic assumption of a great excess
of the ligand in relation to the protein is not satisfied here.
3.5.2. The Gibbs Free Energy of Binding
The Gibbs free energy of binding of cap analogues to eIF4E was calculated from Eq.
1-9. The binding constants (Kas) were used for unsymmetrical dinucleotide and
mononucleotide cap analogues, and the microscopic binding constants (Kas (micro) = 0.5 ⋅
Kas) in case of the symmetrical cap analogues (m 7 Gpppm 7 G, m 7 Gppppm 7 G, GpppG)
because of entropic effects.
3.5.3. Osmotic Stress and Electrostatic Interactions
Increase of the KCl concentration at constant pH and temperature is accompanied by
an increasing osmolality (2ϕ[KCl]) and a decreasing activity coefficient of water (aw):
2ϕ⋅
[ KCl]
log( a w ) = − , Eq. 3-28
ln10⋅
Ω
where: ϕ = 0 . 91±
0.
01,
an osmotic coefficient of KCl approximately constant from 0 to
0.5 M KCl; 189 Ω the number of moles of free solvent water in 1 kg of the solution. The
latter is not assumed to be constant, but the presence of other buffer components of the
molecular masses μi at the concentrations ci, and the hydration number of KCl ( ν ≈ 5),
190
are taken into account:
1000 − μ KCl[
KCl]
− ∑μ
ic
i
Ω =
− ν ⋅[
KCl]
Eq. 3-29
μ
H O
2
Keeping constant Ω = 55.
5 yields overestimation of the results concerning the number of
the water molecules that are exchanged between eIF4E-cap complex and the bulk solvent
(ΔN) by ~ 18% (see next page).
3.5.3.1. Davies-Stockes-Robinson Electrostatic Screening Approach
Binding of two ions of opposite charges (protein z1 and ligand z2) is screened by the
ionic atmosphere of the excess salt and other ionized components of the solution. The
influence of ionic strength and osmotic stress on the activity coefficients of the interacting
species leads to the expression for log(Kas) in function of [KCl]: 191,192
35

2Az1z
2 I
log( K as ) = log( K as ( 0))
+ + ΔN
log( a w ) , Eq. 3-30
1+
a B I
where ionic strength:
j
2
I = [ KCl]
+ ∑c
iz i , Eq. 3-31
zi is a charge of the i-th species, aj is a sum of radii of interacting species, and ΔN is a
number of water molecules taken up (or released, if ΔN < 0) to the macromolecular
surfaces upon complex formation. The temperature-dependent coefficients at 20°C are:
A = 0.
50585,
B = 0.
32789.
192
3.5.3.2. Wyman Linkage Analysis
Hydration effects as well as ionic interactions are considered stoichiometrically: 193
0
log( K as ) = log( K as ) − c ⋅ log( a KCl)
+ ΔN
⋅ log( a w ) , Eq. 3-32
where c is the total number of all ions released from macromolecular surfaces upon
binding. Thermodynamic activity of KCl (aKCl) is used. Assuming aKCl = [KCl] leads to
underestimation of the Kas value extrapolated to 1 M KCl by ~ twofold. Kas 0 corresponds to
the value of Kas, when the thermodynamic effects of ion release (log([KCl]) = 0 at 1 M
KCl) and of the hydration (log(aw) = 0 at 0 M KCl) cancel each other out. Kas 0 occurs at the
KCl concentration of approximately 0.3 M. The ion release does not influence the resultant
ionic strength, since the protein and the ligand are present at nano- to micromolar
concentrations, while [KCl] ranges from 0.05 M to 1 M, and the buffer contribution to I at
pH 7.2 is ~ 0.017 M.
The contribution of the electrolyte effect (ΔG°el) to the stability of the cap-eIF4E
complex can be calculated from the derivative SKas: 118
as:
SK
as
⎛ ∂ log K as ⎞
≡ ⎜ ⎟
⎝ ∂ log[ KCl]
⎠
T,
p
2φ[
KCl]
= −c
− ΔN
Ω
36
Eq. 3-33
Δ ° = −SK
RT ln[ KCl]
, Eq. 3-34
G el as
and the entropy of the electrolyte effect was calculated as:
ΔG°
el
Δ S°
el = − . Eq. 3-35
T

3.5.4. Protonation Equilibria
When the binding event is accompanied by protonation and deprotonation of two
specifically interacting residues at constant temperature, ionic strength and osmotic stress,
these processes interfere to produce "bell-like" shaped dependence of the binding constant
on pH. In case of 7-methylGTP, the population was considered as a mixture of cationic
(Lc) and zwitterionic (Lzw) forms:
= [ L ] [ L ] . Eq. 2-36
[ L]
c zw +
The protein was assumed to exist in equilibrium of two states, one with Glu103 protonated
(Glu) and the other with Glu103 deprotonated (Glu), leading to:
−
[ Pact
] = [ Pact
( Glu)]
+ [ Pact
( Glu )] . Eq. 3-37
When the cationic form of cap binds to eIF4E with deprotonated Glu103, the pH-
independent association constant, for the species in their appropriate ionic states, can be
expressed as:
where:
and
−
pH−ind
[ LcPact
( Glu
K as
−
[ Lc
] 0[
Pact
( Glu
[ P
act
c
( Glu
)]
= Eq. 3-38
)]
−
c
c
act
−
0
)] = [ L P ( Glu )] + [ P ( Glu )]
Eq. 3-39
[ L ] = [ L P ( Glu )] + [ L
act
−
c
]
0
act
37
−
0
. Eq. 3-40
The populations of both the free protonated ligand ([Lc]0) and the free deprotonated protein
([Pact(Glu − )]0) that actually do not form the complex, are in ionic equilibrium defined by
the effective acidic dissociation constants KL and KP, the former for the ligand in the
presence of the protein, and the latter for Glu103 in the presence of the cap analogue:
and
K
K
+
[ Lzw
][ H ]
L = Eq. 3-41
[ Lc
] 0
P
[ Pact
( Glu )] 0[
H ]
= Eq. 3-42
[ P ( Glu)]
act
−
The experimentally observed association constant is:
K
+
[ LcPact
( Glu )]
= Eq. 3-43
−
−
([ L]
− [ L P ( Glu )])([ P ( Glu )] −[
L P ( Glu )])
as −
c act
act
c act
−

and after transformation may be expressed as a function of pH:
log( K
as
pH−ind
as
pH−pK
L
P
) = log( K ) − log( 1+
10 ) − log( 1+
10 ) , Eq. 3-44
38
pK −pH
where log(Kas pH-ind ), pKL and pKP are fitted parameters. The optimal pH for binding is:
pH opt L P
= ( pK + pK ) / 2 . Eq. 3-45
From the pH-dependence of log(Kas), the observed free energy change for the eIF4E-
cap association at a given pH can be regarded as:
pH−ind
ΔG
° ( pH)
= ΔG°
+ ΔG°
+ ΔG°
L
P
, Eq. 3-46
where ΔG°L and ΔG°P denote energies of bringing of the interacting ligand and the protein
residue at a given pH to the appropriate ionic states, for which the binding energy would
equal ΔG° pH-ind :
pH−ind pH−ind
as
Δ G°
= −RT
ln( K ) ; Eq. 3-47
pH−pK
L
Δ G°
= RT ln( 1+
10 ) ; Eq. 3-48
L
pK
pH
Δ G°
= RT ln( 1+
10 ) . Eq. 3-49
P
P −
3.5.5. Thermodynamics
The temperature dependence of Kas was analysed according to the van't Hoff isobaric
equation (Eq. 1-14). 194 The molar heat capacity change ( Δ C ) and the characteristic
temperatures at which
the fitting.
195 196 o
Δ G ,
o
o
Δ S = 0 (TS) or H vH
Eq. 1-9, 1-12, and 1-11, respectvely.
where
o
p
Δ = 0 (TH) were obtained as free parameters of
o
o
Δ S , and the van't Hoff enthalpy change H vH
Discrimination between the linear van't Hoff equation:
ln K
as
o
Δ H vH ,
Snedecor's F-test 197 .
o
o vH
Δ were calculated from
ΔS
ΔH
= − , Eq. 3-50
R RT
o
Δ S = const, and the non-linear model (eq. 4) was based on the statistical
3.5.5.1. Coupling Between Binding and Conformational Transition of Ligand
Thermodynamic parameters describing intramolecular base stacking of dinucleotide
cap-analogues in the cationic form, i.e. entropy ( ΔS°
1 ) and enthalpy ( 1 ΔH°
) changes 198 ,
were used for calculating the following quantities at four temperatures: stacking/unstacking

equilibrium constants ( 1 K ), contributions to enthalpy ( Δ H ), entropy ( Δ S ), and heat
o
psst
capacity ( Δ C ) changes, which result from an induced shift in the self-stacking
equilibrium, and intrinsic enthalpy ( Δ H ) and entropy ( Δ S ) changes of the 7-
39
o
0
methylGpppG − eIF4E association, according to the equations for the case of mandatory
coupling of cap-eIF4E binding to cap stacking. 122 It is assumed that the entropy changes
are approximately additive.
1
⎛ o
⎜ 1ΔS
1ΔH
K = exp −
⎜
⎝ R RT
o
o
⎞
⎟
⎠
o
sst
o
0
o
sst
Eq. 3-51
o 1ΔH
⋅1
K
ΔHsst = −
Eq. 3-52
1+
K
1
o
o
1ΔH
⋅1
K
ΔSsst = −R
ln(
1+
1K
) −
Eq. 3-53
T
( 1+
K)
1
( ) 2
o 2
o ( 1ΔH
) ⋅1
K
ΔCp = −
Eq. 3-54
sst 2
RT 1+
1K
o
0
o
cal ion
o
sst
ΔH = ΔH
− − ΔH
Eq. 3-55
o o o
ΔS0 = ΔS
− ΔSsst
Eq. 3-56
3.5.5.2. Enthalpy-Entropy Compensation in Congener Series – General Model 199
For a system in which the number of states with an energy from U to U+δU is
ω(U)δU, the mean energy (which is approximately the enthalpy of the system) is:
∫ U ⋅ ϖ(
U)
⋅ e
E = U =
∞
∫ ϖ(
U)
⋅ e
−∞
−U
kT
−U
kT
dU
dU
Eq. 3-57
If some energy levels within energy range from U' to U'+δU are significantly
perturbed by ΔU > 3 kT, then the change in the energy due to the perturbance is:
where
ΔE ≈ (E − U')⋅P(U')dU, Eq. 3-58
P(
U'
)
−U'
kT
= ϖ(
U'
) ⋅ e is the probability of finding the system in a state with energy U'
in the unperturbed system. Large and complex protein systems have many closely spaced
energy levels, i. e. P(U')dU

ΔG ≈ kT⋅P(U')dU. Eq. 3-59
The entropy change is then:
TΔS ≈ (E − U' − kT)⋅P(U')dU. Eq. 3-60
Enthalpy-entropy compensation can be thus described by a compensation temperature (Tc)
that is related to the difference between the mean energy and the energy of the perturbed
states:
T c
ΔH°
ΔE°
T
= ≈ ≈
Eq. 3-61
ΔS°
ΔS°
RT
1 −
E°
− U°
'
3.6. Isothermal Titration Calorimetry
Microcalorimetry provides a direct route to thermodynamic characterization of
bimolecular equilibrium interactions. 200 When heat is generated or absorbed within the
sample cell a compensation appears in the feedback power to equilibrate the temperatures
in the sample cell and in the reference cell. The time integral of the power deflection is a
measure of the heat. The molar calorimetric enthalpy (ΔH°cal) and the association constant
could be determined in one experiment by direct measurement of the heat of interaction
when one component is titrated into the other. However, two conditions must be
simultaneously satisfied so that determination of Kas is reliable and accurate: 201
1. A product of the number of the interacting molecules ([Pact] expressed in M) and the
association constant (Kas expressed in M -1 ) should be in the order of 100.
2. Each injection should have at least 40 μJ of heat exchanged with the cell of the
microcalorimeter.
It was entirely impossible to meet these conditions for eIF4E and those cap analogues,
which were described by the non-linear van't Hoff plots. Numerical simulations of such
experiments for m3 2,2,7 GTP with added Gaussian noise at the lowest possible level showed
that fitting of the binding curve would be hopeless. Thus, a modified "single injection"
method was proposed to obtain the most accurate values of the molar calorimetric
enthalpies at several temperatures.
40

3.6.1. Calorimetric Measurements for m 7 GpppG
ITC experiments were run on OMEGA Ultrasensitive Titration Calorimeter
(MicroCal, MA, U.S.A.), calibrated by 18-crown-6 titration with BaCl2. * The jacket of the
microcalorimeter was filled with dry nitrogen to prevent condensation on the outer surface
of the cells below room temperature. A refrigerated circulated bath (7 °C) was connected
to the microcalorimeter to keep the surroundings of the cells cooler than the temperature of
a given experiment. The reference cell was filled with deionized water. Slow stirring at 240
rpm was applied to avoid protein precipitation. The system was allowed to equilibrate until
a stable baseline was observed (≥ 30 min), before an automated titration was initiated.
Suitable buffers (50 mM Hepes/KOH, 100 mM KCl, 1 mM EDTA) were prepared to
keep pH 7.20 ± 0.02 at 288.1, 293.1, 298.4, and 303.2 K. The protein sample buffer was
exchanged by 4-fold centrifugation on 5 kDa Centricon filters (Millipore, MA, USA).
After last centrifugation, the flow-through buffer was collected to dissolve 7-methylGpppG
and to make control measurements of heat of the ligand dilution in the buffer. The
concentration of the injected ligand was 1.00 ± 0.07 mM in each case. Samples were
degassed, then filtered through 0.22 μm filter (Millipore, USA) directly before using. The
main part from each protein solution was used for ITC and the remaining part for the
control fluorescence titration. This ensured consistency for all the measurements. The
active protein concentrations at four temperatures were 8.97 μM, 7.42 μM, 3.61 μM and
5.35 μM, respectively.
3.6.1.1. Modified "Single Injection" Experiment
Low solubility of eIF4E (28-217) hampered also direct determination of Kas for 7-
methylGpppG by the ITC measurements. The very first injections into the eIF4E solution
results in ~ 35 μJ of the evolved heat, so at the verge of the instrument sensitivity, ~ 40
μJ. 201 The subsequent heat signals decrease with the course of the titration due to the
negative value of ΔH°, thus becoming indiscernible from the noise. The titrations were
performed several times for different injection volumes (data not shown) but
unsuccessfully. However, it was possible to determine the calorimetric enthalpies by a
modified "single injection" method. The ligand solution was injected into the calorimetric
cell (1386 μl volume) filled with eIF4E solution. Next, the 7-methylGpppG solution was
* Calibration was done by Dr. Ma³gorzata Wszelaka-Rylik (Inst. Phys. Chem. PAS, Warszawa)
41

injected into the buffer to measure the heat of dilution. Each experiment consisted of the
main 40 μl injection, preceded by two 1 μl injections to calculate the correction for the
initial outflow from the syringe, and followed by two 4 μl injections to check the protein
saturation with the ligand. It was therefore possible to determine the total emitted heat in
the most reliable way.
3.6.2. Calorimetric Data Treatment
After integration of all signals, the corresponding values from the protein titration
with the ligand, and from the buffer titration with the ligand were subtracted from each
other to yield the total calorimetric enthalpy
o
Δ H . As the heat which is exchanged at the
42
μl injections,
which was apparently decreased by the common instrumental artifact (leakage from the
syringe) during the baseline equilibration.
Calorimetric
of the enthalpies:
o
p
o cal
3.6.2.1. Buffer Ionization Heats
o
ion
o o
o o
Δ Hion
, Hcal ion = ΔHcal
− ΔHion
Δ − (see below).
o Buffer ionization heats ( Δ Hion
) of Hepes at pH =7.2 were calculated as:
−pKa
10
−pKa
−pH
o
H−diss
ΔH =
⋅ ΔH
Eq. 3-62
10 + 10
o from the molar ionization heat for Hepes, H H−diss
o temperature-dependence of H H−diss
Δ = +20.95 kJ⋅mol -1 at 298.2 K and
o
Δ estimated as δ Δ H H−diss
/δT = +0.0648 kJ⋅mol -1 K -1 , 202
and pKa = 7.35 ± 0.05 for 7-methylGpppG. 177 The pKa changes were negligible over the
temperature range used.
3.7. NMR Spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy 203 is concerned with the magnetic
energy of nuclei in a strong, static, magnetic field of strength Bo, (2 T to 21 T), and the
transitions in the wave region of 2 - 900 MHz. Significance of the NMR spectroscopy in
dynamic studies of molecules is related to interaction of a nucleus with surrounding

electrons (chemical shift) and other nuclei via the electrons (scalar coupling). The former
leads to shifts of the resonance frequencies of the nuclei located in different chemical
environment since the magnetic field is shielded by the electrons to B = Bo(1-σ), where σ
is the shielding constant. Scalar coupling results in splitting of the resonance lines
according to values of the coupling constants for the nuclear pairs.
Formation of molecular complexes can be followed by changes of the chemical shifts
of the nuclei directly engaged in the interactions. The total chemical shift can be
represented as a sum of local effects (diamagnetic, d, and paramagnetic, p, contributions)
and long-range effects, direct anisotropy (a) of the surrounding chemical bonds, ring
currents (r), electric field (e) and solvent (s):
δ = δd(local) + δp(local) + δa + δr + δe + δs
43
Eq. 3-63
Substantial deshielding effect observed for protons upon formation a hydrogen bond
(high value of δ) arises from an interplay of the increased electron density in the vicinity of
the proton (increase of δd(local)) and predominating effects of the additional restraint
placed on field-induced electron circulations by the acceptor (increase of δp(local)) and its
electric field effect (δe). Stacking of π-electron rings is usually accompanied by an upfield
shift of the protons attached to the rings (δr), since the additional magnetic field from the
circulating π-electrons reinforces Bo in the planes of the rings.
To detect the NMR signal of the exchangeable protons in aqueous solution, e.g. H(8)
of m 7 G, H2O instead of 2 H2O must be applied. The strong water signal in the 1 H NMR
measurements needs to be suppressed.
3.7.1. NMR Spectra Recording
1 H NMR spectra of tryptophan N-acetylamid, a dodecapeptide DGIEPMWEDEKN,
and cap analogues were recorded on a VarianUNITYplus 500 MHz spectrometer, in 1/15
M phosphate buffer, pH 5.6 or 5.2, with sodium 3-trimethylsilyl-[2,2,3,3- 2 H4]propionate
(TSP) as internal standard, and 10% 2 H2O for spin locking. The assignment of protons in
the cap analogues and tryptophan were made on the basis of the splitting patterns of the
proton resonances and their chemical shifts values (accuracy of ± 0.001 ppm). The
unambiguous assignment for the H(4)/H(7) and H(5)/H(6) proton pairs in the tryptophan
indole ring were done earlier from analysis of the cross-peaks in 2D ROESY spectrum by
Dr. Ryszard Stolarski. 204 An effective method (~1000-fold suppression) of a binominal

1331 experiment was used for water suppression. 205 It tailors the excitation profiles so that
there is zero net excitation of water. The hard pulse sequence was p1-τ-p2-τ-p3-τ-p4 with
the length ratio 1 : 3 : 3 : 1. The sum of the pulse lengths was 50 μs giving rotation of the
proton magnetization by π/2 at B1 of 35 dB (attenuation of the maximal B1 strength). The
offset of the maximal excitation in respect to the carrier frequency put on the water signal,
determined by the period τ, was 1500 Hz for maximal excitation in the aromatic region.
3.7.2. NMR Data Analysis
3.7.2.1. Concentration Dependence
Observed differences of 1 H chemical shifts (Δδ = δ(mixture) - δ(free)) of tryptophan
N-acetylamid or of the dodecapeptide tryptophan (concentration ctrp) due to stacking upon
titration with a cap analogue at 298 K depend on the total concentration of the cap
analogue (ccap) according to equation:
Δδ
= Δδ
u
c
⋅
c
u
trp
+ Δδ
st
⎛
⎜
c
⋅ 1−
⎜
⎝
c
u
trp
⎞
⎟ , Eq. 3-64
⎟
⎠
where Δδu is the difference of the chemical shift of the unstacked tryptophan proton (fixed
as 0), Δδst is the difference of the chemical shift of the stacked tryptophan proton (fitted as
a free parameter), and equilibrium concentration of the unbound tryptophan proton (cu) is
determined by a fitted association constant (K):
c
u
2
2
K ⋅(
ctrp
− ccap
) −1+
K ⋅(
ctrp
− ccap
) + 2⋅
K(
ctrp
+ ccap)
+ 1
= Eq. 3-65
2⋅
K
3.7.2.2. Temperature Dependence
Temperature dependence of observed differences of 1 H chemical shifts of tryptophan
N-acetylamid at 1 mM in the presence of 29.8 mM 7-methylGMP, and of the
dodecapeptide tryptophan at 2.8 mM in the presence of 17.2 mM 7-methylGpppG is
described by equation:
Δδ
= Δδ
st
⎛ ⎞
⎜
cu
⋅ 1 − ⎟ , Eq. 3-66
⎜ ⎟
⎝
ctrp
⎠
where cu is defined above, but the association constant is temperature dependent according
to the van't Hoff isobaric equation:
44

⎛ ΔS°
ΔH°
vH ⎞
K = exp⎜
− ⎟ , Eq. 3-67
⎝ R R ⋅T
⎠
R – the gas constant, T – absolute temperature. The standard molar entropy change (ΔS°)
and enthalpy change (ΔH°vH) are fitted, constant parameters or treated as functions of
temperature. Then the fitted parameter is the heat capacity change ( Δ C ) that is involved
through Eq. 1-11 and 1-12.
3.8. Dynamic Light Scattering
Light is scattered by interactions of electrons of molecules dissolved in a solution
with the incident radiation. The scattering is quasi-elastic. The oscillating electric field
causes a vibration of electrons turning them into oscillating dipoles which reemit radiation.
The power spectrum is broadened in the frequency domain due to the Doppler effect on the
Brownian motion of scattering macromolecules and hence due to their diffusion coefficient
(D). D is related to the size of the molecules, for instance to the hydrodynamic radius (Rh)
for approximately globular proteins. Fluctuations in the intensity of light scattered by a
small volume of a solution in the microsecond time-scale are described by an
autocorrelation function (g1(t)) which is registered by the instrument. For small
monodispersed particles and homogeneous spheres the normalized autocorrelation function
of scattered electric field is: 206
g
1
( t)
v
−DQ
2
t
= e , Eq. 3-68
where Q v is the scattering vector resulting from the difference of the scattered and the
incident wave vectors, t is the time interval for displacement of the scattering particle. D is
related to the reciprocal of the characteristic decay time (Γ):
2
DQ v
Γ =
Eq. 3-69
For a continuous polydispersed systems with the Γ distribution function (G(Γ)):
∞
−Γt g1
( t)
= a ∫ G(
Γ)
e dΓ
, where a = const. Eq. 3-70
0
An inverse Laplace transform generates the distribution of decay rates (G(Γ)), from which
the diffusion coefficients distribution and next the particle sizes distribution can be
determined using the Stokes-Einstein equation:
45
o
p

k BT
D = Eq. 3-71
6πηR
h
Molecular weight calculations are performed by means of a logarithmic function
based upon experimental data on several well-characterized proteins, provided by the
manufacturer of the DLS equipment.
The DLS measurements were run on a DynaPro-801 Molecular Size Detector
(Protein Solutions Inc., Charlottesville, VA) for eIF4E (residues 33-217) at a concentration
of 1 mg/ml, in the absence and in the presence of 50-fold excess of m 7 GTP, p-Cl-bz 7 GTP,
m3 227 GTP, m 7 GpppG, m 7 GppppG, or m 7 Gppppm 7 G, at 20ºC, in the standard buffer.
3.9. Statistical Analysis
Goodness of fits was described by the R 2 value, which was not less than 0.999 and 0.99 for
binding isotherms of cap analogues and peptides, respectively:
∑ ( y exp − yfit
)
2
R = 1−
, Eq. 3-72
2
∑(
y − y)
exp
2
where yexp are measured values, yfit are fitted values, and y is the mean of the measured
values. Other R 2 values are given in the text.
Statistical analysis was done on the basis of the runs test and the P value 197 which
denotes the probability that distribution of the fitting residuals of the y-values is random
and does not depend on the x-values.
Discrimination between two models of different numbers of degrees of freedom (ν1
and ν2) was based of the statistical two-parameter Snedecor's F-test. 197 The test allows for
a consistent and systematic assessment whether the results obtained from more
complicated model with a greater number of fitted parameters are statistically important in
comparison with the simpler model. The F-ratio quantifies the relationship between the
relative decrease in sum-of-squares and the relative decrease in degrees of freedom:
F =
2
2
( ∑( y − y ) − ∑(
y − y ) )
exp
fit _1
exp
fit _ 2
( ν1
− ν2
) ν2
46
∑(
y
exp
− y
fit _ 2
)
2
Eq. 3-73
The P(ν1, ν2) value in this case is the probability that the improvement of the fit with the
greater number of fitted parameters (i. e. with smaller ν) expressed by the F-ratio could be
obtained randomly. The significance level was assumed as P(ν1, ν2) < 0.1 or < 0.05.

Errors of reported values (one standard deviation) were calculated according to the
appropriate propagation rules 207 on the basis of the numerical uncertainty resulting from
the fitting. Thanks to avoidance of linear transformations of the fluorescence titration data,
their experimental uncertainties were constant within the whole range of x-axis and almost
always negligibly small (less than the size of graphical symbols). Hence, they did not
influence much the final results (Kas). Experimental errors were taken into account on
integration of the calorimetric data and on analyses of thermodynamic parameters.
Regressions were performed by means of linear or non-linear, least-squares method,
using PRISM 3.02 from GraphPad Software Inc., U.S.A. or ORIGIN 6.0 from Microcal
Software Inc., U.S.A.
3.10. Additional Information − Crystallography
The crystallographic structure of murine eIF4E (residues 28-217) in complex with 7-
methyl-GpppG to which the thesis often refers was resolved by Drs. Joseph Marcotrigiano
and Stephen K. Burley (The Rockefeller University, N.Y., U.S.A.) similarly as reported
previously 75 and was published in a joint paper. 1 Atomic coordinates have been submitted
to the protein databank, accession code: 1L8B.
47

4. Results and Discussion
4.1. Methodological Aspects of Studies of Non-Enzymatic
Protein
Biophysical analysis of intermolecular interactions aimed at rational design of
therapeutic agents requires reliable measurements of the protein-ligand association in
solution as a necessary counterpart to high resolution structural studies. For eIF4E, such
consistent information was lacking. The association constants have been changing
significantly with each new publication due to several neglected sources of errors, both
experimental and numerical. First, the lack of quantitative control of the protein activity.
All association constants (Kas) reported in the previous studies were derived assuming
100% activity of the protein. 5,156,161-164 As eIF4E is known to be a highly unstable protein,
the prevailing assumption that entire population of eIF4E is active at every conditions was
groundless. Second, the presence of practically unremovable contamination of affinity-
purified eIF4E with the cap-analogue that was used for the elution from the cap-affinity
column. Such eIF4E can be up to 60% m 7 GTP-bound, even after application of ionic
exchange chromatography 208 or repeated buffer exchange on a 5 kDa NMWL filter. Third,
dilution of a sample in the course of the titration, inner filter effect, and contribution of a
free ligand to total fluorescence, were only partially eliminated in the previous
publications. Dilution was considered in most of them, 5,156,161-163 while the inner filter
effect and estimated emission of a free ligand were taken into account properly in the
single paper. 5 The results of studies on eIF4E fused with GST, which molecular mass is
comparable with that of eIF4E, were useful only for qualitative interpretation. 164
All above-mentioned experimental problems are rigorously analysed herein.
Purification of eIF4E via refolding from inclusion bodies guarantees that the protein is cap-
free. Since eIF4E is a non-enzymatic protein, it is impossible to determine the specific
activity of each sample from independent experiments. The active protein concentration
([Pact]) is thus introduced as a free parameter in the numerical analysis. Addition of the
next fitting parameter requires a novel, very precise measuring method of fluorescence
quenching. Interactions of the apo-protein with cap analogues are studied by the time-
synchronized titration, yielding the goodness of fit R 2 = 0.999-0.9999, what assures the
48

accuracy enough to extract the lacking information on the actual protein activity from each
specific titration. Only this approach, combining a rigorous gathering of experimental data
with simultaneous check-up on the protein activity, provides results without systematic
deviation from the theoretical model (P 0.05), and leads to concordance among Kas and
other fitted parameters.
4.1.1. Solution of Spectroscopic Difficulties
Absorption
0.012
0.009
0.006
0.003
0.000
250 280 310 340 370
λ [nm]
Figure 4-1. Absorption (left) and fluorescence (right, λex = 280 nm) spectra of eIF4E (solid line,
0.2μM) and m 7 GTP (dotted line, 4μM) at 20°C. Typical eIF4E fluorescence quenching spectra
upon increasing concentration of m 7 GTP (0.7nM−4μM). Fluorescence spectra for higher
concentrations of m 7 GTP exhibit an increasing emission of free m 7 GTP present in solution.
All fluorescent species in solution are explicitly included in the numerical analysis.
The complete overlapping of the absorption spectra for eIF4E and the cap analogues (Fig.
4-1) makes it impossible to excite the protein selectively. The emission spectra are also
overlapped; therefore the contribution from the increasing concentration of the free cap
analogues to the total fluorescence in the course of the titration must not be neglected. This
makes the spectroscopic analysis of intermolecular interactions complicated. Subtraction of
the free cap fluorescence is groundless, since the equilibrium concentration of the free
ligand depends on the association constant to be determined. The spectra overlapping leads
to erroneous attribution of the signal plateau to the saturation state, while the apparent
plateau originates from two effects that cancel each other out: the quenching of the
49
100
80
60
40
20
0
Fluorescence a. u.

intrinsic protein fluorescence and the increasing emission of the free cap analogues. Both
effects are now taken into account. As a result, the observed maximal fluorescence
quenching is different for various cap analogues (Fig. 4-2), and smaller than the calculated
intrinsic eIF4E fluorescence quenching (Q, see Eq. 3-27, p. 34), which is the same for
different cap analogues (~ 65% for the fresh protein). The association constants determined
from the titration curves with the neglected free ligand emission would be erroneous,
especially for weakly binding and strongly emitting ligands. The present method of data
analysis eliminates these problems.
Fluorescence (a. u.)
Residuals
100
80
60
40
20
1
-1
10 -3 10 -2 10 -1 10 0 10 1 10 2
cap concentration (μM) (μ
Figure 4-2. Titration curves for m 7 GTP ( ), m 7 GpppG ( ), and m3 2,2,7 GTP ( ) at 20°C, and
fitting residuals. An increasing fluorescence signal at higher cap concentrations originates from
emission of the free-cap.
50

4.1.2. Activity of Non-Enzymatic Protein
Quantitative control of the protein activity belongs to the fundamental state-of-the-art
biochemical methods usually applied to enzymatic proteins, and was to date impossible to
do with non-enzymatic proteins. Determination of the actual concentration of the active
eIF4E factor upon each titration was the key, which made it possible to interpret the results
of the binding studies in a quantitative manner.
For previously frozen samples of apo-protein, the active fraction decreases down to
even less than 10 % (Table 4-1, Fig. 4-3). The inactive fraction aggregates and precipitates,
in spite of low protein concentration of the stored stock solution (< 0.5 mg/ml), the
presence of 10 % (v/v) glycerol, 0.5 mM EDTA, and flash freezing of the aliquots (from
10 μL to 50 μL). This is in contrast to the cap-saturated eIF4E that is stabilized by the
ligand (see 4.2.3.1.6.1., p. 82). The experiments reported in this thesis were performed
temporarily with frozen protein and then repeated with freshly prepared eIF4E (not frozen,
stored at 4 °C), for which the quantity of active protein was satisfactory and varied mostly
from 80 % to 113 % (± 12%) of the concentration estimated from the absorption spectra
(Table 4-1). The statistics of the results proves that determination of the Kas values is
reliable, and is unaffected by both the actual concentration of the active protein in the
sample and the accompanying amount of the inactive protein. This indicates that the
inactivation by freezing, e. g. by cold denaturation, 137 or by elevated temperatures during
titrations do not partially diminish the affinity of the whole protein population, but removes
a part of the population from the binding reaction, while the native protein molecules still
retain their specific affinity.
51

Table 4-1. Concentration of the active eIF4E protein ([Pact]) in the cuvette during fluorescence
titration with m 7 GTP, equilibrium association constants (Kas), and percentage of the active protein
in respect to the total concentration estimated from absorbance of the fresh or frozen stock solution
([Ptotal]), at 20 °C, pH 7.2, 100 mM KCl.
[Pact] (μM) Kas ⋅ 10 -6 (M -1 ) [Pact]/[Ptotal] (%)
Fresh protein
0.0283 ± 0.0023 115 ± 14 28
0.0405 ± 0.0091 179 ± 103 110
0.1026 ± 0.0053 106 ± 19 59
0.1426 ± 0.0028 143 ± 18 93
0.1551 ± 0.0037 72.3 ± 6.9 80
0.1720 ± 0.0057 113 ± 24 105
0.1798 ± 0.0014 103.2 ± 5.1 90
0.1861 ± 0.0074 76 ± 15 113
0.1893 ± 0.0056 108 ± 23 89
0.2049 ± 0.0013 113.3 ± 6.0 103
0.2258 ± 0.0067 158 ± 47 98
0.2990 ± 0.0075 95 ± 23 60
Frozen protein
0.0005 ± 0.0053 89 ± 26 0.2
0.0167 ± 0.0013 190 ± 19 4.0
0.0239 ± 0.0012 83.3 ± 4.6 8.1
0.0254 ± 0.0025 129 ± 18 20
0.0265 ± 0.0037 70 ± 10 6.8
0.0385 ± 0.0020 157 ± 23 21
0.0466 ± 0.0035 111 ± 19 18
0.0677 ± 0.0054 93 ± 24 53
0.0720 ± 0.0022 75.9 ± 6.9 19
0.0762 ± 0.0034 170 ± 44 42
0.0867 ± 0.0029 118 ± 15 43
0.0967 ± 0.0140 125 ± 71 76
0.1896 ± 0.0180 73 ± 34 94
52

Active protein (%)
N
100
50
10
0
0.0 0.1 0.2 0.3 0.4 0.5
8
6
4
2
0
Total protein concentration (μM)
70 90 110 130 150 170 190
K as ⋅ 10 -6 (M -1 )
(a) (b)
(c) (d)
Figure 4-3. Statistics of titration results obtained for frozen ( ), and fresh protein () from one
purification batch, used within several subsequent days, at 20°C. (a) Active protein fraction
([Pact]/[Ptotal]) vs total protein concentration during fluorescence titration. The frozen protein tends
to precipitate the more the higher is the concentration of the solution, while [Pact]/[Ptotal] of the fresh
samples seems to be clustered about 100% activity. (b) Values of association constant (Kas) for
m 7 GTP binding to the active fraction of eIF4E are invariant irrespectively of the presence of
various amount of inactive protein in the frozen or fresh samples. (c) Distribution (N) of Kas values
obtained from 26 repeats of titration experiments. (d) Gaussian distributions of the Gibbs free
energy of binding of m 7 GTP to frozen ( , thin solid line) and fresh ( , broken line) eIF4E;
summary ( , thick solid line, = -45.11 kJ/mol, SD = 0.87 kJ/mol).
The influence of the concentration of active protein as fitting parameter on quality of
final results is illustrated in Fig. 4-4. Titration of a frozen sample with m 7 GTP in the
nanomolar concentration range, i.e. where fluorescence of the ligand is negligible at the
spectral bandwidths suitable for observation of the protein fluorescence changes, allows for
analysis of the protein activity in the simple case with φlig-free = 0.
53
Kas (M -1 )
N
10 9
10 8
10 7
16
12
8
4
0 20 40 60 80 100 120
Active protein (%)
0
-47 -46 -45 -44 -43
ΔG° (kJ/mol)

Fluorescence (a. u.)
Residuals
800
600
400
200
8
0
-8
(a) (b)
10 -3 10 -2 10 -1
m 7 GTP (μM)
10 -3 10 -2 10 -1
Figure 4-4. Binding isotherms for interaction of m 7 GTP with frozen (a) and fresh (b) eIF4E with
protein concentration fixed as 100% (broken lines, as residuals) or treated as a free parameter of
the fitting (solid lines, and as residuals).
It is clear that the fit with the fixed protein concentration determined from
absorbance before freezing of the sample ([Pact] = 0.295 μM) does not satisfy the assumed
model, with R 2 = 0.2957 and P = 0.00027. The apparent association constant attains an
accidental value of 18 ± 130 ⋅ 10 6 M -1 , while the fit with the active protein concentration
dealt as a free parameter shows an excellent goodness of fit (R 2 = 0.9996 and P = 0.27) and
yields Kas = 107.1 ± 7.8 ⋅ 10 6 M -1 but ([Pact] = 0.0266 ± 0.0014 μM. This indicates that the
active fraction is only ~ 8 %! Attempts for determination of Kas in the full range of the
ligand concentration with the fixed [Pact] value give still worse, worthless results.
Moreover, the parameter describing the actual concentration of the active protein is crucial
not only for the evidently inactivated, frozen samples, but can also change the results
significantly even when the fitted curve is apparently "nice" (which is often the case in
many literature reports, especially in those using a linear scale for ligand concentrations).
Fig. 4-4(b) presents a slight difference between two fits: with [Pact] fixed as 0.2 μM and
with [Pact] fitted as 0.1798 ± 0.0014 μM. Although the graphical difference between solid
(R 2 = 0.9999) and broken (R 2 = 0.9990) lines is almost indistinguishable at the first sight,
and the protein activity is pretty high (90%), the fit assuming the fixed [Pact] value yields a
function with a systematic deviation from the experimental data, which is revealed by the
residuals and the P value of < 0.0001. The latter parameter indicates that it is only less than
0.01% chance that this deviation is not caused by fixing of [Pact] at the level of 100%. The
Fluorescence (a. u.)
Residuals
54
100
80
60
40
10 -3 10 -2 10 -1 10 0 10 1
2
0
-2
m 7 GTP (μM)
10 -3 10 -2 10 -1 10 0 10 1

Kas value obtained with free [Pact] is 103.2 ± 5.18 ⋅ 10 6 M -1 . Neglect of this small, 10%
difference in the protein activity upon non-linear fitting with fixed [Pact] results in as many
as 2-fold greater Kas of 188 ± 26 ⋅ 10 6 M -1 . Hence, the protein activity must be controlled
upon each individual titration. This is especially important for strongly binding ligands,
such as m 7 GTP, p-Cl-bz 7 GTP or m 7 Gppppm 7 G (Fig. 4-5). The temperatures at which the
activity of eIF4E drops by 50% (34 – 41 °C) are in the physiological range, as estimated
from the Boltzmann sigmoidal curves. These results could suggest that the unstable eIF4E
protein is usually complexed with another macromolecule (mRNA 5' cap or other proteins)
in the living cell to avoid inactivation in the apo state, which is consistent with the
observation that most of the eIF4E population is blocked by 4E-BPs, and biological
potency of eIF4E is regulated by its accessibility. 42
Active protein (%)
Active protein (%)
100
50
0
100
50
0
(a) 0 (b)
0 5 10 15 20 25 30 35 40 45
Temperature (°C)
(c) (d)
0 5 10 15 20 25 30 35 40 45
Temperature (°C)
Figure 4-5. Temperature dependence of the active fraction of fresh protein ( , , , ) obtained
upon titration with ligands of different affinity for eIF4E. (a) m 7 GTP, Kas = 108.7 ⋅ 10 6 M -1 ; (b)
m 7 Gppppm 7 G, Kas = 47.0 ⋅ 10 6 M -1 ; (c) p-Cl-bz 7 GTP, Kas = 44.6 ⋅ 10 6 M -1 , results for frozen protein
at 20°C is shown for comparison (); (d) m 7 GpppC, Kas = 3.86 ⋅ 10 6 M -1 ; the Kas values at 20 °C.
55
Active protein (%)
log(% of active protein)
100
50
10
2
0
0 5 10 15 20 25 30 35 40 45
Temperature (°C)
-10
0 5 10 15 20 25 30 35 40 45
Temperature (°C)

For the ligands that bind to the protein less strongly (Kas < 10 7 M -1 ), the
concentration of active protein looses its numerical importance. Determination of [Pact]
becomes inaccurate due to weakly pronounced curvature of the fitted function F([L]) in the
ligand concentration range close to [Pact].
The population of active protein is also ionic strength-dependent (Fig. 4-6). Relative
stabilization is achieved at the ionic strength characteristic for the intracellular fluid (~200
mM). Determination of the active fraction of protein as a function of [KCl] was necessary
to obtain association and dissociation rate constants from the kinetic traces registered
during stopped-flow experiments for interaction of eIF4E with m 7 GpppG. 4 Single-
relaxation fits yielded overall kinetic constants and dual-relaxation fits provided evidence
for the two-step character of the complex formation.
Active protein (%)
100
50
0
0 100 200 300 400
KCl (mM)
Figure 4-6. Dependence of the average active fraction of fresh protein on KCl concentration after
storage by ~6 hours at 25 °C. Results obtained from titrations of eIF4E with m 7 GTP to control the
protein activity during kinetic stopped-flow experiments with use of m 7 GpppG. 4
The knowledge about the actual concentration of active non-enzymatic protein is
crucial for normalization of every quantity being determined experimentally, analogically
as specific activity of enzymes is used. 209 In particular, long-lasting thermodynamic
measurements performed by means of techniques other than emission spectroscopy require
concurrent control fluorescence titrations so that the results are free from systematic errors
caused by temperature-dependent protein inactivation.
56

Fluorescence (a. u.)
Figure 4-7. Determination of protein active fractions at 30 °C (303.2 K, , broken lines) and 15 °C
(288.1 K, , solid lines). Titration curves determine both K as and [Pact] as free parameters of the
fitting (Eq. 3-26, p. 34). [Pact] is graphically represented by the point where the curve for
hypothetical infinite Kas would attain maximal quenching Qmax (Eq. 3-27, p. 34).
Changes in the amount of active eIF4E incubated at 288 K and 303 K during a time
corresponding to duration of isothermal calorimetric titration are shown in Fig. 4-7. Total
eIF4E concentration was 0.2 μM at both temperatures. Beside obvious differences between
the association constants, the control fluorescence experiments have shown that eIF4E
retained 89.7 % activity at 288 K but only 53.5 % at 303 K. Further determination of the
standard molar binding enthalpies and heat capacity changes had to be based on this
information.
100
80
60
40
303K
Qmax Q max 288K
10 -3 10 -2 10-1 10 0 101 [Pact] 20
303K [Pact] 288K
m 7 GTP (μM)
The methodology of determination of the active protein fraction was initially
designed to resolve the problem of activity control of the non-enzymatic eIF4E translation
factor. 4,6 However, it was further proposed to study stoichiometry of ligand binding to an
enzymatic oligoprotein and proved successful in finding that a hexameric enzyme (PNP)
binds only three substrate molecules per one hexamer. 210,211
57

4.1.3. Incorrectness of Data Linearization in Case of Strong Interactions
Independently from skipping the fundamental problem of the protein activity, the
previous reports were based mostly on linearized forms of the equilibrium equation which
are not suitable for the analysis of strong interactions. The commonly used Eadie-Hofstee
linear transformation requires an assumption that protein concentration is negligible in
comparison with ligand concentration over the entire course of the titration. 156,161-163 This is
not satisfied for molecular systems of a higher affinity. When Kas ~ 10 μM -1 , saturation of
~ 40% occurs already for [L] [Pact], at typical [Pact] ~ 0.1 μM. This makes the
transformed data non-linear, even after corrections for dilution, inner filter effect, and free
ligand emission. Additionally, the experimental uncertainty is hidden in the x-axis (Fig. 4-
8). Inverse representation of the data reveals the huge errors which are always neglected
during the Eadie-Hofstee analysis.
ΔΔF (a. u.)
400
300
200
100
0
2500 5000 7500 10000
ΔF/[L] (a. u./μM)
Figure 4-8. (a) Eadie-Hofstee transformation of experimental data from the same fluorescence
titration as in Fig. 4-4(a), apparent Kas = 39 ± 12 ⋅ 10 6 M -1 and (b) inverse Eadie-Hofstee
transformation of these data, apparent Kas = 17.2 ± 5.0 ⋅ 10 6 M -1 .
A modified linear Eadie-Hofstee representation 5 requires the active protein
concentration and the maximal quenching (ΔFmax) as known constants (Fig. 4-9). Even
though they are guessed properly, the most important data points, which should determine
the binding constant, are compressed by the transformation into a very narrow numerical
range (the stronger binding the more narrow the range). This leads to loss of their
numerical importance, and consequently, to significantly biased Kas values.
ΔF/[L] (a. u./ μM)
58
15000
10000
5000
0
-5000
-10000
0 100 200 300
ΔF (a. u.)

[m 7 GTP]/ΔF (μM/a. u.)
0.0006
0.0004
0.0002
0.0000
0.00 0.01 0.02 0.03
1/(ΔF max-ΔF) (1/a. u.)
Figure 4-9. Modified linear Eadie-Hofstee representation 5 of the data from Fig. 4-4(a) and Fig. 4-8.
Although the required constant parameters ([Pact] and ΔFmax) of the linear data transformation are
taken from the previous fit with the free active protein concentration, shown in Fig. 4-4(a), the
apparent Kas value is significantly decreased, 72.3 ± 1.2 ⋅ 10 6 M -1 , due to contraction of the most
important data at the low concentrations of the ligand to a very narrow range of the x variable.
The non-linear analysis is free from the systematic errors caused by the linear
transformations. The methodological improvements afford possibilities for a reliable
comparison of the results for the protein from different purification batches, previously
frozen or not, from experiments at different temperatures, ionic strengths, and pH. The Kas
values up to 10 8 M -1 , accompanied by the systematic and self-consistent structure-affinity
relationship, provide an exact, quantitative test for the proper fold of the renatured protein.
This assures for the first time that the equilibrium association constants reflect the true,
intrinsic affinity of eIF4E for mRNA 5' cap.
59

4.2. Molecular Mechanism of Recognition of mRNA 5' Cap
Structure by eIF4E Cap-Binding Protein
4.2.1. Affinity of Cap Analogues for eIF4E
4.2.1.1. Equilibrium Binding Constants
Kas values determined for a wide class of cap analogues (Table 4-2) vary from 10 8 M -
1 for specific 7-substituted cap analogues to 10 2 M -1 for 7-unsubstituted ligands (Scheme 2-
1). The human and murine proteins have the same binding affinities for eIF4E. The murine
protein was used for the further studies because of technical matters, i. e. more efficient
expression and purification. However, thanks to almost 100% identity, the conclusions
drawn from the data are common for both proteins.
The substantial differences of the Kas values follow structural modifications of cap,
m 7 , 2 , 2
e.g. a 760-fold reduction of Kas for 3 GTP and up to a 5000-fold drop of Kas for GTP
as compared to m 7 GTP. The presence of any N(7)-substituent enhances binding, but the
methyl substituent appears to be optimal. Among several elements important for specific
eIF4E-cap binding, the negative electrostatic charge of the phosphate chain, which depends
both on the number of phosphate groups and the presence of a second nucleotide, is of
primary importance. The decrease of Kas with single step-wise reduction of the phosphate
Fluorescence (a. u.)
100
80
60
40
GMP
GDP
GTP
m 7 GMP
m 7 GDP
m 7 GTP
10 -3 10 -2 10 -1 10 0 10 1 10 2
20
Concentration of cap analogue (μM)
Figure 4-10. Comparison of binding isotherms for eIF4E complexes with unmethylated and
methylated at N(7) guanosine mono-, di-, and triphosphates, at 20°C, pH 7.2, 100 mM KCl.
60

Table 4-2. Binding free energies (ΔG°) and equilibrium association constants (Kas) for complexes of
murine eIF4E (28-217) and human eIF4E (1-217) with various cap analogues, at 20°C, 50 mM
Hepes/KOH pH 7.2, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA.
Cap-analogue Murine eIF4E (28-217) Human eIF4E (1-217)
ΔG° (kJ/mol) Kas ⋅ 10 -6 (M -1 ) Kas ⋅ 10 -6 (M -1 )
m 7 GTP -45.099 ± 0.088 108.7 ± 4.0 97 ± 30 1.17 ± 0.12 a
m 7 GDP -41.02 ± 0.18 20.4 ± 1.5
m 7 GMP -33.15 ± 0.20 0.806 ± 0.067
m 7 G -20.54 ± 0.63 0.0046 ± 0.0012
et 7 GTP -39.38 ± 0.20 10.41 ± 0.86
bz 7 GTP -40.660 ± 0.059 17.59 ± 0.43
p-Cl-bz 7 GTP -42.93 ± 0.21 44.6 ± 3.8
m 7 , 2
2 GTP
-41.89 ± 0.20 29.2 ± 2.4
m 7 , 2 , 2
3 GTP -28.9 ± 1.4 0.143 ± 0.080
m 7 Gpppm 2’O G -38.75 ± 0.16 8.04 ± 0.51
m 7 GpppG -38.55 ± 0.15 7.39 ± 0.46 9.6 ± 0.8 0.69 ± 0.07 a
m 7 GpppA -37.43 ± 0.27 4.68 ± 0.52
m 7 GpppC -36.41 ± 0.40 3.08 ± 0.51 4.8 ± 0.4 0.48 ± 0.05 a
m 7 Gpppm 7 (micro) b
G -35.28 ± 0.36 1.93 ± 0.28
m 7 Gpppm 7 (macro) b
G 3.86 ± 0.56
m 7 GppppG -44.96 ± 0.11 102.8 ± 4.4
m 7 Gppppm 7 (micro) b
G -41.37 ± 0.15 23.5 ± 1.4
m 7 Gppppm 7 (macro) b
G 47.0 ± 2.7
GTP -24.30 ± 0.11 0.0214 ± 0.0010
GDP -20.71 ± 0.25 0.0049 ± 0.0005
GMP -12.93 ± 3.8 0.0002 ± 0.0003
(micro) b
GpppG -20.66 ± 0.21 0.0048 ± 0.0004
(macro) b
GpppG 0.0095 ± 0.0007
a values for m 7 G-affinity purified full length human eIF4E 5
b microscopic and macroscopic association constants, see text for details.
chain in the series of 7-methylated mononucleotides is followed by the same changes of
Kas for the unmethylated compounds (~5- and ~25-fold for removal of the γ-phosphate and
the β-phosphate, respectively). Removal of the α-phosphate leads to a drastic fall of Kas,
which is 175-fold lower for m 7 G than that of m 7 GMP (Fig. 4-10).
61

The dinucleotide triphosphate cap analogues also bind to eIF4E less strongly (~20-
fold) than m 7 GTP. The identity of the second base seems to be of minor importance.
Generally, either base of the dinucleotide cap analogues penetrate the binding slot of
eIF4E, but since the association constants of unmethylated molecules are far lower than
those of the methylated ones, such dual binding is negligible for asymmetrical cap
analogues. By contrast, for symmetrical molecules (m 7 Gpppm 7 G, m 7 Gppppm 7 G and
GpppG) the observed Kas must be divided by 2 because of entropic effects. Only this
normalized Kas represents a true microscopic association constant Kas (micro) , reflecting the
intrinsic stabilization energy of the complex. The macroscopic Kas, however, corresponds
to the effective biological potency of the cap analogues.
In the pioneering work of Carberry et al., 156 the association constants of two
fundamental cap analogues, m 7 GTP and m 7 GpppG, for eIF4E from human erythrocytes
purified by cap-affinity chromatography were determined as 3.87 ⋅ 10 5 M -1 and 3.70 ⋅ 10 5
M -1 , respectively (23°C, pH 7.6, without KCl in the buffer). Similar Kas values in the range
of 10 5 -10 6 M -1 were reported for recombinant human eIF4E purified by the same
method. 5,161-163 In those studies also no essential difference in affinity between m 7 GTP and
m 7 GpppG was observed. While the association constants reported here for the murine
protein are at variance with these data, they are very close to those obtained for
recombinant full length human eIF4E, which was also purified via refolding from inclusion
bodies without any prior contact with cap (Table 4-2). These Kas values are up to 500-fold
higher than those reported previously.
4.2.1.2. Binding Affinity vs Inhibitory Properties of Cap Analogues
The affinities represented by the present Kas values (Table 4-2) reveal large
differences among cap analogues possessing different functional groups and correspond
well to the inhibitory properties of the cap analogues observed in a rabbit reticulocyte
lysate (Fig. 4-11). In a kinetic model developed for the in vitro translational
system, 157,212,213 the inhibition constant (KI) was determined as the overall dissociation
constant of the cap analogue from the 48S initiation complex. Results of both approaches,
biophysical and biochemical, point out the significance of the same structural features of
the cap responsible for specific recognition, e.g. the presence and type of N(7)-substituent
of guanine, the negative charge density in the phosphate chain, and the N 2 amino group
with at least one proton capable of forming a hydrogen bond. Interestingly, the observed
62

values of KI are ~100 times greater than those of 1/Kas for all cap analogues. This
apparently larger amount of cap analogue required to inhibit translation in vitro than that
necessary to displace eIF4E from the mRNA cap most likely arises from the following
reasons. As opposed to HeLa cells, 41 the eIF4E concentration in reticulocyte lysate is not
limiting for translation, and the majority of eIF4E is not engaged in this process. 214 The
assumption underlying the determination of KI values, that eIF4E concentration is
negligible in comparison with that of a competitive inhibitor, 213 is not applicable here. The
concentration of eIF4E is up to 50-fold higher than previously thought, 215 so much more
cap analogue is required to inhibit translation due to the quadratic form of the equilibrium
equation. Besides, protein synthesis results from a large number of catalytic reactions and
depends not only on the eIF4E activity, e. g. in the in vitro experiments, the mRNA
binding activity of eIF4F complex is enhanced by the presence of eIF4G. 216
KI [μμM]
10 3
10 2
10 1
10 0
10 -2 10 -1 10 0 10 1 10 2
K as [μM -1 ]
Figure 4-11. Correlation between inhibition constants of the cap analogues (KI), obtained from in
vitro translation (30°C 157 ), and their equilibrium association constants (Kas, 20°C; macroscopic Kas
were used for the symmetrical caps) by 4 orders of magnitude. Correlation coefficient r 2 = 0.795.
63

4.2.2. Parsing the Free Energy of eIF4E Binding to mRNA 5' Cap
A comparison of the binding free energies rather than that of the association
constants reflects the biophysical basis of the ligand affinity for the protein. The data
collected for twenty structurally different cap analogues permitted analysis of the influence
of single structural modifications on the cap-eIF4E binding in terms of the standard Gibbs
free energies ΔG° (Table 4-2), which were calculated either from the binding constants Kas,
Δ G° = −RT
ln K as , or from Kas (micro) ,
64
( micro)
as
Δ G° = −RT
ln K , for the symmetrical cap
analogues. Contribution of a given chemical alteration is represented by the corresponding
change of the standard Gibbs energy of association (ΔΔG°), defined individually for each
group of compounds in Table 4-3.
It should be noted here that parsing is generally not applicable for every molecular
system. 152,153 However, direct comparison of the ΔG° values surprisingly but distinctly
showed that the total binding free energy resolved itself into individual contributions from
interactions of the phosphate chain and into common contribution from all interactions of
the m 7 guanosine moiety. This limited additivity is a kind of empirical verification of
correctness of parsing employment in this particular case, and, on the other hand, points to
cooperativity of interactions involving the m 7 guanosine moiety (stacking and hydrogen
bonding) that may not be decomposed into their contributions.

Table 4-3. Changes in the standard Gibbs free energy (ΔΔG°) on eIF4E binding to the
structurally modified cap analogues, at 20°C.
Structural alteration ΔΔG° ΔΔ kJ/mol
replacement of N(7)-methyl for larger substituents:
ΔΔG° = ΔG°(x 7 GTP) − ΔG°(m 7 GTP)
m → x : m → et m → bz m → p-Cl-bz
x 7 GTP +5.73 ± 0.21 +4.44 ± 0.13 +2.18 ± 0.21
methylation at N(7) for n phosphate groups:
ΔΔG° = ΔG°(m 7 Gpn(G)) − ΔG°(Gpn(G))
n: 1 2 3
Gpn → m 7 Gpn −20.2 ± 3.8 −20.29 ± 0.29 −20.79 ± 0.13
GpnG a → m 7 GpnG −17.91 ± 0.25
addition or alteration of the second nucleoside:
ΔΔG° = ΔG°(m 7 GpnY) − ΔG°(m 7 GpnX)
X → Y: none → G G → m 7 G a
G → A G → C G → m 2’O G
m 7 Gp3X → m 7 Gp3Y +6.57 ± 0.17 +3.26 ± 0.38 +1.13 ± 0.29 +2.13 ± 0.42 −0.2 ± 0.2
m 7 Gp4X → m 7 Gp4Y +3.60 ± 0.17
Gp3X → Gp3Y a
+3.64 ± 0.25
successive addition of the 5'-phosphate groups:
ΔΔG° = ΔG°(n+1) − ΔG°(n)
n → n+1: 0 → 1 1 → 2 2 → 3 3 → 4
m 7 Gpn −12.59 ± 0.67 −7.87 ± 0.25 −4.10 ± 0.21
Gpn −7.8 ± 3.8 −3.60 ± 0.29
m 7 GpnG −6.40 ± 0.17
m 7 Gpnm 7 G a
−6.11 ± 0.38
successive addition of the N 2 -methyl groups:
ΔΔG° = ΔG°(n+1) − ΔG°(n)
n → n+1: 0 → 1 1 → 2
2,
2 7
m n m GTP +0.77 ± 0.05 +3.10 ± 0.33
a for symmetrical cap analogues ΔΔG° calculated from the microscopic association constant Kas (micro)
65

4.2.2.1. Energetic Cost of 7-Substituent Alteration
The presence of each type of substituent at the N(7) position of the guanine moiety
produces a comparable effect on the eIF4E binding enhancement (Fig. 4-12), in
comparison with the non-substituted cap analogue, by ΔΔG° of −15 to −21 kJ/mol (Tables
4-2, 4-3). The main gain of the stacking energy is reached irrespective of the nature of the
substituent. This indicates that the delocalized positive charge resulting from N(7)-
substitution is of primary importance. The only direct interaction involving the 7-methyl
group is a non-specific van der Waals contact with Trp166 in the complex with m 7 GDP
(3.93 Å, Fig. 4-13a), which is lost in the complex with m 7 GpppG (4.17 Å, Fig. 4-13b).
Replacement of the methyl group for larger substituent (ethyl, benzyl) can cause steric
hindrance, and can interfere with creation of the water-mediated hydrogen bonds
stabilizing the α-phosphate. Thus, it is energetically unfavourable by ΔΔG° of +2.2 to +5.7
kJ/mol.
Fluorescence (a. u.)
100
80
60
40
20
et 7 GTP
p-Cl-bz 7 GTP
m 7 GTP
bz 7 GTP
10 -3 10 -2 10 -1 10 0 10 1
Concentration of cap analogue (μM)
Figure 4-12. Binding isotherms for eIF4E complexes with N(7)-substituted guanosine
triphosphates, at 20°C, pH 7.2, 100 mM KCl.
66

Trp-166
Trp-166
Glu-103
Trp-102
Glu-103
Trp-102
Arg-112
Arg-112
c
n2
Figure 4-13. Interatomic contacts in (a) m 7 GDP-eIF4E complex, determined on the basis of the
crystallographic structure (PDB ID code: 1EJ1), 75 and (b) m 7 GpppG-eIF4E complex. Hydrogen
bonds, salt bridges and van der Waals interactions are indicated with dotted lines, with the lengths
expressed in Å. Trapped water molecules supporting the hydrogen-bonded network are shown as
magenta spheres. The fragments of eIF4E distant from the cap by 4 Å are shown as spheres.
67
Lys-162
n2
Lys-162
Lys-206
Trp-56
c5'
Trp-56
c5'
(a)
Arg-157
(b)
Arg-157

4.2.2.2. General Separation of the Binding Free Energy
The binding free energy of the 7-methylated analogues association with eIF4E
appears to be totally separated into two groups of interactions: stacking together with
hydrogen bonding and binding through the phosphates. Irrespective of the level of the cap
analogue phosphorylation, methylation at N(7) is accompanied by almost constant binding
free energy change, ΔΔG° about –20.5 kJ/mol (Table 4-3). The gain from N(7)-
methylation slightly decreases for the shorter phosphate chains. This minute effect may
result from the fact that acidic-basic properties of N(1)-H proton slightly depend on the
number of the phosphate groups 177 (Scheme 3-1, p. 23). Therefore, the m 7 G moiety
assumes the required cationic form (see 4.2.3.2., p. 83) more readily in analogues with a
longer phosphate chain. However, both the pKa-shifts 177 and differences among ΔΔG°
values related to methylation of mono-, di- and triphosphates (Table 4-3) are very small,
indicating that the presence of the phosphates influences the charge of m 7 G to a negligible
extent. The charge flow via the ribose σ-bonds does not occur.
Surprisingly, the measured values of ΔG° satisfy the relationship: ΔG°(m 7 Gpn) =
ΔG°(m 7 G) + ΔG°(G pn), for n = 1, 2 or 3. This means that the 7-methylguanosine binding
is separate from binding of the phosphate chain. The possible entropic cost arising from a
change in the degrees of freedom due to linkage of the two moieties is either negligible or
can be partially cancelled out if the bound phosphates facilitate to some extent penetration
of the cap-binding slot by m 7 G. The resultant entropic effect is, however, below the best
experimental accuracy: δ(ΔG°(m 7 Gpn) − ΔG°(m 7 G) − ΔG°(Gpn)) = ± 0.67 kJ/mol. The
total binding free energy of 7-methylguanosine, ΔG°(m 7 G) = −20.54 ± 0.63 kJ/mol (Table
4-2), is the same as ΔΔG° resulting from methylation at N(7), which demonstrates that the
ribose does not contribute to the complex stabilization. The 3D structures show only one
non-specific van der Waals contact between the ribose and Trp56 (Fig. 4-13).
The stabilization energy of the GMP-eIF4E, GDP-eIF4E and GTP-eIF4E complexes
originates only from the interactions involving the phosphate groups, independently
pointing to the minute role for the ribose in stabilization of the complex. It is directly seen
from the comparison of the total binding energy, ΔG°(GMP) = −12.93 ± 3.8 kJ/mol (Table
4-2), with the contribution of the single α-phosphate, ΔΔG° = −12.59 ± 0.67 kJ/mol (Table
4-3). These findings reveal that although unmethylated guanosine phosphates are
potentially capable to form three Watson-Crick like hydrogen bonds, such bonds are not
68

created. It is only when the guanine moiety possesses a substituent at the N(7) position that
the hydrogen bonds are formed. As shown above, the positive charge at m 7 G determines
the cap affinity for eIF4E being a cause of the stacking enhancement.
The most striking conclusion is that the cation-π stacking enhancement is a
precondition for the hydrogen bond formation, and a double role should be attributed to the
presence of the conserved tryptophans 56 and 102 in the eIF4E binding site: stabilization
of the 7-methylguanine ring by stacking itself and enabling 7-methylguanine to form the
hydrogen bonds with the carboxylate oxygen atoms of Glu-103 and the backbone amino
group of Trp-102 (Fig. 4-13). Consequently, the system of m 7 G-eIF4E works according to
the "all-or-nothing" rule.
4.2.2.3. Relation of Stacking - Hydrogen Bonding Cooperativity to Other Cap-
Binding Molecules
Previous studies using small model peptides showed that the stacking interactions of
N(7)-methylated guanine with the aromatic rings of Trp, Tyr, Phe are stronger in
comparison with the unmethylated base. However, the difference in the Kas values was
lesser than 1.5-fold. 217 On the other hand, spectroscopic studies on the interaction between
cap analogues and small model peptides, containing Trp and Glu, showed simultaneous
formation of hydrogen bonds between 7-methylguanine and the Glu carboxyl side group,
and stacking with the Trp indole ring. 218-220 These investigations alone, however, do not
explain why compounds containing unmethylated guanine moiety, capable to form three
hydrogen bonds involving O 6 , N(1) and N 2 , do not competitively inhibit translation 157 and
are unable to bind eIF4E as well as an other cap-binding protein, the mRNA 5' cap-specific
viral methyltransferase VP39. 221,222 Enhanced stacking of the methylated, positively
charged bases with two parallel aromatic side chains of VP39 (Tyr22 and Phe180) plays a
dominant role in the cap-recognition, 221 and hydrogen bonding is of secondary
importance. 223 The single- and double-substitutions of Tyr22 and Phe180 by more strongly
stacking tryptophans were shown to be associated with increased affinity for 7-
methylguanosine by factors of 10 and 50, respectively. 224 The structural requirements for
the specific cap recognition were qualitatively examined by means of the fluorometric
titration applied to GST-fused eIF4E and His-tagged VP39, with amino acid substitutions
in the cap-binding sites of the proteins. 164 The results suggest that, in order to permit the
selective binding of m 7 GDP, eIF4E and VP39 require both the aromatic residues and a
69

glutamic acid in a specific mutual orientation. Although VP39 and eIF4E do not share a
common phylogenetical ancestor, they use a similar mode of cap binding.
Application of the exact quantitative approach makes it possible now to elucidate this
strict stacking/hydrogen bonding cooperativity. The observed entirely different affinity of
the 7-methylated nucleotides as compared with their unmethylated counterparts does not
arise from different acidic/basic properties of the two classes of compounds, as was
suggested previously, 156 but originates from their different stacking ability.
4.2.2.4. Energetic Cost of Methylation of the N(2)-Amino Group
Large ΔΔG° of +13 kJ/mol that accompanies substitution of the second amino-proton
m 7 , 2 , 2
for the methyl group at N 2 of guanine in 3 GTP points to disruption of possibly more
than one hydrogen bond between eIF4E and the cap. If one subtracts the steric and
hydrophobic costs (ΔΔG° of about +3 kJ/mol, as observed for the methyl substitution of
m 7 , 2
2
the first amino-proton in GTP , Table 4-3), the resulting energy (about +10 kJ/mol)
suggests breaking of not only the Glu103–COO...HN 2 –m 7 GTP hydrogen bond but also the
second hydrogen bond, Glu103–COO...HN(1)–m 7 GTP, most likely due to steric effects of
the bulk dimethylamino group, which can move the acceptor group of Glu103 away.
4.2.2.5. Energetic Cost of the Second Nucleoside Addition and Modification
All of the dinucleoside triphosphates bind to eIF4E less strongly than m 7 GTP, by
ΔΔG° of +6.6 to +10 kJ/mol, and even with lesser strength than m 7 GDP (Tables 4-2, 4-3).
The decreased affinity suggests that the second nucleoside does not bind tightly to eIF4E.
Independently from the affinity studies, the same conclusion arises from the cocrystal
structure of m 7 GpppG bound to eIF4E. Figures 4-13b and 4-14 show the only part of
m 7 GpppG, which can be detected from the crystallographic data. There is no defined
electron density for the second guanine nucleoside. 1
The presence of the unbound fragment of the cap analogue can destabilize the
remaining intermolecular contacts. Addition of the second guanosine to GTP gives
disadvantageous entropic effect by ΔΔG° of about +3.6 kJ/mol, while the second
guanosine attached to m 7 GTP results in ΔΔG° about +6.6 kJ/mol (Table 4-3). This larger
value, together with the former finding that GTP is bound only through the phosphates (see
4.2.2.2., p.68), can indicate that the interactions of the 7-methylguanine moiety in
70

Figure 4-14. Stereo view of the geometry of the eIF4E cap-binding site with the only part of
m 7 GpppG, which was detected by crystallography. 1 Molecular electrostatic potential of the eIF4E
surface at pH 6.0 in the absence of the ligand was calculated by Protein Explorer 225 (red and blue
represent negatively and positively charged regions, respectively). The ligand is represented as
sticks (carbon – green, other atoms – CPK colours). Water molecules are shown as magenta balls.
m 7 GpppG are also disturbed by the unbound second nucleoside "hanging" on the
phosphate chain, by ΔΔG° of about +3 kJ/mol. The crystallographic structure of the
m 7 GpppG-eIF4E complex shows clearly, that the hydrogen bonds with Glu103 (donor-
acceptor distances > 3Å) and the van der Waals interaction of the 7-methyl group with
Trp166 (distance > 4Å) are destabilized (Fig. 4-13b).
In the other crystal complex of m 7 GpppA with full length human eIF4E, 48 the second
adenosine is visible in the electron density map. It is stabilized by a direct hydrogen bond
between adenosine N 6 -amino group and the backbone carbonyl of Thr205. Guanosine
possesses a hydrogen acceptor at C(6), which precludes formation of this bond, and the
protein loop containing Thr205 is disordered. The binding free energy (ΔG°) of m 7 GpppA
is slightly less than that of m 7 GpppG (Table 4-2), which was recently confirmed by
subsequent studies. 74 Hence, the energetic gain from the additional stabilization contact of
the adenosine does not compensate for the entropic cost related to the ordering of the C-
terminal loop.
71

Methylation of the second guanine at N(7) decreases the affinity of tri- and
tetraphosphate dinucleotide caps to the same extent, ΔΔG° about +3.4 kJ/mol, consistent
with attenuation of the negative charge of the phosphate chain (see below) and, at least
partially, with the entropic cost of ordering the larger molecule. Exchange of the second
base for adenine or cytosine is energetically less favourable. A minute advantageous effect
in comparison with m 7 GpppG appears after methylation of the second ribose at O(2'),
which is however in the range of the experimental error.
4.2.2.6. Contributions of Phosphate Groups and Trapped Water Molecules
Data in Table 4-3 show a clear accordance of the ΔΔG° values related to the
phosphate interactions between two series of compounds: 7-methylated and nonmethylated
guanosine phosphates. Addition of the phosphate groups one after another is accompanied
by formation of direct or water-mediated hydrogen bonds and salt bridges, yielding the
same free energies in the two groups of compounds. This finding shows again that there is
no influence of the positive charge resulting from N(7)-methylation on the charge
distribution at the phosphate chain. ΔΔG° for each step agrees with the number of
intermolecular contacts revealed by the crystallographic structures of the eIF4E-cap
complexes (Fig. 4-13). The α-phosphate forms a direct salt bridge with Nχ2 of Arg157 as
well as three indirect hydrogen bonds with Nε1 of Trp102, Nε1 of Trp166 and Nχ2 of
Arg112 via five water molecules trapped in the cavity of the binding centre. All water-
mediated hydrogen bonds satisfy a requirement of tetrahedral geometry of the water
molecules and the Pα-oxygen anion. These five bridging water molecules support the same
hydrogen-bonded network both in the eIF4E-m 7 GDP complex 75 and the eIF4E-m 7 GpppG
complex, and are present also in the crystallographically independent second complexes in
the asymmetric units. It should be noted here that such specific network of water molecules
in the protein interior is sometimes very rigid, and the water molecules buried in the
internal cavities increase the protein stability. 226 Moreover, the internal water scaffold
which is directly involved in ligand binding contributes to the protein specificity attained
by the evolutionary specialization. 227
The total free energy of the α-phosphate stabilization is ΔΔG° = −12.59 ± 0.67
kJ/mol (Table 4-3). The β-phosphate group makes a salt bridge with Nζ of Lys162 and a
hydrogen bond with Nε of Arg157, with the total ΔΔG° = −7.87 ± 0.25 kJ/mol, i.e. about
72

−4 kJ/mol per one direct bond. This indicates average ΔΔG° of about –3 kJ/mol per one
water-mediated hydrogen bond in case of the α-phosphate. The latter value is slightly less
than typical magnitudes of hydrogen bonds and salt bridges in a solvent accessible region
in proteins (–5.4 ± 2.5 kJ/mol) 141-143 due to the entropic cost of ordering the water
molecules. The γ-phosphate of m 7 GpppG is stabilized by an additional, bifurcated salt
bridge with Nζ of Lys206 of a similar energy about –3.9 kJ/mol (Fig. 4-13b).
Extension of the phosphate chain by the δ-moiety in the dinucleotide cap analogues
gives an energetic gain of about −6.3 kJ/mol, what cancels out the energetic cost of the
second guanosine addition to m 7 GTP (Table 4-3). Most likely the δ-phosphate does not
interact directly with eIF4E but the four-membered chain secures the necessary
conformational freedom for the second nucleoside which is not bound, enabling the
remaining m 7 Gppp-part of the cap to bind tightly. Thus, the presence of the δ-phosphate in
m 7 GppppG leads to formation of the same intermolecular contacts as for m 7 GTP (ΔG° =
−45.099 ± 0.088 kJ/mol) and restores the affinity of the dinucleotide cap analogue to the
same level (ΔG° = −44.96 ± 0.11 kJ/mol). However, it cannot be excluded that the δ-
phosphate is additionally stabilized, and the entropic cost due to loss of some
conformational degrees of freedom of the second nucleoside partially cancels the
stabilization effect, thus leading to accidental agreement.
4.2.3. Interactions in Context of Environment
Interpretation of structural and of thermodynamic data obtained under a single set of
solution conditions is complicated by the fact that ligand binding by proteins is an
exchange reaction. Functional groups of both protein and the ligand are capable of
interacting not only with each other, but also with water and solutes. Hydrogen bonds with
water and Coulombic interactions of phosphates with cations may be comparable to the
protein-ligand interactions that replace them in the specific complex. In order to evaluate
the role ions and water upon the eIF4E-cap complex formation the dependence of the Kas
values of several cap analogues on salt concentration and pH have been investigated.
73

4.2.3.1. Interaction of eIF4E-Cap in the Presence of Electrolyte
The process of eIF4E-cap binding has been examined regarding the salt effects in
two complementary ways: according to Davies-Stockes-Robinson electrostatic screening
theory 191,192 and according to stoichiometric Wyman linkage analysis. 193
4.2.3.1.1. Davies-Stockes-Robinson Electrostatic Screening Approach
4.2.3.1.1.1. Electrostatic Effects
Fluorescence (a. u.)
100
80
60
40
10 -3 10 -2 10 -1 10 0 10 1 10 2
m 7 GppppG (μM)
Figure 4-15. Exemplary eIF4E binding isotherms for m 7 GppppG at different KCl concentrations
added to Hepes 50 mM, pH 7.2, at 20 °C (, 20 mM; , 100 mM; , 201 mM; , 500 mM).
At elevated ionic strength (I) the interaction is strongly attenuated (Fig. 4-15, Table
4-4). Screening of the electrostatic attraction between eIF4E and the cap analogues of
increasing negative charges, i.e. m 7 GDP, m 7 GpppG, m 7 GTP and m 7 GppppG, is
qualitatively shown in Fig. 4-16, on the basis of the simple Brønstes-Bjerrum-Debye-
Hückel limited law. ∗ This approximation, although too far-reaching, is useful to show a
certain experimental regularity. The negative slope of the regression line is approximately
proportional to the charge at the phosphate chain, assuming that the terminal phosphates in
the mononucleotide cap analogues are not fully ionized at pH 7.2 (pKa phosph ~6.1-6.5,
depending on the number of phosphate groups 178 ).
∗ A linear dependence of logKas on the square root of ionic strength (I 1/2 ); assumptions: very low salt
concentration (< 0.01 M), dissociation rate constant the same for each I value, point charges (aj = 0), no
hydration (ΔN = 0). None of the assumptions is fulfilled in the case of macromolecular interactions at the salt
concentration close to physiological conditions, hence no quantitative conclusions can be drawn.
74

log Kas - log Kas(0)
0
-1
-2
-3
-4
m 7 m
GppppG
7 m
Gppp
7 Gpp
m 7 GpppG
0.0 0.2 0.4 0.6 0.8
I 1/2 (M 1/2 )
Figure 4-16. The ionic strength-dependent decrease of the relative affinity for eIF4E of four
selected cap-analogues with increasing negative charges. The approximate slopes are the steepest
the more negative are the charges of the phosphate chains. Kas(0) is the value of the association
constant at I = 0, extrapolated on the basis of the linear regression.
Table 4-4. Equilibrium association constants for binding of eIF4E to the cap analogues at 20 °C,
pH 7.2, in Hepes/KOH 50 mM (pKa = 7.5), and at the concentration of added KCl as listed below.
Ionic strength was calculated taking into account contribution from partially ionized buffer. The
slopes and the correlation coefficients (r 2 ) have been obtained by linear regression based on the
Brønstes-Bjerrum-Debye-Hückel limited law.
[KCl] m 7 GDP m 7 GpppG m 7 GTP m 7 GppppG
(mM) Kas ⋅ 10 -6 (M -1 )
5.3 1900 ± 3200
20 40.7 ± 5.6 34.4 ± 3.8 1000 ± 460 1112 ± 530
50 17.9 ± 1.0 192 ± 23
54 20.2 ± 2.4 263 ± 32
104 20.4 ± 1.5 7.39 ± 0.46 121.0 ± 3.0 102.8 ± 4.4
150 3.92 ± 0.18 73.0 ± 4.0
201 4.94 ± 0.71 2.99 ± 0.32 21.5 ± 1.9 27.6 ± 3.1
250 1.96 ± 0.13 36.5 ± 1.5
300 1.21 ± 0.18
350 0.88 ± 0.10 3.98 ± 0.79
500 1.83 ± 0.31 0.47 ± 0.12 3.85 ± 0.33 1.83 ± 0.33
slope -2.58 ± 0.12 -3.619 ± 0.044 -4.47 ± 0.14 -5.08 ± 0.29
r 2
0.9553 0.9895 0.9592 0.9901
75

Monovalent cations were reported to significantly increase in vitro translation in
rabbit reticulocyte lysate and K + appeared to have the most suitable radius to support
translation. 228 As the efficiency of eIF4E-mRNA 5'-cap binding decreases at higher K +
concentration, the positive effect caused by K + should rather be attributed to the regulation
of other translation components.
Figure 4-17. Determination of hydration number ΔN from KCl-dependence of the eIF4Em
7 GpppG complex formation, at 20°C. Analysis according to electrostatic screening theory (solid
line) and stoichiometric cation release with the cation number c = 1 (broken line).
An extended analysis, including finite dimensions of the interacting molecular
spheres and the hydration effects revealed by the osmotic stress, has been performed for
m 7 GpppG ∗ (Fig. 4-17). The optimal result has been obtained within Davies approximation
for the effective ionic diameter a j = 8 Å, which describes the sum of the radii of the
interacting species, i.e. three-membered phosphate chain of m 7 GpppG and the group of
four positively charged amino acids localized at the entry of the eIF4E cap-binding slot
(Arg112, Arg157, Lys162 and Lys206, Fig. 4-13). The product of mutually attracting
charges is z = −11.
54 ± 0.
95e.
Previous studies showed that the resultant protein charge
z1 2
log Kas (m 7 GpppG)
8
7
6
5
10 -2 10 -1 10 0
KCl (M)
is only +0.58 e, while a dipole moment is substantial (579 D). 4 Hence, the cap recognizes
directly the binding site of eIF4E and not the protein as a whole (Fig. 4-18), and the charge
∗ The extended, non-linear analysis required very accurate experimental data for Kas. Those were only
available for m 7 GpppG. The binding constants of the remaining cap analogues were determined with too
much scattering due to the greater extent of wear of the xenon impulse lamp of the spectrofluorometer and
thus insufficient signal-to- noise ratio (< 100:1).
76

localized on the guanine moiety does not by itself play any important role in the first step
of the encounter. The negatively charged phosphate chain serves as a molecular anchor,
enabling the cap to form further contacts within the binding site. This conclusion is in a
very good agreement with our independent finding that the free energies corresponding to
the phosphate chain interactions are separate from the energy of stacking together with
hydrogen bonds (Table 4-3).
77
Figure 4-18. Molecular electrostatic potential
of the eIF4E surface at pH 6.0 75 calculated by
Protein Explorer 225 in the absence of the cap
analogue. The ligand is represented as green
sticks.
4.2.3.1.1.2. Two-Step Mechanism of the Cap-eIF4E Association
Taken together, the above results provide a molecular interpretation of the two-step
mechanism observed for the cap-eIF4E association by means of stopped-flow
spectroscopy 4 . The first step can be now attributed to the anchoring the phosphate on the
external basic amino acids, and the second step to the interactions deeply inside the m 7 G-
binding slot, i.e. cooperative cation-π stacking and hydrogen bonding with Trp56, Trp102
and Glu103. As all these residues are located at the flexible loops, 75 this second step of the
complex formation is expected to involve a conformation change.
The two-step model could be verified by mutant analysis, a mirror reflected
methodological approach to the current cap analogue studies. The mutations eliminating all
phosphate interactions should yield the equilibrium Kas for m 7 GTP close to that for m 7 G
(Table 1) and considerably decrease the kinetic encounter rate constant k+1. The complete
cap-slot mutations should retain only the phosphate binding affinity, with Kas as for GTP
and the k+1 unchanged. However, some mutations of crucial residues can change not only
the nature of amino acids, but also the internal structure of the protein (local or even global

fold), thus obscuring the investigated effect. By contrast, the studies based on the
chemically modified cap analogues, where each chemical alteration can be individually
controlled, are free from such pitfalls.
4.2.3.1.1.3. Relation to the "Clamping Cycle"
The analysis of electrostatic screening confirms a biological role for a putative
phosphate bridge connecting Ser-209 with Lys-159. It is thought to "clamp" the previously
bound mRNA chain 229 when eIF4E is phosphorylated by the eIF4G-associated Mnk1
kinase. 230,231 As the phosphate chain of the cap is electrostatically steered toward the
binding site, the mechanism of a "clamping cycle", where the phosphorylation happens
after the cap-eIF4E binding, 229 is most likely. Otherwise, if the phosphorylation at Ser-209
occurred prior to the cap binding, it would attenuate the electrostatic attraction between
eIF4E and the cap. Therefore, the affinity of the cap to the previously phosphorylated
protein would be significantly decreased. Recent data showing the effect of eIF4E
phosphorylation on the affinity of cap for eIF4E 73,74 confirm this conclusion.
4.2.3.1.1.4. Osmotic Stress
Molecular crowding of the solution causes a shortage of the free water molecules.
The Kas at elevated salt concentration decreases more than it could be explained by
electrostatic screening only (Fig. 4-17), indicating that the binding of the cap to eIF4E
requires additional hydration of the molecular surfaces. It should be noted that, although
the activity of water at the maximal salt-concentration in the present study was not very far
from unity (aw ~0.97 at 0.5 M KCl), the hydration effects appear quite distinctly, and the
considerable number of water molecules that are taken up to the cap-eIF4E complex has
been detected: ΔN = 68 ± 16.
The association constant of m 7 GpppG extrapolated to the optimal hydration
conditions, i.e. without osmotic stress and electrostatic screening, is
K
as
( 0)
9
−1
= 1.
12 ± 0.
18⋅10
M . It is 150-fold greater than Kas measured in 100 mM KCl,
and is an evidence of the strong electrostatic contribution to eIF4E-cap binding. On the
other hand, the association constant extrapolated to the pseudostandard state of 1M KCl is
K
as
( 1)
4
−1
= 4.
8 ± 1.
9 ⋅10
M . In spite of strong electrostatic screening, the intrinsic
thermodynamic preference of eIF4E for the methylated cap is still efficient. This
78

completely screened interaction of eIF4E with m 7 GpppG at 1 M KCl has exactly the same
association constant as m 7 G, the analogue insensitive toward electrostatic interactions,
K
as
7
4
−1
( m G)
= 4.
6 ± 1.
2 ⋅10
M (Table 4-2). Hence, at ionic strength of 1 M all the
interactions with the phosphates are lost.
4.2.3.1.2. Wyman Linkage Analysis
In terms of Wyman linkage analysis, stoichiometrical ionic interactions are taken
into account in the same manner as excluded volume effects. The most probable process
accompanying the eIF4E-cap binding in addition to the change of preferential hydration is
potassium cation release 118 from the phosphate chain of the cap analogue:
cap( c⋅K + ) + eIF4E + ΔN⋅H2O capeIF4E( ΔN⋅H2O) + c⋅K +
4.2.3.1.2.1. Electrolyte Effect
The number of K + released from the ligand, c = 0.826 ± 0.092 (Fig. 4-17), indicates
that possibly one K + is displaced from the phosphate chain of m 7 GpppG upon binding to
eIF4E. Cation release (polyelectrolyte effect) was extensively studied for the entropy-
driven B-DNA interactions with oligocations. 118 Large contribution of the counterions
displacement into bulk solvent was also reported for anthracycline antibiotic binding to
DNA. 120 Because the cap analogues lacked substantial axial charge distribution, such as
that observed in the helical B-DNA polyanion, the cation dilution effect is much less
pronounced. The contribution of the electrolyte effect to the stability of the m 7 GpppG-
eIF4E complex at 100 mM KCl is ΔG°el = −6.40 ± 0.63 kJ/mol, in comparison with the
total free energy of m 7 GpppG binding upon these conditions, ΔG° = −38.55 ± 0.15 kJ/mol
(Table 4-2), so the entropic contribution to the association driving force, resulting from the
cation release, is rather moderate (ΔS°el = + 21.8 ± 2.1 J/mol⋅K). The association constant
calculated from the stoichiometrical model and extrapolated to 1M KCl is the same as that
derived from the approach of the continuous screening, Kas(1) = 4.45 ± 0.34 ⋅ 10 4 M -1 .
Relatively high Kas ~10 4 of m 7 GpppG at 1M KCl and the moderate influence of the
electrolyte effect on the binding distinguish the electrostatically attracting cap-eIF4E
system from the counterion release-driven DNA-oligocation system, for which the
corresponding Kas value was within an order of magnitude with unity. 118
79

4.2.3.1.2.2. Osmotic Stress
The number of water molecules that have to be taken up from the bulk is ΔN = 95 ±
18. Keeping the constant value of c = 1 does not cause significant worsening of the fit,
according to the Snedecor's F-test (P = 0.11), and gives ΔN = 63.5 ± 9.3. Both of these
values agree within the numerical accuracy with the hydration effect (ΔN) derived from the
complementary model of electrostatic screening, so the two theoretical treatments indicate
the same extent of water participation in the cap binding event.
4.2.3.1.3. Comparison of the Two Approaches
In respect of the direct interaction with the electrolyte, the Snedecor's F-test
determines that the screening theory is better (R 2 = 0.9967) than the cation release
approach (R 2 = 0.9906 for c = 1), on the significance level of P = 0.0155. Hence, the
description in terms of continuous screening by the ionic atmosphere of the excess salt is
more relevant. The possible stoichiometric interaction with the counterion contributes to
the screening, and additionally provides the advantageous entropic effect to the binding.
4.2.3.1.4. Reasons for KCl-Dependence of Internal Rearrangement Rate Constants
Significance of the electrostatic forces in kinetics of the eIF4E-cap complex
formation was also shown by the fluorescence stopped-flow measurements. 4 The encounter
rate constant (k+1) measured experimentally and confirmed by Brownian dynamics
simulations was markedly diminished by the ionic strength (from 4.58 ± 0.18 ⋅10 8 M -1 ⋅s -1 at
50 mM KCl to 0.68 ± 0.04 ⋅10 8 M -1 ⋅s -1 at 350 mM KCl), and the dissociation rate constant
(k-1) was slightly increased (from 30 ± 8 s -1 to 45 ± 7 s -1 , respectively). However, estimates
of the second-step rate constants for the internal rearrangement of the initial complex (k+2
and k−2) revealed surprisingly analogous ionic strength dependence (from 124 to 73 s -1 , and
from 1 to 12 s -1 , respectively). This intriguing effect could not be explained when
neglecting the excluded volume effects. Now, in light of the osmotic stress analysis, the
decrease of the second-step rate constant k+2 and the increase of k−2 is readily explicable in
terms of the conformational change, upon which the significant number of water molecules
have to hydrate the protein surface, which becomes more difficult at the elevated osmotic
stress.
80

4.2.3.1.5. Discussion of Hydration Effects
For several years increasing attention was paid both to the activity of water and to the
ions that can participate in the intermolecular interactions. 117,126,232,233 Additional hydration
can accompany the ligand binding by the proteins that show simultaneous conformational
changes. For instance, the hydration of haemoglobin by 60–65 water molecules occurs
with the transition from the deoxy T to fully oxygenated R state. 119 Only one chlorine
anion is released upon this transition. Recently, hydration changes that accompany the
binding of several intercalators to DNA were exhaustively studied 121 . It was found that
from 0 to 30 water molecules are taken up during complex formation, depending on the
intercalator type. On the other hand, the entropy-driven association of lac repressor to lac
operator DNA is accompanied by the release of ~200 water molecules. 114 In the cap-eIF4E
system, the additional hydration similar to that accompanying the haemoglobin
oxygenation is observed. The total number of water molecules taken up from the bulk
solvent (~ 65) is much greater than that trapped inside the cap-binding centre, found by
crystallography (9 and 16, in the complex with m 7 GDP 75 and m 7 GpppG, 1 respectively, Fig.
4-19), and apparently comparable with the amount of total water bound per one protein
molecule. 75,91 It should be noted, however, that the water found in the X-ray diffraction
structures is usually an integral part of the macromolecular structure and is located in its
internal cavities. The water taken up from the bulk solvent during the complex formation
refers not only to the trapped water but also to the changes of the first-layer hydration shell
of the molecular surfaces. 234 Such peripheral water is highly unstructured, due mainly to
fluctuations of the water molecules imposed by the protein hydrophobic groups.
Figure 4-19. 16 water molecules (magenta spheres, van der Waals radii) were found by
crystallography in the eIF4E-m 7 GpppG complex deeply inside the cap-binding pocket. Molecular
solvent accessible surfaces (beige for eIF4E and bluish for m 7 GpppG, probe radius of 1.4 Å) show
that these water molecules are tightly closed in the protein cavity by the ligand which serves as a
molecular stopper, and thus cannot be regarded as a part of the dynamic first-layer hydration shell.
81

4.2.3.1.6. Conformational Change of eIF4E upon Cap Binding
Such extensive hydration of eIF4E would respond to exposure of additional ~450 Å 2
of the molecular surface area to contact with water 232 . The exposure of the surface requires
a substantial conformational change of the protein. This hypothesis is supported by several
independent facts, i.e. by the rearrangement of the encounter complex, 4 by the profound
(~65%) quenching of the intrinsic fluorescence of eIF4E upon cap binding, by significantly
different fluorescence quenching patterns when comparing interactions of apo-eIF4E and
cap-saturated eIF4E with the 4E-BP1 and eIF4G peptides (see 4.3., p. 86), and by
deaggregation of eIF4E molecules (residues 33-217) forced by the cap analogues binding,
observed by means of Dynamic Light Scattering (Fig. 4-20). Conformational changes of a
homologous plant eIF(iso)4F complex upon binding of m 7 GpppG were also observed by
means of the fluorescence stopped-flow measurements. 235
4.2.3.1.6.1. Deaggregation of eIF4E Induced by Cap Binding
The apo-form of eIF4E with an N-terminal tail shorter by 5 amino acids than that
used for the fluorescence studies formed huge aggregates and the solution was totally
polydispersed, as checked by DLS. Only rough estimates of the molecular weight (MW)
and the hydrodynamic radius (Rh) of the eIF4E aggregates could be registered. After
addition of one of cap analogues (m 7 GTP, p-Cl-bz 7 GTP, m3 227 GTP, m 7 GpppG,
m 7 GppppG, or m 7 Gppppm 7 G) to the eIF4E samples progressive deaggregation with the
time of incubation with the cap analogue has been observed. Exemplary results are shown
in Fig. 4-20. The estimates of the molecular weight and the hydrodynamic radius of the
aggregates dropped after several hours down to the well defined, reliably measured values.
The efficiency of the process correlated with the affinity of the cap analogue for eIF4E.
After 24 hours of incubation with m 7 GTP the protein molecules was sufficiently
monodispersed, characterized by MW = 23.3 kDa and Rh = 2.36 nm, which corresponded
to the monomeric state of the protein in the complex with m 7 GTP (MWcx ≈ 22.2 kDa,
dimensions: 41 Å × 36 Å × 45 Å, as determined by crystallography 75 ), while eIF4E in the
presence of m3 227 GTP was polydispersed, and the apo-protein remained still totally
aggregated. Hence, deaggregation of eIF4E has occurred under the influence of interaction
with cap.
82

Autocorrelation
coefficient
1.6
1.4
1.2
1.0
10 -6
10 -5
10 -4
Delay time (μs)
(a) 10 (b)
5
Figure 4-20. Deaggregation of eIF4E (33-217) at 1 mg/ml induced by the cap analogue binding.
(a) The autocorrelation function becomes well defined with the time of incubation with the 50-fold
excess of m 7 GTP ( , apo-protein; , after 1-hour incubation; , after 24-hour incubation). (b) The
estimates of the molecular weight (MW, ) and of the hydrodynamic radius (Rh, ) of non-specific
protein aggregates decrease systematically in the time of incubation with m 7 GTP by several orders
of magnitude to the final values that are characteristic for the single eIF4E-cap complex.
4.2.3.2. Ionic Equilibria in eIF4E-Cap Complex
7-methylguanosine is a mixture of positively charged and zwitterionic forms (due to
dissociation of N(1)-H, pKa ~7.24-7.54), the proportions being slightly dependent on the
secondary substituents and the length of the phosphate chain. 177 The drop of the pKa value
by approximately 2 units, which accompanies the 7-substitution of guanosine was
suspected to have a biological significance. The zwitterionic form of 7-methylated guanine
was initially postulated to be responsible for interaction with eIF4E. 156 In contrast,
structural data indicated that the cationic form of m 7 GDP binds to eIF4E, because of the
spatial distances suitable for formation of a hydrogen bond between solvent accessible
Glu103 and N(1)-H of 7-methylguanosine in the crystal structures (PDB ID codes: 1EJ1,
1AP8) 75,76 . This hydrogen bond is also unambiguously present in the ternary complex of
eIF4E bound to m 7 GDP and eIF4GII peptide, although the crystals were obtained at pH 8.5
(PDB ID code: 1EJH) 91 . However, the N(1)-H proton was not directly observed during the
NMR experiment, 76 so the issue was still not entirely resolved.
10 -3
10 -2
The affinity of eIF4E for m 7 GTP as a function of pH (Table 4-5, Fig. 4-21) shows
the optimal binding at pH 7 . 24 ± 0.
13,
close to the physiological pH of 7.4, and lower than
the pH 7.6 reported for the full length eIF4E from human erythrocytes, purified by m 7 G-
affinity chromatography. 156 The observed pH-dependence of Kas is rather flat, providing a
wide pH-range of efficient cap-eIF4E binding. From the Wyman model, 193 comprising one
site to be protonated and one site to be deprotonated, two effective acidic dissociation
83
Rh (nm), MW (kDa)
10 6
10 4
10 3
10 2
10 1
10 0
MW
R h
0 6 12 18 24
Time of incubation with m 7 GTP (h)
23.3
2.36

constants have been determined: = 7.
99 ± 0.
14 for N(1)-H of the ligand base moiety
pK L
in the presence of the protein, and = 6.
49 ± 0.
13 for the hydroxyl group of Glu103 in
pK P
the binding site of eIF4E, in the presence of the ligand (the errors of pKs are numerical).
Taking into account experimental uncertainties as well as simplifications of the model, the
real accuracy of pKs is close to 1 pH unit. Making the number of protonated and
deprotonated sites as free parameters of the fitted function leads to negligible improvement
of the results, on the basis of the statistical Snedecor's F-test. The elevated effective pKL in
relation to the pKL value in the absence of the interacting counterpart shows that m 7 G
exists in a cationic form in the eIF4E-bound state and donates N(1)-H proton for the
hydrogen bond with the deprotonated carboxyl group of Glu103. Such an increase of pKL
caused by the chemical microenvironment of the negatively charged Glu103 was
postulated on the basis of the crystallographic structure. 75
Table 4-5. Association constants for m 7 GTP
binding to eIF4E as a function of pH, at
20 °C, 100 mM KCl.
pH Kas ⋅ 10 -6 (M -1 )
6.32 53.7 ± 4.4
6.56 74.4 ± 7.7
6.78 96.9 ± 8.4
7.01 97.9 ± 8.4
7.20 108.7 ± 9.2
7.42 102.3 ± 6.5
7.61 82.1 ± 11.5
7.84 74.2 ± 4.3
8.02 74.7 ± 6.7
log Kas (m 7 GTP)
84
Figure 4-21. Binding affinity of m 7 GTP to
eIF4E as a function of pH, at 20°C.
6.0 6.5 7.0 7.5 8.0 8.5
The free energy of binding between the species in their appropriate ionic states (pH-
independent ΔG°) is: ΔG° pH-ind = −45.765 ± 0.017 kJ/mol, and the energetic costs of
keeping the cationic state of m 7 GTP and the anionic state of Glu103 in the eIF4E binding
site are very small: ΔG°L = +0.368 ± 0.11 kJ/mol and ΔG°P = +0.435 ± 0.12 kJ/mol,
respectively. Even after the increase of the pKP of Glu103 to ~ 6 units inside the protein,
pKL and pKP are separated enough to allow tight m 7 GTP-eIF4E binding at pH 7.2,
characterized by ΔG° = −45.099 ± 0.088 kJ/mol (Table 4-2) very close to ΔG° pH-ind .
8.5
8.0
7.5
7.0
pH

In summary, the decrease of pKa of guanosine as a result of methylation at N(7) is a
side-effect, which must be cancelled by the microenvironment of the protein binding site.
Instead, the biological importance of the 7-methylation consists in strong enhancement of
stacking between the cationic m 7 G and the aromatic side chains (cation - π stacking),
which stabilizes the base moiety enough to allow formation of the three hydrogen bonds in
the binding site (see 4.2.2., p. 64).
4.2.4. Conclusions
The results reveal individual role of various structural elements of the cap in the two-
step process of eIF4E-cap recognition. The 5'-phosphate chain is the primary anchor to
eIF4E. The eIF4E-cap binding is accomplished by the specific contacts of three phosphates
and m 7 G moiety but not of the second guanosine. The biological importance of
methylation of the first guanosine at N(7) has been elucidated by showing that the
thermodynamic origin of the strong discrimination between 7-methylated and
unmethylated counterparts (5000-fold difference in Ka at 20°C) consists in absolute
cooperativity of cation-π stacking and hydrogen bonding during the second step of the
complex formation. The enhanced stacking between the cationic m 7 G and the aromatic Trp
side chains is necessary to stabilize effectively the base moiety, which is a precondition for
formation of three hydrogen bonds inside the cap-binding slot. eIF4E selects precisely
between the 7-monomethylguanine and 2,2,7-trimethylguanine analogues (760-fold
difference in Kas). Possibilities of cation release and water exchange that could accompany
the binding have been also checked. The estimated water uptake of ~65 water molecules to
the cap-binding slot and to the hydration shell of the complex is relevant to conformational
rearrangement of entire eIF4E upon the second step of cap binding.
85

4.3. Molecular Interactions within Ternary Complexes
Involving eIF4E, a Cap Analogue, and an eIF4E-Binding Motif
from eIF4G or 4E-BP1 Proteins
The fluorescence affinity measurements have been extended to include ternary
complexes which consisted of eIF4E, a cap-analogue, and a synthetic peptide
encompassing the eIF4E recognition motif from the mammalian proteins eIF4GI, eIF4GII
and 4E-BP1. The 4E-BP1 peptides were either unphosphorylated or monophosphorylated
at Ser65 and diphosphorylated at Ser65/Thr70. The eIF4G and 4E-BP1 proteins bind to the
convex dorsal surface of eIF4E, on the opposite side of the protein in respect to the cap-
binding slot (Fig. 4-22). Application of the precise synchronized titration method allowed
to test the putative cooperation between the two distant binding sites of eIF4E, 166,167 and
the influence of phosphorylation at Ser65 and Thr70 on regulation of 4E-BP1 binding to
eIF4E.
(a) (b)
Figure 4-22. The dorsal surface of eIF4E coloured according to the molecular electrostatic
potential 225 in the absence of ligands (red – negative, blue – positive). The attached peptide is
shown in the ribbon representation and corresponds to the eIF4E recognition sequence of (a) 4E-
BP1 (yellow, at pH 7.5) or (b) eIF4GII (orange, at pH 8.5). 91 The peptide tyrosine is shown as balls
and sticks. Trp73 of eIF4E is shown in the van der Waals spacefill representation and coloured
green. Orientation of the two aromatic residues is almost ideally perpendicular. A fragment of
bound m 7 GDP (oxygens of the β phosphate group, red spheres) is visible in the bottom right part of
each figure.
86

4.3.1. eIF4E Fluorescence Quenching Pattern upon Peptide Binding
Association of the eIF4GI, eIF4GII or 4E-BP1 peptides with eIF4E leads to
quenching of the eIF4E intrinsic fluorescence, mostly at longer wavelengths (355 – 360
nm) than quenching upon association with cap (330 – 340 nm). This is caused primarily by
interaction of the peptides with the water accessible Trp73 of eIF4E. This amino acid is
thought to be the most important residue of the phylogenetically invariant dorsal surface
region, since it interacts with three peptide amino acid side chains: Leu(5), Leu(6) and
Gln(9) of eIF4GII, or Leu(5), Met(6) and Arg(9) of 4E-BP1 91 (Fig. 4-23, numbering in
relation to the invariant Tyr). Mutation of Trp73 to Ala was found to prevent productive
interactions with 4E-BP1 and eIF4GI in a biological assay. 230
(a)
(b)
Figure 4-23. interatomic contacts between Trp73 of eIF4E (green sticks) and the peptides in the
crystal structures. 91 The peptide amino acid side chains that interact with Trp73 are shown as thick
sticks in the CPK colours. (a) Leu(5), Leu(6) and Gln(9) of the eIF4GII peptide; (b) Leu(5), Met(6)
and Arg(9) of 4E-BP1. Remaining parts of the peptides are represented by the backbone. The
hydrogen bond and the van der Waals bonds with their lengths are drawn as dotted lines.
87

To analyse the eIF4E binding affinity of the peptides and check whether cap-binding
and 4E-BP/eIF4G-binding centres are cooperative, cross-titration experiments with each
peptide have been performed (Fig. 4-24). Comparison of the emission band changes for
saturation of eIF4E with a cap analogue or with an oligopeptide, and with both of them is
shown in Fig. 4-25. Titration curves for formation of eIF4E binary and ternary complexes
with m 7 GTP and the oligopeptides are presented in Fig. 4-26, 27, 28. Irrespective of the
succession of the cap and the peptide added, the total final quenching after saturation of
both eIF4E binding sites is always identical. However, the partial quenching (Q) of the
total initial fluorescence signal, which corresponds to saturation of one of the eIF4E
binding sites, with and without the prior saturation of the other, is different (Fig. 4-25,
Table 4-6). The fluorescence quenching upon titration with m 7 GTP of the peptide-saturated
eIF4E is < 60 %, while the apo-protein reveals ~ 65 % of quenching. The quenching
resulting from the peptides binding is much smaller, ~ 12 % for m 7 GTP-saturated eIF4E
and ~ 19 % for the apo-protein. The differences between Q(apo) and Q(sat) are ~ 3 % for
the unphosphorylated 4E-BP1 peptide, ~ 9 % for the phosphorylated 4E-BP1 peptides and
~ 7 % for eIF4G peptides. This succession- and structure-dependent behaviour of the
quenching patterns supports previous conclusions pointing to the conformational change of
eIF4E upon cap binding, and a possible conformational change upon the peptide binding.
Fluorescence (a. u.)
100
80
60
40
20
0
0 10 20 30 40 50 60 70
Time (min)
eIF4E + 4GI + m 7 GTP
eIF4E + m 7 GTP + 4GI
Figure 4-24. A typical time course of the fluorescence intensity for the cross-titration experiment
of the ternary complex formation, at 20°C.
Data points ( ): 0-5 min, initial baseline; 6-38 min, titration with eIF4GI peptide; 39 min,
intermediate baseline; 40-71 min, titration with m 7 GTP; 72-76 min, final baseline.
Data points ( ): 0-5 min, initial baseline; 6-37 min, titration with m 7 GTP; 38 min, intermediate
baseline; 39-71 min, titration with eIF4GI peptide; 72-76 min, final baseline.
88

Fluorescence (a. u.)
ΔΔ F (%)
100
80
60
40
20
0
300 320 340 360 380 400
20
0
-20
-40
-60
Wavelength (nm)
Figure 4-25. (a) Fluorescence emission spectra excited at 290 nm of apo-eIF4E (——), eIF4E in
the binary complex with the 4E-BP1 P-Ser65 peptide (-----), eIF4E in the binary complex with
m 7 GTP (− − −), eIF4E in the ternary complex with m 7 GTP and the peptide (− - −), and of the pure
peptide (). Application of the emission wavelength of 355 nm allows for elimination of the
direct fluorescence signal originating from peptide tyrosine during titration. (b) Differential spectra
corresponding to saturation of the eIF4E-m 7 GTP complex with the 4E-BP1 P-Ser65 peptide (− - −),
saturation of apo-eIF4E with the peptide (-----), saturation of the eIF4E-peptide complex with
m 7 GTP (− − −), saturation of apo-eIF4E with m 7 GTP(− - - −), and saturation of apo-eIF4E with
both m 7 GTP and the peptide (——).
89
(a)
300 320 340 360 380 400
Wavelength (nm)
(b)

Table 4-6. Equilibrium association constants (Kas) and partial quenching (Q) of total initial
fluorescence upon formation of the binary complexes of the apo-form of eIF4E with the eIF4GI,
eIF4GII and 4E-BP1 peptides, and of the ternary complexes (m 7 GTP-eIF4E-peptide) of previously
m 7 GTP- or peptide-saturated eIF4E, at 20°C. For reference the association constant of m 7 GTP to
apo-eIF4E Kas = 108.7 ± 4.0 ⋅ 10 6 M -1 (Table 4-2), quenching Q ~ 65 %.
Peptides: eIF4GI eIF4GII
Kas ⋅ 10 -6 (M -1 ) for the peptides
eIF4E(m 7 GTP-saturated) 21.43 ± 2.15 5.35 ± 0.20
eIF4E(apo) 13.65 ± 0.86 6.51 ± 0.19
Kas ⋅ 10 -6 (M -1 ) for m 7 GTP
eIF4E(peptide-saturated) 120.5 ± 7.2 137.9 ± 15.8
Fluorescence quenching Q (% of total initial fluorescence)
eIF4E(m 7 GTP-saturated) 12.1 ± 0.9 13.6 ± 2.1
eIF4E(apo) 18.3 ± 1.2 20.6 ± 2.5
Peptides: 4E-BP1 4E-BP1 4E-BP1
(P-Ser65) (P-Ser65/Thr70)
Kas ⋅ 10 -6 (M -1 ) for the peptides
eIF4E(m 7 GTP-saturated) 9.47 ± 0.39 4.77 ± 0.40 5.72 ± 0.35
eIF4E(apo) 10.59 ± 0.84 5.91 ± 0.44 5.74 ± 0.34
Kas ⋅ 10 -6 (M -1 ) for m 7 GTP
eIF4E(peptide-saturated) 101.6 ± 7.3 107.4 ± 5.2 101.0 ± 8.6
Fluorescence quenching Q (% of total initial fluorescence)
eIF4E(m 7 GTP-saturated) 15.1 ± 3.6 10.2 ± 5.5 11.3 ± 4.6
eIF4E(apo) 18.2 ± 9.5 18.8 ± 9.0 20.2 ± 1.4
4.3.2. Cooperativity in Ternary Peptide-eIF4E-Cap Complexes
In general, the image of interactions within ternary complexes is unsymmetrical,
since binding of one ligand (e. g. the peptide) can modify the protein affinity for the other
(the cap analogue), but this is not necessarily accompanied by the evident influence in the
reverse direction. Hence, there is no simple thermodynamic cycle that would describe
binding of the two ligands to eIF4E.
90

4.3.2.1. Cooperativity of Cap and eIF4G Peptides Binding
The fluorometrically determined values of the association constants have been used
to address the cooperativity problem by a direct quantitative comparison. All the eIF4E-
peptide Kas are in the order of 10 7 M -1 (Table 4-6). The Kas values for binding of cap-
saturated eIF4E to eIF4GI and eIF4GII peptides (21.43 μM -1 and 5.35 μM -1 , respectively)
are close to those derived by Isothermal Titration Calorimetry 91 (ITC) (37 μM -1 and 6.7
μM -1 , respectively). The peptide which binds the tightest to eIF4E is eIF4GI and its
interaction is few but unambiguously influenced by the apo- or cap-saturated state of
eIF4E, i.e. an 1.6-fold increase of Kas has been observed for the eIF4GI peptide binding to
cap-saturated eIF4E. A similar effect, albeit to a lesser extent, appears also for the eIF4GII
peptide. Reversely, a slight enhancement of the m 7 GTP-affinity (only up to 1.3-fold,
quantitatively beyond one standard deviation but within two standard deviations) has been
noted after previous saturation of eIF4E with eIF4GI or eIF4GII peptides.
Fluorescence (a. u.)
100
80
60
40
20
Figure 4-26. Titration with m 7 GTP of apo-eIF4E () and of the eIF4E-eIF4GI peptide complex
(); 20 °C, excitation wavelength 290 nm, emission wavelength 355 nm.
The case for the cooperativity is based on several experimental results:
1. The eIF4GI protein was found to strongly enhance the crosslinking effectivity of eIF4E
to the mRNA 5'-cap structure. 216
10 -3 10 -2 10 -1 10 0 10 1
m 7 GTP (μM)
2. Mutation of Trp166 to Ala in the cap-binding slot resulted in the protein which was
unable to bind either eIF4G or 4E-BPs at its dorsal surface. Hence, it can be concluded
91
eIF4E+eIF4GI
eIF4E

Fluorescence (a. u.)
Fluorescence (a. u.)
100
90
80
10 -3 10 -2 10 -1 10 0 10 1
eIF4GI peptide ( μM)
100
90
80
(a)
(b)
Figure 4-27. (a) Titration with the eIF4GI peptide of eIF4E ( ) and of the eIF4E-m 7 GTP complex
( ). (b) Titration with the eIF4GII peptide of eIF4E ( ) and of the eIF4E-m 7 GTP complex ( ). 20
°C, excitation wavelength 290 nm, emission wavelength 355 nm.
that this cap-binding residue plays a role in maintaining the internal structure of eIF4E
necessary for recognition of the eIF4G proteins and of 4E-BPs. 236
3. Among nine crystallographically independent structures of the m 7 GDP-eIF4E binary 75
and ternary 91 complexes, and the m 7 GpppG complex, 1 there are two cases where the C-
terminal loop (206-213) located in the vicinity of the cap-binding site has fully ordered
backbone, and the N-terminus (28-35) close to the eIF4G/4E-BP binding site is
concurrently ordered. In the remaining cases, both the loop and the N-tail are
92
eIF4E+m
eIF4E
7 GTP
eIF4E+m
eIF4E
7 GTP
10 -3 10 -2 10 -1 10 0 10 1
eIF4GII peptide ( μM)

unordered. These could indirectly suggest a possibility of cooperation between the two
distant binding sites.
On the other hand:
1. Comparison of the crystallographic structures of binary and ternary complexes revealed
no important structural differences within the eIF4E-cap binding slot in the absence or
presence of either the eIF4GII or 4E-BP1 peptide. 75,91
2. Preliminary fluorescence study suggested no significant changes of the association
constant for binding of eIF4E with the cap analogue in the presence of a 17-aminoacid
mammalian eIF4GII peptide. 237
Solution of the apparent discrepancies came from two sets of experiments.
1. Recently, it was shown by the gel-shift experiments and m 7 GTP-Sepharose binding
that a 17-mer fragment of yeast eIF4GI exerted a negligible influence on the eIF4E-cap
interaction, while complete eIF4GI and its larger fragments produced distinct
enhancement of the eIF4E-cap binding. 168
2. Finally, the quantitative results reported herein confirm that the putative positive
cooperativity is possible, and that the minimal eIF4E-binding motifs of the eIF4G
proteins can be alone insufficient to induce such spatial changes which could be
evident in the crystal structures of the cap-binding site. 91
4.3.2.2. Cooperativity of Cap and 4E-BP1 Peptides Binding
In case of the 4E-BP1 peptides, the affinity for the apo-protein is the same as that for
the cap-saturated eIF4E (Table 4-6, p. 90). The Kas values for the unphosphorylated 17-mer
peptide 4E-BP1 (9.47 ⋅ 10 6 M -1 and 10.59 ⋅ 10 6 M -1 ) agree well with Kas calculated from
ITC 91 (20 ⋅ 10 6 M -1 ). ITC for the full length 4E-BP1 yielded Kas 7-fold higher (67 ⋅ 10 6 M -
1 ). In terms of the binding free energy, however, this difference between ΔG° for the full
length protein (−43.93 kJ/mol) and the peptide (−39.16 kJ/mol) is small, what indicates
that majority of the intermolecular contacts required for stabilization of the 4E-BP1-eIF4E
complex are accomplished in the model system with participation of the short peptide.
Binding of full length 4E-BP1 and 4E-BP2 to eIF4E was reported to enhance the
cap-eIF4E association, as suggested from m 7 GTP-affinity chromatography and Surface
Plasmon Resonance 166 (SPR). The reversed cooperativity, i.e. an increase of the 4E-BP2
affinity for the cap-saturated eIF4E in comparison with apo-eIF4E was also found using
SPR. 238 The differences in the fluorescence quenching patterns upon formation of the
93

ternary complexes (Fig. 4-25, 26, 27, 28, Table 4-6), and arguments discussed above for
the eIF4G peptides points to a putative cooperation between the two binding sites.
However, the binary 4E-BP1 peptide-eIF4E and cap-eIF4E interactions do not affect the
association constant values for the further formation of the ternary peptide-eIF4E-cap
complexes, within experimental errors. No measurable affinity changes for the 17-mer and
25-mer fragments of 4E-BP1 could reflect the inability of the peptides to completely
mimic the influence of full length 4E-BP1 on the conformation of the eIF4E protein. It is
worth noting here that free 4E-BP1 is unfolded in solution unless gets bound to eIF4E. 239
4.3.3. Regulation of 4E-BP1 Activity by Phosphorylation
Phosphorylation of specific serine and threonine residues of 4E-binding proteins
modulates their affinity for eIF4E. 229 A two-step mechanism of 4E-BP1 phosphorylation
was proposed, involving phosphorylation on Thr37 and Thr46 as a priming event for
subsequent phosphorylation of Ser65 and Thr70. 92 Recently, it was shown by
phosphopeptide mapping that both of these phosphorylation events were insufficient to
disrupt the 4E-BP1 binding with eIF4E, 3 and the possible sources of disagreement with the
previous publications were thoroughly discussed. 93-95
The fluorescence quenching experiments for cap-free and cap-saturated eIF4E
confirm these results (Table 4-6). The monophosphorylation of the 4E-BP1 peptide at
Ser65 causes only ~ 2-fold decrease in the affinity for eIF4E (ΔΔG° less than +1.7 kJ/mol),
and diphosphorylation at both Ser65/Thr70 does not reduce the affinity further. Mono- and
diphosphorylated 4E-BP1 peptides still retain the high ability to interact with eIF4E, with
Kas about 5 ⋅ 10 6 M -1 and ΔG° about −38 kJ/mol. Thus, phosphorylation of both Ser65 and
Thr70 is insufficient to abolish the eIF4E binding.
However, several authors reported substantial reduction of the eIF4E association
with 4E-BP1 phosphorylated at Ser65 in vitro. 93-95 A nearly two orders of magnitude
decrease of Kas was observed by means of SPR due to phosphorylation at Ser65 of the full
length 4E-BP1 mutant in which alanine had been substituted at four out of the five
Thr/Ser-P sites, leaving Ser65 susceptible to phosphorylation. 94 A value of Kas for the
unphosphorylated mutant was determined as 278 ⋅ 10 6 M -1 . ITC yielded a lower Kas value
of 67 ⋅ 10 6 M -1 for the full length 4E-BP1, 91 but at this level of the equilibrium constants
94

Fluorescence (a. u.)
Fluorescence (a. u.)
Fluorescence (a. u.)
100
90
80
100
(a)
10 -3 10 -2 10 -1 10 0 10 1
90
80
100
(b)
4E-BP1 peptide ( μM)
Figure 4-28. () Titration of eIF4E with (a) the 4E-BP1 peptide, (b) the 4E-BP1 P-Ser65 peptide,
(c) the 4E-BP1 P-Ser65/Thr70 peptide. () Titration of the eIF4E-m 7 GTP complex with the
peptides, as indicated above. 20 °C, excitation wavelength 290 nm, emission wavelength 355 nm.
95
eIF4E+m
eIF4E
7 GTP
eIF4E+m
eIF4E
7 GTP
10 -3 10 -2 10 -1 10 0 10 1
90
80
4E-BP1 P-Ser65 peptide ( μM)
(c)
eIF4E+m 7 GTP
eIF4E
10 -3 10 -2 10 -1 10 0 10 1
4E-BP1 P-Ser65/Thr70 peptide ( μM)

(corresponding to ΔG° about –46 kJ/mol) the 4-fold difference between the Kas values
determined by two different methods is not very significant (ΔΔG° about 3.7 kJ/mol). Even
taking into account the 75-fold drop of the association constant to 3.7 ⋅ 10 6 M -1 upon
phosphorylation at Ser65 according to SPR results, 94 the corresponding energy difference
ΔΔG° would be about +10.5 kJ/mol, which is not much in comparison with the remaining
binding free energy of the monophosphorylated protein (ΔG° about –36.8 kJ/mol).
Discrepancies could partially arise from some experimental problems with
unphosphorylated 4E-BP1 protein, which lacks folded structure in solution, 239 so the
cysteine residues are not protected against the oxidation. Many times the presence of the
4E-BP1 peptide dimers in the stock solution (see 3.2., p. 24) resulted in apparently much
higher values of the association constants for the 4E-BP1 peptide than reported in Table 4-
6 (up to 2 orders of magnitude, data not shown).
The peptides seem not to be the perfect mimetic models for all properties of the full
length protein. However, the previous studies by phosphopeptide mapping demonstrated
that full-length 4E-BP1 monophosphorylated on Ser65 could bind to eIF4E efficiently,
while no binding could be detected for 4E-BP1 deleted in the eIF4E-binding site. 3 Hence,
the peptides are good models for investigations how single phosphorylation modulates the
intermolecular interactions.
4.3.4. Concluding Remarks
Experiments based on the fluorescence titration studies reconcile the biochemical,
sometimes contradictory, observations that were done hitherto. The results suggest that
cooperation between cap- and eIF4G/4E-BP-binding sites of eIF4E is possible but hardly
detectable for the short peptides. The mRNA cap can be caught more tightly by eIF4E in
complex with the full length eIF4Gs, and eIF4GI can bind more efficiently to eIF4E in
complex with RNA.
4E-BP1 monophosphorylated at Ser-65 still possesses the high, submicromolar
affinity for eIF4E. Diphosphorylation at Ser-65 and Thr-70 of 4E-BP1 has been also
proved insufficient to abrogate 4E-BP1 binding with both apo-eIF4E and cap-saturated
eIF4E. Thus, phosphorylation of additional residues, most likely the priming sites Thr37
and Thr46, 92 is required to release 4E-BP1 from eIF4E.
96

4.4. Thermodynamics of mRNA 5' Cap Binding by eIF4E
Hydrophobic interactions are widely believed to make important contributions to the
stability of protein-ligand complexes. A lot of studies were devoted to evaluation of their
structural and thermodynamic basis for the purpose of drug design. Considering that salt
bridges, cation-π stacking, and hydrogen bonds play a dominant role in mRNA 5' cap −
eIF4E binding 75 , this molecular system is significantly distinct from the hydrophobic ones
which are usually a focus of the thermodynamic studies. The cap − eIF4E binding is also
accompanied by other processes, i.e. the conformational change of the protein and the
solvent effects, like additional water uptake to the complex and partial protonation (see
4.2.3., p. 73).
In an effort to elucidate the structural features of mRNA 5' cap analogues responsible
for the observed eIF4E binding energetics, thermodynamic investigations have been
undertaken. The aim of the present chapter is to analyse the specific binding in terms of
quantitative thermodynamic parameters determined independently by the van't Hoff
method and isothermal titration calorimetry (ITC). Such quantitative data are of primary
importance for rational design of new cap analogues of potential therapeutic activity since
the high eIF4E cellular level is relevant to malignancy and apoptosis 13 .
In this chapter the absolute temperature scale will be used, T (K) = t (°C) + 273.16.
4.4.1. Van't Hoff Analysis of Binding of eIF4E to Cap Analogues
Fluorescence titration experiments were performed to obtain values of equilibrium
association constants for nine structurally altered cap analogues, m 7 GMP, m 7 GDP, m 7 GTP,
m3 2,2,7 GTP, bz 7 GTP, p-Cl-bz 7 GTP, m 7 GpppG, m 7 GpppC, and m 7 Gppppm 7 G in the
temperature range of 280 – 314 K. Exemplary binding isotherms for eIF4E association
with m 7 GpppG at six selected temperatures are presented in Fig. 4-29. The equilibrium
association constants (Kas) and the corresponding standard molar free energies of
formation of the complexes (ΔG°) are gathered in Table 4-7.
97

Table 4-7. Association constants (Kas ± SD) and standard molar free energies (ΔG° ± SD) for
binding of selected mononucleotide and dinucleotide cap analogues to eIF4E at different
temperatures (T), at pH 7.2.
m 7 GMP
T (K)
279.0
280.2
283.9
288.2
293.2
297.9
301.2
304.2
306.3
309.7
313.2
m 7 GDP
T (K)
280.2
283.2
286.2
286.7
287.7
288.7
289.2
293.2
297.8
304.2
309.7
313.2
⋅10 −6 (Μ −1 )
Κ as ⋅10
Y SD
2.734 0.605
2.492 0.201
1.260 0.390
1.430 0.090
0.766 0.090
0.870 0.160
0.780 0.110
0.475 0.157
0.580 0.181
0.473 0.250
0.838 0.328
⋅10 −6 (Μ −1 )
Κ as ⋅10
Y SD
74.33 10.57
74.58 6.75
46.64 4.08
38.25 3.36
43.18 6.05
38.57 4.16
37.50 4.46
20.40 1.54
17.48 3.15
10.48 1.32
5.04 0.74
6.00 1.70
98
ΔG° (kJ/mol)
Y SD
-34.38 0.51
-34.31 0.19
-33.15 0.73
-33.96 0.15
-33.02 0.29
-33.87 0.46
-33.97 0.35
-33.06 0.84
-33.79 0.79
-33.64 1.36
-35.51 1.02
ΔG° (kJ/mol)
Y SD
-42.22 0.33
-42.68 0.21
-42.01 0.21
-41.61 0.21
-42.05 0.34
-41.92 0.26
-41.93 0.29
-41.02 0.18
-41.29 0.45
-40.88 0.32
-39.73 0.38
-40.64 0.74

m 7 GTP
T (K)
281.7
283.6
286.0
288.2
290.6
293.2
295.1
296.5
297.9
299.3
301.2
303.4
305.2
309.0
311.4
313.5
m3 2,2,7 GTP
T (K)
281.3
284.9
286.2
290.2
293.0
296.1
298.6
301.6
304.4
309.7
313.2
Kas ⋅ 10 -6 (M -1 )
Y SD
389.4 45.6
333.5 157.4
352.3 97.1
161.4 35.7
139.7 61.8
116.4 13.0
112.3 22.4
105.3 16.8
54.1 12.1
50.7 10.0
67.7 13.6
44.6 6.8
63.0 8.4
28.5 7.2
17.1 2.5
16.6 9.3
Kas ⋅ 10 -6 (M -1 )
Y SD
0.291 0.155
0.175 0.098
0.155 0.084
0.129 0.110
0.143 0.080
0.112 0.145
0.112 0.100
0.138 0.113
0.126 0.040
0.209 0.087
0.290 0.408
99
ΔG° (kJ/mol)
Y SD
-46.32 0.27
-46.27 1.11
-46.79 0.66
-45.28 0.53
-45.31 1.07
-45.47 0.25
-45.47 0.49
-45.53 0.39
-44.10 0.55
-44.14 0.49
-45.15 0.50
-44.43 0.38
-45.56 0.34
-44.10 0.65
-43.11 0.37
-43.32 1.47
ΔG° (kJ/mol)
Y SD
-29.42 1.24
-28.60 1.32
-28.44 1.29
-28.38 2.07
-28.92 1.36
-28.62 3.18
-28.85 2.23
-29.68 2.04
-29.72 0.80
-31.54 1.07
-32.75 3.67

z 7 GTP
T (K)
281.6
289.1
291.2
293.2
299.2
304.2
309.7
313.2
p-Cl-bz 7 GTP
T (K)
281.4
283.7
285.9
287.4
288.8
290.8
292.0
293.2
297.0
301.6
304.7
308.5
309.1
312.0
313.8
Kas ⋅ 10 -6 (M -1 )
Y SD
58.60 6.80
32.94 3.20
24.20 1.90
17.50 0.50
12.88 0.40
9.88 0.95
7.46 1.03
4.26 0.96
Kas ⋅ 10 -6 (M -1 )
Y SD
91.2 13.2
84.8 12.9
80.0 12.4
113.3 39.6
57.3 4.6
51.5 4.7
85.0 9.8
47.9 7.8
76.6 17.4
29.9 3.8
32.4 4.1
29.7 5.4
16.4 3.5
21.3 4.2
19.7 4.0
100
ΔG° (kJ/mol)
Y SD
-41.87 0.27
-41.60 0.23
-41.16 0.19
-40.65 0.07
-40.72 0.08
-40.74 0.24
-40.74 0.36
-39.75 0.59
ΔG° (kJ/mol)
Y SD
-42.88 0.34
-43.06 0.36
-43.25 0.37
-44.31 0.83
-42.90 0.19
-42.93 0.22
-44.32 0.28
-43.10 0.40
-44.82 0.56
-43.16 0.32
-43.81 0.32
-44.13 0.46
-42.69 0.55
-43.77 0.51
-43.82 0.53

m 7 GpppG
T (K)
m 7 GpppC
280.6
283.9
288.2
293.2
293.2
297.6
301.4
304.9
309.8
312.6
T (K)
280.0
284.3
287.4
290.2
292.8
296.6
299.4
304.0
308.6
313.1
m 7 Gppppm 7 G
T (K)
281.4
283.8
287.8
290.4
292.7
293.2
296.8
302.2
304.9
308.7
312.6
⋅10 −6 (Μ −1 )
Κ as ⋅10
Y SD
24.94 1.05
23.70 4.35
12.94 1.20
7.39 0.46
6.50 0.50
5.13 0.81
4.01 0.61
2.29 0.52
2.41 0.27
2.56 0.22
⋅10 −6 (Μ −1 )
Κ as ⋅10
Y SD
12.25 1.22
8.11 0.82
7.09 0.86
5.16 0.64
2.76 0.48
3.39 0.52
2.44 0.50
2.42 0.43
1.27 0.56
2.74 1.80
micro
Kas ⋅10 -6 (M -1 )
Y SD
125.85 46.68
75.00 20.50
48.70 5.45
35.70 5.20
23.95 1.95
23.48 2.40
20.00 2.10
10.72 1.10
6.62 1.57
5.88 1.07
4.91 0.27
101
ΔG° (kJ/mol)
Y SD
-39.73 0.10
-40.08 0.43
-39.23 0.22
-38.55 0.15
-38.24 0.19
-38.22 0.39
-38.10 0.38
-37.12 0.58
-37.84 0.29
-38.35 0.22
ΔG° (kJ/mol)
Y SD
-37.99 0.23
-37.60 0.24
-37.69 0.29
-37.29 0.30
-36.10 0.42
-37.07 0.38
-36.61 0.51
-37.15 0.45
-36.06 1.13
-38.58 1.71
ΔG° (kJ/mol)
Y SD
-43.63 0.87
-42.78 0.64
-42.35 0.27
-41.98 0.35
-41.34 0.20
-41.37 0.25
-41.48 0.26
-40.67 0.26
-39.81 0.60
-40.00 0.47
-40.04 0.14

Fluorescence (a. u.)
100
80
60
40
10 -3 10 -2 10 -1 10 0 10 1
m 7 GpppG (μM)
Figure 4-29. Quenching of eIF4E intrinsic fluorescence upon titration with 7-methylGpppG.
Increasing fluorescence intensity at higher concentration of 7-methylGpppG originates from the
free ligand in solution. Binding isotherms for different temperatures: 312.6 K, 304.9 K, 297.6 K,
293.2 K, 288.2 K, 280.6 K. Titrations were performed in 50 mM Hepes/KOH (pH 7.2), 100 mM
KCl, 1 mM DTT, 0.5 mM EDTA.
The results of the fluorescence titrations have been analysed by the van't Hoff
method. For the symmetrical cap analogue, m 7 Gppppm 7 G, the microscopic association
constant was taken into the analysis. Standard molar enthalpy changes (ΔH°) and standard
molar entropy changes (ΔS°) at 293 K determined from the van’t Hoff dependencies are
collected in Table 4-8. The association of eIF4E to mRNA 5' cap at 273 K has been found
to be generally enthalpy-driven for the whole series, and entropy-opposed or –driven,
depending on the cap analogue (Fig. 4-30). Strong, specific interactions may be attributed
to fairly high enthalpy of association, from −50 to −81 kJ⋅mol -1 , while for the less specific
ligands ΔH° is −17 to −36 kJ⋅mol -1 . One exception among the most tightly binding
analogues is p-Cl-bz 7 GTP that has a small binding enthalpy of only –38.5 kJ/mol but a
large positive binding entropy of +16.7 J/mol⋅K. The binding entropy of the remaining cap
analogues ranges from +40 to −136 J⋅mol -1 ⋅K -1 .
The enthalpy change plays a more pronounced role for binding of the analogues with
longer phosphate chains, which is entropy-opposed. An enthalpy decrease that results from
comparison of the binding enthalpies for m 7 GMP and m 7 GDP equals ΔΔH°m7GMPm7GDP =
−25.9 ± 8.4 kJ/mol, and elongation of the phosphate chain to three members gives the
further enthalpy decrease of ΔΔH°m7GDPm7GTP = −12.4 ± 4.6 kJ/mol. These enthalpy
differences, when divided by the number of hydrogen bonds or salt bridges formed by the
102
312.6 K
304.9 K
297.6 K
293.2 K
288.2 K
280.6 K

β and γ phosphate groups (2 and 1, respectively), yield almost equal values of the unitary
enthalpy change per one bond, ΔΔH°B ≈ −12.8 ± 9.6 kJ/mol, which is a typical value. 240
Although in general the binding entropy cannot be theoretically decomposed into
individual atom-atom interactions, in this case the entropic penalties, that equal
ΔΔS°m7GMPm7GDP = −61 ± 11 and ΔΔS°m7GDPm7GTP = −30 ± 16 J/mol⋅K, appear to yield
an average of ΔΔS°B ≈ −30 ± 19 J/mol⋅K per bond. This observation can serve as an
indicator that binding of the subsequent phosphate group is approximately independent
from binding of the previous one, i. e. there is no visible effect associated with linkage of
the phosphates, arising from a change in the number of degrees of freedom.
The binding entropy is more conducive to association of the analogues possessing
more or larger substituents at the guanine moiety. For the larger and larger substituents in
the series of triphosphates: 7-methyl-, 7-benzyl, 7-para-chloro-benzylGTP, the ΔS° value
increases subsequently by +46 ± 18 and +69 ± 20 J/mol⋅K. This is caused most likely by
expulsion of several water molecules from the depth of the cap-binding slot into the bulk
solvent, proportionally to the N(7)-substituent volume. The large and electronegative
chloride atom at the N 7 -benzyl ring can attenuate the van der Waals interaction with Trp-
166 and destroy the specific water network inside the eIF4E cap-binding centre due to
steric and electrostatic effects (Fig. 4-13, p. 67). Consequently, the binding enthalpy for p-
Cl-bz 7 GTP is significantly less negative than that for bz 7 GTP by ~ 18 kJ/mol. The higher
affinity of p-Cl- bz 7 GTP for eIF4E arises from the more favourable entropy change.
ΔΔH°° (kJ/mol)
50
0
-50
-100
-150
-45 -40 -35 -30
ΔG° (kJ/mol)
Figure 4-30. Enthalpic (ΔH°) and entropic (−TΔS°) contributions to Gibbs free energy (ΔG°) of
eIF4E binding to structurally different cap analogues at 293 K.
103
-TΔ S°° (kJ/mol)
50
0
-50
-100
-150
-45 -40 -35 -30
ΔG° (kJ/mol)

m 7 , 2 , 2
3
In the case of GTP , the relative contribution of the hydrophobic interactions to
ΔG° increases due to fewer hydrogen bonds stabilizing the complex (see 4.2.2., p. 64) and
much weaker cation-π stacking 241 which itself would provide a large enthalpic component
(discussed below, 4.4.9.3., p. 124). Hence, the thermodynamic driving force for GTP
becomes more entropic.
104
m 7 , 2 , 2
3
Table 4-8. Standard molar enthalpy changes (ΔH°) and entropy changes (ΔS°) at 293 K obtained
from the van't Hoff isobaric equation for binding of eIF4E to selected mRNA 5' cap analogues.
Standard molar Gibbs free energy changes (ΔG°) calculated from the association constants at 392
K are shown for reference, pH 7.2.
Cap analogue ΔH° ΔS° ΔG°
(kJ·mol -1 ) (J·mol -1 ·K -1 ) (kJ·mol -1 )
m 7 GMP a
m 7 GDP b
m 7 GTP b
bz 7 GTP b
p-Cl-bz 7 GTP b
m3 2,2,7 GTP a
m 7 Gpppp(m 7 G)
m 7 GpppG a
m 7 GpppC a
a, c
-36.0 ± 7.9 -9.3 ± 3.4 -33.15 ± 0.20
-61.9 ± 2.9 -69.8 ± 10.5 -41.031 ± 0.179
-74.3 ± 3.6 -98.7 ± 12.1 -45.109 ± 0.090
-56.5 ± 3.8 -52.7 ± 13.0 -40.669 ± 0.060
-38.5 ± 4.6 +16.7 ± 15.5 -42.94 ± 0.21
-16.6 ± 2.5 +40.3 ± 12.7 -28.94 ± 1.36
-81 ± 54 -136 ± 88 -41.377 ± 0.146
-65 ± 31 -91 ± 58 -38.555 ± 0.152
-50 ± 28 -45 ± 29 -36.42 ± 0.40
a temperature-dependent ΔH° and ΔS° (non-linear van’t Hoff plot)
b constant ΔH° and ΔS° (deviation of van’t Hoff plot from linearity not detected, P(ν1, ν2) > 0.6)
c the microscopic association constant (Kas micro ) has been taken into account
Careful examination of the van’t Hoff plots revealed that the linear character is lost
for the analogues of moderate affinity for eIF4E (Fig. 4-31). The thermodynamic
parameters that are related to the non-linearity, i. e. standard heat capacity change under
constant pressure (ΔC°p), and the critical temperatures where ΔH° = 0 and ΔS° = 0 (TH and
TS, respectively) are gathered in Table 4-9. The large
o
Δ C p values resulting from the non-
linearity are surprisingly positive, from +1.66 up to +5.12 kJ⋅mol -1 ⋅K -1 for the m3 2,2,7 GTP -
eIF4E association, and TH is higher than TS. The results derived from the non-linear fitting
are statistically unambiguously superior to those derived from the linear one on the
significance level (P) resulting from the Snedecor's F-test, from 0.049 to even less than
0.0001. Involvement of the next fitting parameter that was related to a possible linear
dependence of ΔC°p on temperature did not render further improvement of the results.

ln Kas
ln Kas
22
20
18
16
14
12
10
22
20
18
16
14
12
10
m 7 GTP
m 7 GDP
m 7 GMP
m 7 m
GpppC
7 m
GpppG
7 Gppppm 7 G
3.2 3.3 3.4 3.5 3.6
T -1 ⋅ 10 3 (K -1 )
(a)
(c)
105
bz 7 p-Cl-bz
GTP
7 GTP
2,2,7
m3 GTP
3.2 3.3 3.4 3.5 3.6
T -1 ⋅ 10 3 (K -1 )
(b)
Figure 4-31. Dependence of van’t Hoff
plots for eIF4E - cap binding on structural
alterations of the cap analogue:
(a) elongation of the phosphate chain,
(b) replacement of the 7-methyl group for
larger substituents and dimethylation of 2amino
group,
(c) change of the second base and number of
the phosphate groups.

Table 4-9. Critical temperatures TH (where ΔH° = 0), TS (ΔS° = 0), and standard molar heat
capacity changes under constant pressure (ΔCp°) obtained from fitting of the non-linear van't Hoff
equation to the equilibrium association constants for selected mRNA 5' cap analogues, at pH 7.2.
The relative decrease in sum-of-squares (F) for the number of degrees of freedom (ν2) in
comparison with that for the linear model (ν1), and the probability of random improvement of
goodness of fit P(ν1, ν2). Calculated Gibbs free energies at TS, ΔG°TS = ΔH°TS, and at TH, ΔG°TH =
−TH⋅ΔS°TH.
Cap analogue TH TS ΔCp° F P(ν1, ν2)
(K) (K) (kJ·mol -1 ·K -1 )
m 7 Gpppp(m 7 G) a 342.0 ± 16.0 318.0 ± 7.8 +1.66 ± 0.57 8.54 0.019
m 7 GpppG 327.1 ± 15.2 307.4 ± 6.0 +1.92 ± 0.93 6.36 0.040
m 7 GpppC 310.0 ± 6.2 297.6 ± 2.1 +2.96 ± 1.25 5.65 0.049
m 7 GMP 306.9 ± 4.8 294.20 ± 1.68 +2.62 ± 0.97 7.34 0.027
m3 2,2,7 GTP 296.41 ± 0.43 290.86 ± 0.69 +5.12 ± 0.48 115 TH, which is most often
attributed to burial of solvent accessible hydrophobic molecular surface upon complex
formation. 131
To confirm validity of the exceptional results derived from the van't Hoff method for
interaction of eIF4E with the cap analogues, direct calorimetric measurements have been
o carried out. The standard enthalpy changes ( Δ Hcal
) have been determined for one selected
cap analogue, m 7 GpppG, at four different temperatures by means of Isothermal Titration
Calorimetry (ITC) (Fig. 4-32). In order to control the activity of eIF4E during the ITC
106

experiment, concurrent fluorometric titrations have been run with use of the same protein
samples, incubated at a given temperature for the same time. The synchronised
measurements of the protein intrinsic fluorescence quenching allowed to determine the
actual concentration of the active protein ([Pact]) at each temperature. [Pact] at 288.1, 293.1,
298.4, and 303.2 K was 8.97, 7.42, 3.61, and 5.35 μM, respectively. The enthalpy
estimates for the assumed 100 % protein activity (10 μM), and those corrected for the
active protein concentration determined from fluorometric titrations, are shown in
comparison with the van't Hoff enthalpy change (Fig. 4-33). The corrected calorimetric
enthalpies yield a slope of
the result of the van't Hoff analysis (
o
Δ C p = +1.966 ± 0.061 kJ⋅mol -1 K -1 (Table 4-10), which confirms
o
Δ C p = +1.92 ± 0.93 kJ⋅mol -1 K -1 ).
Table 4-10. The van't Hoff and calorimetric thermodynamic parameters for binding of m 7 GpppG to
eIF4E in Hepes buffer at pH 7.2
ΔH
ΔH
o a
cal
o b
ion
o
cal ion
d
ΔH
−
o
ΔH
vH
o
ΔS e
Temperature (K)
288.1 293.1 298.4 303.2 ΔCp°(kJ⋅mol -1 K -1 )
Enthalpy change (kJ⋅mol -1 )
−65.5 ± 3.2 −54.4 ± 4.0 −47.3 ± 8.4 −35.5 ± 1.7 +1.966 ± 0.061 f
+8.53 +8.66 +8.81 +8.94 +0.0273 ± 0.0003 f
c −74.0 ± 3.2 −63.1 ± 4.0 −56.1 ± 8.4 −44.4 ± 1.7 +1.941 ± 0.059 f
−75 ± 36 −65 ± 31 −56 ± 26 −46 ± 22 +1.92 ± 0.93 g
Entropy change (J⋅mol -1 ⋅K -1 )
−124 ± 71 −91 ± 58 −57 ± 47 −26 ± 40
a
directly measured total enthalpy changes
b o
buffer ionization heats at pH 7.2; estimated δ Δ Hion
= ± 0.09
c o
o
net calorimetric enthalpy changes, Δ Hcal−ion = Δ Hcal
− ΔH
d van't Hoff enthalpy changes
e entropy changes from the van't Hoff method
f calculated from linear temperature-dependence of corresponding
g calculated from non-linear van't Hoff dependence
107
o
ion
o
ΔH
x

Power ( μ J⋅ s -1 )
0
-2
-4
-6
-8
-10
-12
-14
0
-2
-4
-6
-8
-10
-12
-14
Time (s)
Time (s)
Figure 4-32. Modified "single injection" ITC curves at 288.1 K (left panels) and 303.2 K (right
panels). Each experiment consisted of a main 40 μl injection, preceded by two 1 μl injections
enabling to calculate a correction for the instrumental artifacts, and followed by two 4 μl injections
to check the protein saturation with the ligand. Measured heat of mixing of 7-methylGpppG with
eIF4E solution (—,—), and of 7-methylGpppG dilution in buffer (—,—) (upper panels). Calculated
reaction heat of binding of 7-methylGpppG to eIF4E at pH 7.2 in Hepes buffer (—) (bottom
panels).
4.4.3. Protonation Equilibrium of m 7 GpppG
o A systematic positive shift of the calorimetric enthalpies ( Δ Hcal
) in comparison with
o their van't Hoff counterparts ( Δ H vH ) appears from the data (Fig. 4-33, Table 4-10).
Although the numerical uncertainty of
the difference between
288.1 K
500 1000 1500
o
Δ H vH is about ± 30 kJ⋅mol -1 , the average value of
o o
Δ Hcal
and Δ H vH of about +9.7 kJ⋅mol -1 seems to be well specified.
Differences between the calorimetric and the van't Hoff enthalpy estimates were the
subject of empirical and theoretical analyses for various association processes 244-250 . The
observed discrepancies were ascribed to contributions of usually unknown molecular
108
303.2 K
500 1000 1500

Δ H° cal (kJ ⋅mol -1 )
Figure 4-33. Total calorimetric enthalpies corrected for ligand dilution, assuming 100 % protein
activity ( ,------), and those corrected also for active protein concentration, ( ,——). Temperature
dependence of the van't Hoff enthalpy is shown for comparison ( ).
transitions or coupled processes, other than the net complex formation, to
erroneous apparent values of
0
-20
-40
-60
-80
283 288 293 298 303 308
o
Δ H vH and
Temperature (K)
109
o
p
o
cal
Δ H , and/or
Δ C , arising from the experimental noise. In the
case of cap − eIF4E binding the latter obscuring effect is eliminated, as testified by the
accordance of the van't Hoff and calorimetric
o
p
Δ C values. This approximately constant
difference can be analysed in terms of the protonation equilibria 251 . 7-methylGpppG exists
as a mixture of cationic (58%) and zwitterionic (42%) forms at pH 7.2 (pKa = 7.35 ± 0.05
for the N(1)-H proton of 7-methylguanine moiety 177 , Scheme 3-1, p. 23). Several
contradictory conclusions were reported regarding the ionic state of the mRNA 5' cap that
binds to eIF4E most tightly. Initially, the zwitterionic form of 7-methylguanine was
postulated to interact with eIF4E 155,156 . In contrast, the crystallographic and NMR
structures revealed the spatial distances suitable for formation of a hydrogen bond between
Glu103 and N(1)-H of 7-methylguanine 1,48,75,76 , pointing to the cationic form of 7-
methylguanine. This hydrogen bond is also present in the ternary 7-methylGDP − eIF4E −
eIF4GII peptide complex at pH 8.5 91 . However, the N(1)-H proton has not been directly
observed by NMR 76 . The binding studies as a function of pH showed the upward shift of
pKa of N(1)-H inside the eIF4E binding site, suggesting that the tightest binding is
accomplished through the cationic form of 7-methylguanosine (see 4.2.3.2., p. 83). The
eIF4E − cap association that is accompanied by the partial protonation of the ligand must
be equilibrated by additional deprotonation of the buffer to keep the cation-zwitterion
equilibrium of the free ligand at constant pH. As shown in Table 4-10, the calculated

o
ion
contribution of the Hepes ionization heat ( Δ H ) to the total reaction heat is in a very good
agreement with the difference between
o
cal
Δ H and
110
o
Δ H vH . This demonstrates the linkage
between 7-methylGpppG − eIF4E binding and concomitant protonation of the residue
which has pKa = 7.35 in the unbound state. Taking into account the net calorimetric
enthalpy changes related to cap binding by eIF4E,
o
cal ion
Δ H − = Δ Hcal
− Δ Hion
,
o
o
the calorimetric heat capacity change corrected for buffer ionization is
0.059 kJ⋅mol -1 K -1 .
o
p
Δ C = +1.941 ±
The coupling of the binding process with the acidic-basic equilibrium of the ligand is
nonmandatory 122 , since the zwitterionic form of 7-methylGpppG is also able to bind to the
protein via weaker interactions of 7-methylG moiety and almost unchanged interactions of
the phosphate chain. It was shown that the protein-induced shift in the two-state transition
of the ligand could contribute to the observed heat capacity change 122 . This contribution
may be either positive or negative, depending on the unknown values of the intrinsic
enthalpy changes and equilibrium constants that describe binding of the cationic and the
zwitterionic forms of 7-methylGpppG to eIF4E independently.
4.4.4. Conformational Equilibrium of Cap Analogues
Association of eIF4E with the mRNA 5' terminus is also directly coupled with
intramolecular self-stacking of the cap. The question arises whether and to how extent this
coupled process influences binding to eIF4E. Self-stacking provides for additional
o o enthalpic ( Δ Hsst
) and entropic ( Ssst
o psst
C
Δ ) contributions, and an apparent molar heat capacity
change ( Δ ), resulting from an induced shift in the conformational equilibrium of the
ligand upon binding to the protein. 122 As only the unstacked form of dinucleotide cap
analogues can penetrate the eIF4E cap-binding slot, the coupling is mandatory.
The known thermodynamic parameters describing self-stacking of the cationic form
(pH 5.2) and zwitterionic form (pH 9.0) of some dinucleotide cap analogues at 293 K 198
gave a possibility to assess the influence of this coupled process on the cap − eIF4E
complex formation. On the basis of these parameters, required values of equilibrium
constants (1K) and standard molar enthalpy changes (1ΔH°) for self-stacking at pH 7.2 have
been calculated, and next the contributions from self-stacking to the observed
thermodynamic parameters have been determined (Table 4-11). Both
o o
Δ Hsst
and Ssst
Δ for

each dinucleotide analogue are positive and relatively large. They reduce the negative
values of the intrinsic thermodynamic parameters ( Δ H , Δ S , Table 4-12) to a substantial
extent, leading to the apparently less negative values of the observed ΔH°cal-ion, ΔH°vH and
ΔS°, as reported above (Table 4-8). In case of m 7 GpppG, the Δ H and Δ S values change
by 12 – 17 %, and even by 17 – 46 %, respectively, depending on temperature.
Table 4-11. Equilibrium constants (1K) and standard molar enthalpy changes (1ΔH°) for
intramolecular self-stacking of dinucleotide cap analogues; and apparent contributions to standard
molar enthalpy (ΔH°sst), entropy (ΔS°sst), and heat capacity (ΔCp°sst) changes of eIF4E association
with cap analogues, resulting from the self-stacking equilibrium shift that is induced by the
mandatory coupling of unstacking to the binding, at 293 K.
Cap analogue 1K 1ΔH° ΔH°sst ΔS°sst ΔCp°sst
(kJ·mol -1 ) (kJ·mol -1 ) (J·mol -1 ·K -1 ) (J·mol -1 ·K -1 )
pH 5.2 a
m 7 GpppG 1.40 -15.2 ± 0.5
m 7 GpppC
pH 9.0
0.18 -12.0 ± 0.8
a
m 7 GpppG 1.58 -18.6 ± 0.7
m 7 Gppp(m 7 G)
pH 7.2
0.48 -21.1 ± 0.9
m 7 GpppG b
111
o
0
1.47 -16.6 ± 0.4 +9.89 ± 1.17 +30.5 ± 2.6 -93.2 ± 10.4
m 7 Gppp(m 7 G) c 0.32 -15.8 ± 0.7 +3.85 ± 0.59 +10.81 ± 1.38 -64.5 ± 8.3
m 7 GpppC d
0.18 -12.0 ± 0.8 +1.83 ± 0.44 +4.87 ± 0.82 -26.1 ± 5.8
a data from 198
b values calculated from pKa = 7.35 ± 0.05 177 , 1K and 1ΔH° at pH 5.2 and 9.0
c values estimated from the assumptions: pKa = 7.2 for both ends, no stacking for the double
cationic form, stacking for the zwitterionic-cationic form the same as for the double zwitterionic
form (pH 9.0)
d values estimated from the assumptions: 1K and 1ΔH° the same as for the cationic form (pH 5.2)
Table 4-12. Calculated standard molar enthalpy (ΔH°0), entropy (ΔS°0), and Gibbs free energy
changes (ΔG°0) at 293 K, pH 7.2, for intrinsic binding of the unstacked dinucleotide cap analogues
to eIF4E, at 293 K, pH 7.2.
Cap analogue ΔH°0 ΔS°0 ΔG°0
(kJ·mol -1 ) (J·mol -1 ·K -1 ) (kJ·mol -1 )
m 7 Gpppp(m 7 G) b
-85 ± 54 -147 ± 88 -42 ± 60
m 7 GpppG -75 ± 31 -122 ± 58 -39 ± 35
m 7 GpppC -52 ± 28 -50 ± 29 -37 ± 29
b microscopic association constant has been taken into account
o
0
o
0
o
0

Table 4-13. Calculated critical temperatures TH 0 and TS 0 (where ΔH°0 = 0 and ΔS°0 = 0,
respectively) and standard molar heat capacity changes under constant pressure (ΔCp°0) for the
intrinsic binding of eIF4E to the unstacked dinucleotide cap analogues, at pH 7.2.
Cap analogue TH 0 TS 0 ΔCp°0
(K) (K) (kJ·mol -1 ·K -1 )
m 7 Gpppp(m 7 G) a
342 ± 35 319.2 ± 18.5 +1.73 ± 0.57
m 7 GpppG 330 ± 23 311.5 ± 12.5 +2.01 ± 0.93
m 7 GpppC 310.6 ± 11.8 298.1 ± 3.6 +2.99 ± 1.25
a microscopic association constant has been taken into account
While enthalpy and entropy changes of binding of the dinucleotide cap analogues to
eIF4E are profoundly affected by the coupling between the binding and the unstacking, the
intrinsic binding free energy for the unstacked caps ( Δ G , Table 4-12) are only negligibly
enhanced in comparison with the resultant ΔG° values (constant within SD). The negative
o
Δ C psst contributions to the resultant heat capacity changes are very small and almost
temperature-independent. Assuming constant
112
o
0
o
Δ C psst values, they encompass a range from
–26 to −93 J⋅mol -1 K -1 for the three analogues (Table 4-11), and shift negligibly the heat
capacity changes of the overall association with eIF4E from the intrinsic values for the
o
p0
unstacked caps ( Δ C , Table 4-13) to the resultant values reported in Table 4-9. The
critical temperatures TS0 and TH0 are also not significantly changed. Although the
indirectly determined contributions from self-stacking as well as the intrinsic
thermodynamic parameters for eIF4E binding to the unstacked cap are charged by
significant uncertainties, they can serve as general indicators whether and how the complex
formation is influenced by the coupled equilibrium of cap self-stacking.
The determined small
o
Δ C psst values are very close to a value found as a contribution
due to the coupling of adenine base unstacking to binding between single-stranded
dA(pA)34 and the E. coli SSB protein, −62.8 ± 2.5 J⋅mol -1 K -1 per one stack 252 . This
suggests that unstacking of the dinucleotide mRNA 5' cap analogue, i.e. between 7-
methylguanosine and guanosine moieties linked via the symmetric 5'-5' triphosphate
bridge, can be energetically considered in a similar way as oligodeoxyadenylates
unstacking that accompanies specific binding of proteins to single-stranded DNA.

4.4.5. Could the Positive Values of ΔCp° Result from Protein
Deaggregation ?
Two eIF4E (28-217) molecules are sticked together through the 4E-BP and eIF4G
binding hydrophobic dorsal surface in the crystallographic asymmetric unit. 75,91 On the
other hand, binding of cap forces the eIF4E (33-217) deaggregation (see 4.2.3.1.6.1., p.
82). Therefore, some additional non-polar surface area would be exposed to water due to
the binding-induced deaggregation. One could thus suspect that the positive
113
o
p
Δ C is related
only to this side effect. Hence, the question arises whether the possible eIF4E (28-217)
aggregation – deaggregation equilibrium coupled to the cap binding could be responsible
for the observed positive heat capacity changes.
The eIF4E (28-217) protein was filtered through 100 kD pore size immediately
before experiments, and the probability of appearance of dimers in a dilute eIF4E solution
was minimal. DLS measurements of this apo-eIF4E (28-217) showed that it was
monodispersed. 253 Moreover, the main argument against this hypothesis is that then the
putative deaggregation-related heat capacity change should be maximal for the strongest
binding analogue (m 7 GTP), for which the deaggregation was the most effective. The
situation is entirely opposite, since m 7 GTP and the other most tightly binding compounds
yield
o
p
Δ C = 0. Hence, it's impossible that the phenomenon which was independently
detected by means of both emission spectroscopy and calorimetry at the eIF4E
concentration of ~ 0.1 μM and ~ 10 μM, respectively, could be a trivial one, resulting from
a series of systematic experimental mistakes and wrong interpretation.
4.4.6. Enthalpy-Entropy Compensation for Individual Cap Analogues
Having verified the surprisingly positive and unusually high value of the heat
capacity change for m 7 GpppG, one can ask about its consequences regarding the
mechanism of cap – eIF4E interaction. As a result of the non-zero values of
o
p
Δ C , which
are large in comparison with ΔS°, temperature-dependent enthalpy-entropy compensation
occurs, i.e. the values of both the linear
o
Δ H vH term and the logarithmic TΔS° term in the
expression for ΔG° increase with temperature, with nearly identical slopes (Fig. 4-
34). 118,196 The large positive
o
p
ΔC values for the eIF4E − cap association cause that the
binding is enthalpy-driven at temperatures below TS (Table 4-9), while the entropy is

unfavourable, confirming the importance of electrostatic stabilization of the complex.
Then, the interaction changes its thermodynamic character to both enthalpy- and entropy-
driven between TS and TH, and finally, above TH the association becomes entropy-driven
and enthalpy-opposed.
Although the ΔG° function attains its maximum at TS, the stabilization of the
complex does not decrease rapidly but is still efficient. For the two analogues which are of
frequent occurrence as natural mRNA 5' termini, m 7 GpppG and m 7 GpppC, the maximal
values are about −38 and −37 kJ⋅mol -1 , respectively (Table 4-9). The fact of occurrence of
the non-zero heat capacity changes for the two natural dinucleotide cap analogues has three
salient consequences:
1. the interaction with eIF4E is both enthalpy and entropy favourable at biological
temperatures (~ 310 K, 37 °C);
2. the affinities of the two analogues for eIF4E are almost equal at ~ 310 K;
3. the free energies of the complexes stabilization are relatively temperature-invariant
over this range.
On the contrary, the results obtained for the mononucleotide triphosphates show that
thermodynamic driving forces make a distinction between the monomethylguanosine cap
(m 7 GTP) and the trimethylguanosine cap ( m GTP
7 , 2 , 2
3
114
) at the biological temperature range.
The association constant of the former is ~ 110-fold greater than that of the latter at 310 K
(37 °C). The temperature-dependent thermodynamic parameters for m GTP
7 , 2 , 2
3 are ΔH°310K
= +69.6 ± 6.9 kJ/mol and ΔS°310 K = +326 ± 33 J/mol⋅K. It is worth noting that due to the
positive heat capacity change the m GTP
7 , 2 , 2
3 binding enthalpy attains the same absolute
value as that for m 7 GTP but with the opposite sign, in spite of the fact that both analogues
have the charged triphosphate chains. Hence, elevation of temperature at this range causes
an increase of the eIF4E affinity for m GTP
7 , 2 , 2
3 and an affinity decrease for m 7 GTP. This
phenomenon diminishes the predominance of m 7 GTP affinity in relation to m GTP
7 , 2 , 2
value of ~ 57 already at 313 K (40 °C).
3 to a

Contributions to ΔΔG°° (kJ⋅⋅mol -1 )
Contributions to ΔΔG o (kJ⋅mol -1 )
20
-30
-40
-50
-60
-70
-80
0
-20
-40
-60
-80
-100
TΔS°
m 7 GpppG
ΔH° cal-ion
m 7 GTP
ΔG o
ΔH o
T S
TΔS o
ΔG°
280 290 300 310 320 330
Temperature (K)
ΔH° vH
280 290 300 310 320 330
Temperature (K)
T H
Figure 4-34. Temperature-dependent enthalpy-entropy compensation that accompanies the binding
of m 7 GpppG, m 7 GpppC, and m3 2,2,7 GTP to eIF4E. Theoretical non-linear fit (—) to the binding free
energies ΔG° (, , ), the entropic (− − −), and the van't Hoff enthalpic (----) contributions to
ΔG° as well as the calorimetric enthalpies corrected for ligand dilution, protein activity and buffer
ionization heat ( ) with the linear regression (—) are plotted as a function of temperature. ΔS° = 0
at TS and ΔH° = 0 at TH. Linear contributions to ΔG° of m 7 GTP () are shown for comparison.
115
Contributions to ΔG o (kJ⋅mol -1 )
Contributions to ΔG o (kJ⋅mol -1 )
60
30
0
-30
-60
-90
150
100
50
0
-50
-100
m 7 GpppC
TΔS o
ΔG o
ΔH o
280 290 300 310 320 330
Temperature (K)
m 3 2,2,7 GTP
TΔS o
ΔG o
ΔH o
280 290 300 310 320 330
Temperature (K)

4.4.7. Biological Implications of Non-Linear van't Hoff Thermodynamics
Thermodynamic features of the process of recognition of the 5' cap structure by
eIF4E can provide an eIF4E-dependent switch for several in vivo metabolic pathways, as
follows:
1. The temperature-invariability of ΔG° for the natural dinucleotide cap analogues is of
special biological importance, because the regulation of eIF4E and its inhibitory
binding protein, 4E-BP1, is affected by heat shock which causes an increase of the
association between eIF4E and 4E- BP1 81 . Increased binding of 4E-BP1 to eIF4E
interferes with the formation of the active eIF4F translation initiation complex 31
during heat shock 41,254-256 . As mRNAs of heat shock proteins are relatively cap-
independent 257,258 , the enhanced inhibition of eIF4E by 4E-BP1 together with the
temperature-invariant cap-binding affinity of eIF4E could provide a mechanism for
the selective up-regulation of the synthesis of heat shock proteins.
2. The distinct thermodynamic parameters of m 7 GTP and m GTP
7 , 2 , 2
116
3 can shift the
biochemical equilibria related to splicing in eukaryotic cells during heat shock. Two
types of capped RNAs exist in eukaryotes, mRNAs and small nuclear RNAs
(snRNAs). Their 5' termini are methylated in the nucleus to yield m 7 GpppN
structure. 259 After nuclear export to cytosol, the cap of snRNAs is further methylated
the at the amino group of the 7-methylguanosine moiety, forming m GpppN
7 , 2 , 2
The trimethylguanosine cap is a fragment of a nuclear localization signal (NLS),
which is responsible for import of the snRN-protein spliceosomal complexes
(snRNPs) into the nucleus, 23,24 where they take part in pre-mRNA splicing. 25 The
trimethylguanosine cap-dependent transport is enhanced by a m GpppN
7 , 2 , 2 -specific
nuclear import receptor, a protein called snurportin1, which binds the trimethyl-
guanosine cap structure approximately three orders of magnitude more strongly than
the monomethylguanosine cap. 260 Since the preference of eIF4E for m 7 GTP vs.
GTP
, 2 , 2
3 is significantly diminuted at higher temperatures, snRNAs could be also
m 7
recruited to the translational machinery, thus contributing to inhibition of translation
initiation. Moreover, the binding of eIF4E to the trimethylguanosine cap of snRNPs
prevents recognition of this structure by snurportin1, hence the nuclear import and
consequently splicing could be disturbed. This competition could also lead to
significant inhibition of protein biosynthesis.
3
3
. 22

3. In some primitive eukaryotes 29,261,262 and in some chordate species 28 the process of
trans-splicing causes that RNAs contain the trimethylguanosine cap structure. Even
~ 70% of the mRNA population has the original cap replaced by 3 GpppN in the
117
m 7 , 2 , 2
C. elegans nematode. Five eIF4E-like translation initiation factors were found in this
organism, and three of them could bind both to m 7 GTP and m GTP
7 , 2 , 2
3
. 55,263,264 The
cellular role for the individual eIF4E isoforms is still not known, although some of
them are essential for viability of the worm embryos, as shown by RNA
interference. 264
4.4.8. Isothermal Enthalpy-Entropy Compensation in Congener Series
The enthalpic and entropic contributions to the Gibbs free energies of binding of
eIF4E to the series of the cap analogues at 293.16 K (Table 4-8) appear to satisfy a linear
relationship with the high correlation coefficient, R 2 = 0.975 (Fig. 4-35). The slope yields a
compensation temperature, Tc = 399 ± 24 K. The result is exactly the same, irrespective of
the data set which is chosen for the dinucleotide cap analogues, i. e. either the couples of
o o the intrinsic ( Δ H 0 , S0
Δ ) or observed (ΔH°, ΔS°) parameters. This means that although the
resultant enthalpy and the entropy of interaction are significantly influenced by self-
stacking, the general thermodynamic property of recognition of mRNA 5' cap by eIF4E is
insensitive to it.
Enthalpy-entropy compensation is a widely reported phenomenon, which is often
called an extra-thermodynamic feature of complex molecular systems that fluctuate or
interact with the aqueous medium. However, several authors made detailed and critical
reviews of compensation in congener series, demonstrating that it is in majority a trivial
observation or an artifact that follows immediately from applied experimental
conditions. 199,265-267 There are three categories of isothermal enthalpy-entropy
compensation that can be detected in a series of homologous compounds:

TΔΔS° (kJ mol -1 )
40
20
Figure 4-35. Isothermal enthalpy-entropy compensation for the mRNA 5’ cap congener series at
293 K. The enthalpy gain is always greater than the entropy loss that accompanies the association
of more and more tightly binding cap analogues, irrespective of whether the binding is described by
a zero () or non-zero () heat capacity change. The linear fitting was performed with weighting by
errors of both TΔS° and ΔH° (Table 4-8). The errors are not shown for clarity.
1. In case of the solvation thermodynamics of a solute series, or the binding
thermodynamics of a ligand series, when the congeners differ in the number or size
of similar substituents, then the linear compensation is a trivial manifestation of the
presence of a single source of additivity. The series of the studied nine cap analogues
contains several sources of additivity: phosphate groups at the ribose ring, methyl
substituents at the 2-amino group of the 7-methylguanine moiety, different aromatic
substituents for the 7-methyl group, and different second nucleosides. Hence, this
case can be ruled out.
2. If the range of binding free energy (ΔG°) that can be observed during experiments is
small in relation to the binding enthalpy (ΔH°), due to some intrinsic features of an
investigated system or due to experimental conditions, then the linear correlation is
simply a consequence of the equation:
ΔH° − TΔS° = ΔG° ≈ constant .
0
-20
-40
-60
m 7 GTP
bz 7 GTP
p-Cl-bz 7 GTP
m 7 GDP
m 7 GpppG
m 7 Gppppm 7 G
-100 -80 -60 -40 -20 0
The ΔG° values of the eIF4E-cap association are always greater than 50 % of the
corresponding ΔH° values (Fig. 4-36), which allows to exclude this case, too.
118
2,2,7
m3 GTP
m 7 GMP
m 7 GpppC
ΔH° (kJ⋅mol -1 )

ΔG° /ΔH°
2.0
1.5
1.0
0.5
0.0
119
Figure 4-36. Proportion of ΔG° to
ΔH° for association of cap analogues
to eIF4E at 293 K
3. True extra-thermodynamic compensation that reflects some additional information
about a system, e. g. about the distribution of energy levels available to the system or
about interactions between components, can occur when the two above mentioned
cases were eliminated. The only problem that remains to be resolved consists in an
answer to the question whether enthalpy-entropy compensation does not result
fortuitously from high correlation between errors of ΔH° and ΔS° obtained by the
van't Hoff method.
-45 -40 -35 -30 -25
ΔG° (kJ⋅mol -1 )
A statistical test has been used to determine the significance of the observed ΔH° vs.
ΔS° plot for the series of the cap analogues. 266,267 The most exact criterion is to check
whether the experimental temperature (Texp) lies outside the 95 % confidence interval for
the compensation temperature: Texp < Tc − 2⋅σ or Tc + 2⋅σ < Texp. The 95 % interval is 351
– 447 K, while the harmonic mean value of the experimental temperatures applied for the
van't Hoff analysis is ~ 297 K, as calculated from Table 4-7. Hence, the difference between
Tc and Texp is even more than 4⋅σ, which unambiguously points to strong, non-trivial,
isothermal enthalpy-entropy compensation.
4.4.8.1. Molecular Interpretation of Isothermal Enthalpy-Entropy Compensation
It is often suggested that enthalpy-entropy compensation is an intrinsic property of
complex systems that have many soft modes of fluctuations, e. g. aqueous solutions and
soluble proteins. A statistical mechanical analysis of an entirely general model of a
significantly perturbed complex system revealed that the compensation temperature
depended on where the energy of the perturbed state (U') lay with respect to the mean

energy of the unperturbed system (E). 199 However, any clear interpretation for the Tc value
was not proposed.
It seems that Tc can be directly related to the difference in stability of the system in
the unperturbed and perturbed states. In other words, the difference between the
compensation temperature and the harmonic mean experimental temperature can be a
measure of the fluctuations of the unperturbed system, which are modulated by the
perturbation. According as Tc is greater or lower than Texp, the fluctuations are silenced or
enhanced, so the perturbation acts either stabilizing or destabilising, respectively. Thus, the
Tc value of 399 ± 24 K shows that the energy (U') of the state of eIF4E, which binds to the
cap structure lies much below the mean energy of the apo-protein (E):
E − U' = 9.66 ± 1.7 kJ/mol.
This rather large value, far exceeding RT (~ 2.5 kJ/mol), and almost comparable with the
stabilization energy of globular proteins of similar size and hydrophobicity (~ 50 kJ/mol), 11
suggests that the apo-eIF4E is a highly fluctuating, unstable protein, and only the specific
cap binding provides enough stiffening of the global protein structure to make it stable on
an usual level. This conclusion is in very good accordance with the observations gathered
on other fields:
1. biochemical: the great instability index calculated from the eIF4E amino acid
sequence; 44
2. biophysical: extensive conformational changes of eIF4E upon cap binding; the
significant decrease of the amount of the active protein even at medium temperature;
aggregation;
3. biological: much lower concentration of eIF4E than that of the other translation
initiation factors; the presence of eIF4E bound to 4E-BP or to eIF4G within the
eIF4F complex in the living cell; regulation of eIF4E biological activity by its
cellular accessibility. 42
In summary, the thermodynamics of the eIF4E association with the cap structure
elucidates basic properties of this unusual protein, which are key to its biological activity.
120

4.4.9. Inquiries about Origins of the Unusual Positive Heat Capacity
Change
Usually observed large, negative values of standard heat capacity change are
characteristic for specific hydrophobic binding between proteins and nucleic acids as well
as for protein folding. 131,134,194,196,242,268-271 The main negative contribution to
121
o
p
Δ C comes
from the hydrophobic effect, i.e. the removal of non-polar and aromatic molecular surface
from water upon complex formation. 131 On the contrary, the reduction of the polar surface
area gives positive contribution to
o
Δ Cp
, although to less extent, 2.3-fold 134 or 1.7-
fold 130,268 , depending on the proposed model. Other contributions to negative
o
ΔCp
comprise changes in soft internal vibrational modes and temperature-dependent
conformational changes which lead to the overall decrease in mobility of residues and to
stabilization of relative arrangement of domains, and/or protein aggregation 131 . For
instance, a large
o
p
Δ C value of −1.58 ± 0.06 kJ/mol⋅K for water mediated antigen –
antibody association is completely unrelated to the hydrophobic effect. Instead, this results
only from an interdomain induced fit of the heavy and light polypeptide chains, which was
proved by resolving of the crystal structures of the antibody in the free and antigen-bound
forms. 243
A process of protein-ligand interaction characterized itself by
o
p
Δ C = 0 can also give
rise to a non-zero, positive or negative heat capacity change, due to thermodynamic
coupling with other possible equilibrium transitions 122,272 : proton uptake or dissociation,
binding of the second ligand, a conformational change of the protein and/or the ligand.
While the positive
o
p
Δ C relevant to the protein unfolding is a common observation,
examples reported for intermolecular interactions are extremely rare and contain few data,
e.g. the formation of the phosphofructokinase tetramer 123 , the interaction of the brain
natriuretic peptide with heparin 273 , cobalt hexamine and spermidine binding to DNA, 274
anion-exchange adsorption of cytochrome b5 and its surface-charge mutants. 275 On the
other hand, it was shown for the interaction of c-Myb DNA-binding domain (R2R3) with
its target DNA, that the heat capacity change can be strongly temperature- and ionic
strength-dependent, leading even to the sign inversion of
o
p
Δ C within some ranges of those
parameters 276 . In each case, Coulombic interactions were involved into the binding
processes.

4.4.9.1. Changes of Solvent Accessible Surfaces Upon Binding
The kinetic studies of the eIF4E − cap interaction by means of stopped-flow
fluorescence spectroscopy as well as Brownian molecular dynamics simulations 4 revealed
two-step character of the complex formation. The first step is the diffusionally and
electrostatically controlled encounter of the protein and the ligand and the second step is
the internal rearrangement of the encounter complex. Detailed analysis of the salt effect on
the equilibrium association constants revealed that an uptake of roughly 65 water
molecules to the first-layer hydration shell is necessary for the complex formation (see
4.2.3.1., p. 74). This large hydration effect is relevant to significant conformational change
of the protein upon the second step of the cap binding (see 4.2.3.1.6., p. 82), and leads to
an increase of the protein solvent accessible surface area. The cooperativity of cap-binding
site with the eIF4G/4E-BP-binding site (see 4.3.2., p. 90) suggests that the hydrophobic
dorsal surface recognized by the eIF4G and 4E-BP1 peptides becomes more accessible
after the cap has been bound. To estimate the upper limit of the positive contribution to
ΔCp° from hydration of the protein hydrophobic surface, one should subtract the number of
water molecules trapped inside the cap-binding centre (at least 16, as detected by
crystallography 1 ) from the total number of waters taken up to the complex (ΔN ≈ 65), since
water in internal cavities is usually stiffly bound. 226,227 Since the surface hydrated by one
water molecule is ~ 9.5 Å 2 , 119,232 then, even if one assumes that all remaining 49 water
molecules hydrate a purely aliphatic surface of ~ 465 Å 2 (which is unlikely), and if one
takes the maximal increment of ΔCp° per 1 Å of aliphatic surface (+2.14 J/mol⋅K 130 ), the
o
p
calculated contribution to Δ C would equal only about +1 kJ/mol⋅K.
Some additional contribution to the positive
122
o
p
Δ C arises from the burial of the
uncharged and charged polar groups in the protein binding centre. Heat capacity changes
arising from dehydration of nucleic acid components were calculated recently on the basis
of polar and apolar accessible surface areas as +52.63 J/mol⋅K for guanine, −36.07 J/mol⋅K
for ribose, and +46.02 J/mol⋅K for a single phosphate group. 277 Neglecting neighbour
effects between base, sugar, and phosphate groups in the entire cap analogue, and the
unknown resultant influence of the guanosine methylation which provides both the
additional aliphatic group and the positive charge to the ring, the estimated range of
contribution from burial of 7-methylguanosine triphosphate would be about +0.2 kJ/mol⋅K.

4.4.9.2. Electrostatic Contributions to Heat Capacity Changes
Careful inspection of the crystallographic 1,48,75 and NMR 76 structural data shows that
stabilization of the mRNA 5' cap – eIF4E complexes is accomplished to great extent by
charge-related interactions and partially complemented by the van der Waals contacts (see
4.2., p. 60). Many charged groups are removed from water upon the association. The
negatively charged 5'-5' phosphate chain of 7-methylGpppG is a primary anchor to the
positively charged Arg112, Lys162, Arg157 and Lys159 side-chains of eIF4E. This
charge-to-charge anchoring makes it possible to form further specific polar contacts in the
narrow binding slot: cation-π sandwich stacking of the 7-methylguanine moiety with
Trp56 and Trp102, and three hydrogen bonds with Glu103 and Trp102.
Hydrophobic area-based models of the heat capacity changes take into account only
short-rang effects, while there are direct electrostatic contributions to heat capacity, which
do not scale with the non-polar and polar surface areas. As shown by the finite-difference
Poisson-Boltzmann method, three components of the total electrostatic contribution to
o
p
Δ C are related to: (1) the rearrangement of water dipoles upon binding, (2) the
redistribution of mobile ions in the solvent upon binding, and (3) the coupling between the
dipolar and ionic terms. 278 The overall contribution of electrostatic interactions to
123
o
p
Δ C due
to dehydration of charged residues upon protein – ligand binding is dominated by the
positive term arising from the dielectric behaviour of water, which is opposed by the
contribution of mobile solvent ions. Therefore, in addition to burial of polar or polarizable
surface, changes in the water structure that accompany dehydration of ionized groups can
also partially account for the observed positive
o
p
Δ C . However, the possible electrostatic
contribution from the phosphate groups and the basic amino acid side-chains is still at least
one order of magnitude too small to explain the huge positive heat capacity changes for the
cap analogues.
The cation - π stacking itself has also a great electrostatic component, resulting from
the attraction between the cation and the quadrupole charge distribution of the aromatic
ring, which dominates the polarizability and dispersive forces. 129,148,149,279,280 In light of the
above mentioned possible explanation of the positive
seemed to be very interesting to check whether the unusual values of
o
p
Δ C by Coulombic contributions, it
stacking of tryptophan with the non-typical, large, 7-methylguanosine cation.
o
p
Δ C could rise from

4.4.9.3. Tryptophan Stacking with Cationic 7-Methylguanosine Moiety
In order to extract information concerning the thermodynamics of the cationic 7-
methylguanosine moiety interactions, NMR spectroscopy has been applied to examine two
model systems: 7-methylGMP and tryptophan N-acetylamid, and a dinucleotide cap
analogue, 7-methylGpppG, interacting with a dodecapeptide of the sequence related to a
part of the eIF4E cap-binding site around Trp102 (DGIEPMWEDEKN). Stacking between
7-methylG and tryptophan shifts the 1 H signals upfield, yielding microscopic equilibrium
association constants (K), listed for the tryptophan protons at 298 K in Table 4-14,
according to the titration curves shown in Fig. 4-37.
Similar microscopic association constants for the individual tryptophan protons,
related to stacking with m 7 GMP suggest that configuration of the two heteroaromatic rings
is almost parallel. The dodecapeptide tryptophan interacts only with the methylated base of
m 7 GpppG, and the complex assumes both parallel and perpendicular orientations of the 7-
methylguanosine moiety in relation to the indol ring in a dynamic equilibrium. 7,281 The
presence of multiple forms of the complexes causes that the chemical environment of the
tryptophan protons is affected in different manner, which yields the microscopic K for
Table 4-14. Microscopic equilibrium association constants for tryptophan protons, related to
stacking of tryptophan N-acetylamid with m 7 GMP at pH 5.6, and to binding of tryptophancontaining
dodecapeptide with m 7 GpppG at pH 5.2; temperature 298 K.
Tryptophan protons: H(2) H(4) H(5) H(6) H(7)
Model system K (M -1 )
Trp(N-aa)-m 7 GMP 15.9 ± 3.8 6.5 ± 1.6 6.7 ± 2.0 5.9 ± 1.8 7.8 ± 1.3
Trp(pept)-m 7 GpppG 242 ± 67 61.8 ± 7.5 51.4 ± 7.2 39.4 ± 9.5 n. d.
a not determined because of too weak changes of H(7) chemical shift with m 7 GpppG concentration
Δδ HTrp (ppm)
0.00
-0.02
-0.04
-0.06
-0.08
H(2)
H(6)
H(5)
H(4)
H(7)
0 5 10 15 20 25 30
m 7 GMP (mM)
Figure 4-37. Differences of 1 H chemical shifts of tryptophan N-acetylamid at 1 mM due to
stacking upon titration with m 7 GMP at pH 5.6 (left); and of the dodecapeptide tryptophan at 2.8
mM due to stacking upon titration with m 7 GpppG at pH 5.2 (right) at 298 K.
Δδ HTrp (ppm)
124
0.01
0.00
-0.01
-0.02
-0.03
-0.04
0 3 6 9 12 15
m 7 GpppG (mM)

Δδ HTrp (ppm)
Figure 4-38. Temperature dependence of differences of 1 H chemical shifts of tryptophan Nacetylamid
at 1 mM in the presence of 29.8 mM m 7 GMP at pH 5.6 (left); and of the dodecapeptide
tryptophan at 2.8 mM in the presence of 17.2 mM m 7 GpppG at pH 5.2 (right).
H(2) about 5-fold greater than K for the remaining protons, except H(7). The H(7) proton
of the dodecapeptide tryptophan exercises a slight downfield shift.
Although stacking is thought of as a hydrophobic interaction, binding of tryptophan
with both cationic 7-methylGMP and 7-methylGpppG is enthalpy-driven and entropy-
opposed, without significant heat capacity changes in the temperature range of 278–320 K,
as determined on the basis of the temperature-dependent differences of 1 H chemical shifts
of the tryptophan protons (Fig. 4-38, Table 4-15). Satisfactory goodness of fit in terms of
the sum-of-squares of deviations (R 2 ) has been obtained when it is assumed that the
stacking is described by the constant values of the van't Hoff enthalpy change (ΔH°vH) and
the entropy change (ΔS°). However, the P-value that refers to the probability of random
distribution of fitting residuals along the fitted curve is slightly improved if a non-zero
o
Δ Cp
value is allowed for the m 7 GMP-Trp interaction (Fig. 4-39). The heat capacity change
estimated in this way is positive but small, from +0.12 to +0.18 kJ/mol⋅K for different
tryptophan protons, charged by the relative numerical error of ~ 100 %. It seems unlikely
that the 7-methylguanosine sandwich stacking between Trp102 and Trp56 within the cap-
binding site of eIF4E could alone provide a positive contribution to
o
p
explain the observed Δ C values of the order of +5 kJ/mol⋅K.
R 2 , P value
-0.02
-0.06
-0.10
-0.14
1.0
0.5
0.0
280 290 300 310 320
Temperature (K)
-1000 -500 0 500 1000
ΔC p° (J/mol⋅K)
H(2)
H(6)
H(5)
H(4)
H(7)
125
Δδ HTrp (ppm)
-0.02
-0.04
-0.06
-0.08
280 290 300 310
Temperature (K)
o
p
H(2)
H(7)
H(6)
H(5)
H(4)
Δ C great enough to
Figure 4-39. Goodness of fit (R2, ) and
probability of random distribution of
fitting residuals (P value, ) for curves
fitted to temperature dependence of
ΔδH(7) of tryptophan N-acetylamid
stacking with m 7 GMP at pH 5.6 with fixed
ΔCp° values indicated in figure.

Table 4-15. Thermodynamic parameters for stacking of mRNA 5' cap analogues with tryptophan
N-acetylamid and the tryptophan-containing dodecapeptide. The van't Hoff enthalpy change
(ΔH°vH) and the entropy change (ΔS°) are approximately constant with temperature. For reference
the parameters of eIF4E-m 7 GpppG interaction at 293 K: ΔH°vH = −65 ± 31 kJ/mol, ΔS° = −91 ± 58
J/mol⋅K (Table 4-8)
Tryptophan protons: H(2) H(4) H(5) H(6) H(7)
Model system
Trp(N-aa)-m
ΔH°vH (kJ/mol)
7 GMP
Trp(pept)-m
-26.6 ± 1.8 -25.87 ± 0.78 -26.3 ± 1.4 -25.7 ± 1.1 -26.44 ± 0.79
7 GpppG n. d. a
-29.6 ± 3.2 -33.8 ± 4.1 -31.8 ± 4.6 -35.1 ± 4.1
ΔS° (J/mol⋅K)
Trp(N-aa)-m 7 GMP -65.8 ± 4.6 -64.0 ± 1.9 -65.1 ± 3.4 -62.4 ± 2.7 -64.1 ± 2.0
Trp(pept)-m 7 GpppG n. d. a
-71.3 ± 8.6 -81 ± 12 -75 ± 13 -89 ± 12
a
not determined due to H(2) and H(6) signal overlapping at higher temperatures
The ΔH°vH values for stacking of the 7-methylG moiety with tryptophan are ~2-fold
greater than those reported for adenine and uracil base stacking (−12.6 ÷ −14.2 kJ/mol per
stack), 252,282,283 and for intramolecular self-stacking of the dinucleotide cap analogues 198
(1ΔH°, Table 4-11). The enthalpy and entropy changes of the m 7 G-Trp stacking seem to
provide a significant contribution to the overall thermodynamic parameters of cap binding
to eIF4E upon translation initiation. The 7-methylG moiety represents a unique example of
a cation which is concurrently a heteroaromatic ring. The large, negative values of both
ΔH°vH and ΔS° for stacking of 7-methylG with the protein tryptophan or with the second
base within the dinucleotide cap should be attributed to the dominating Coulombic
character of the cation - π interactions. 129,148,149,279,280
Figure 4-40. Sandwich stacking of 7-methylG moiety in between two absolutely conserved
tryptophans in the mRNA 5’ cap-binding centres of murine 75 (left) and yeast 76 (right) translation
initiation factor eIF4E. The structure of the human complex 48 is identical to the murine one.
126

The results obtained for the model systems point to a diversity of permissible spatial
structures of the stacked complexes. Similar diversity is observed among the sandwich
configurations in the eIF4E cap-binding site. The structures are becoming more parallel in
the course of evolution, from primitive eukaryotes like yeast 76 to higher ones, like mouse 75
or human 48 (Fig. 4-40).
4.4.9.4. Coupling of Other Intermolecular Equilibria to eIF4E – Cap Binding
The possible origin of the positive heat capacity changes can be also connected to
coupled equilibrium transitions. If coupled equilibria are occurring and/or significant
concentrations of intermediate complexes are present at equilibrium, then measured
association constants may depend on the total concentration of protein and/or ligand. 118,284
The question arises why the Kas values for eIF4E – cap interactions do not depend on the
concentration range (see 4.1.2., p. 51).
The processes that are coupled to the eIF4E – cap binding take place with
participation of environment components. The partial protonation is immediately
equilibrated by buffer ionization (Hepes at 50 mM), and the molar concentration of water
(~ 50 M) and potassium cations (0.1 M) is much greater than those of the protein and the
ligand (nM to μM range). Self-stacking of the dinucleotide cap analogues is weak and the
conformational change is fast.
The intermediate complex life-time can be calculated from the kinetic data obtained
by the stopped-flow method. 4 The encounter rate constant for the association of eIF4E with
m 7 GpppG was k+1 = 1.59 ± 0.07 ⋅ 10 8 M -1 ⋅s -1 (at 150 mM KCl), the rate for dissociation of
the encounter complex was k−1 = 50 ± 8 s -1 , the rate for internal rearrangement to the final
conformation was k+2 = 99 s -1 , and the rate for the reverse process was k−2 = 6 s -1 . Hence,
at typical protein concentration range that was applied for the Kas determination ([Pact]
from 0.01 μM to 1 μM) and at the saturating concentration of the ligand, the equilibrium of
the second step is much more shifted toward the final product than the equilibrium of the
first step that leads to the intermediate state:
k + 2 k + 1 ⋅[
Pact
]
>>
k k
−2
−1
Additionally, the cap analogue concentration is far from the saturation state during the
whole course of the titration, so the inequality is still stronger. Therefore, the only
significant rate-limiting step during the overall association is the diffusionally and
127

electrostatically driven first encounter of eIF4E and cap, and the observed Kas does not
depend on the protein concentration.
Having removed the doubts regarding the lack of influence of the coupled equilibria
on the values of the association constants one can consider putative contributions to
from the thermodynamic coupling.
128
o
ΔCp
1. Self stacking of dinucleotide cap analogues is mandatory coupled to eIF4E
association with cap, and yields a negligible negative contribution to the observed
o
p
Δ C (see 4.4.4., p. 110).
2. Partial protonation of the N(1) position of the cap analogue, the potassium cation
release from the phosphate chain, and uptake of ~ 65 water that accompanies
conformational changes of the protein upon cap binding (see 4.2.3.1., p. 74)
represent the case of non-mandatory coupling, and hence can provide positive
o
p
contributions to the overall Δ C .
In summary, the following effects can together yield the positive
several kJ/mol⋅K: 131
• the hydration of the hydrophobic dorsal surface (up to ~ +1 kJ/mol⋅K),
o
p
Δ C at the level of
• the dehydration of the ligand and protein ionized groups in the binding site (up to ~ 0.4
kJ/mol⋅K for cation-π stacking, and up to ~ 0.5 kJ/mol⋅K for dehydration of the other
charged and uncharged polar surfaces),
• and the thermodynamic coupling with accompanying equilibrium transitions
A kind of difficulty in interpretation is related to the lack of the apo-eIF4E structure,
which is necessary for the complete analysis.
4.4.10. Linear Correlation between ΔCp° and ΔG°
The values of
o
Δ C p appear to correlate linearly with the intrinsic free energy of
the binding (ΔG°0): the stronger the binding the less positive
o
Δ C p (Fig. 4-41). The
apparent correlation can be fortuitous but it can also consist in any causality, as the
correlation coefficient (r 2 ) equals 0.91. The cap analogues of the highest binding affinity,
e.g. 7-methylGTP, do not reveal any observable curvature of the van't Hoff plot.
Systematic ΔC°p dependence on the cap affinity for eIF4E suggests that the common
positive contribution can be compensated to different extent by the negative contribution

elated to the binding specificity. Most probably, this can result from tightening of the
protein global fold upon binding, that yields the negative contribution to
129
o
p
Δ C , similarly as
in the case of the specific antigen-antibody association. 131,243 This hypothesis is supported
by the presence of the non-trivial enthalpy-entropy compensation discussed above. It was
shown that the preferential ligand binding to the lower microstates of a protein led to a
shift in the distribution of the microstates, and, consequently, reduced width of the
distribution implied a decrease in heat capacity upon ligand binding. 122 Thus, for the most
specific cap analogues the negative stabilization effect related to the specificity of the
binding can make the
experimental data. 248
ΔCp°° (kJ/mol⋅K)
6
3
0
-3
-6
-9
o
p
Δ C value too small to be discerned within the noise of the
-70 -60 -50 -40 -30 -20 -10 0 10
ΔG° (kJ/mol)
eIF4E + mRNA 5' cap analogues
DNA + intercalators
L-isoleucine tRNA ligase + substrates
serine proteases + Na +
protein + DNA (specific)
protein + DNA (nonspecific)
Figure 4-41. Correlation of heat capacity changes (ΔC°p) with standard molar binding free energies
(ΔG°) for different intermolecular interactions.
Linear ΔG° - ΔCp° correlation is rarely found, since it requires systematic studies on
a consistent ligand series, while the suitable data obtained by time- and protein-consuming
thermodynamic measurements are rather scattered. Several available examples are
collected in Fig. 4-41. A general tendency is common but only two enthalpy driven
processes involving charged ligands, i. e. specific sodium cation binding by serine
proteases 285 and the eIF4E – cap binding yield remarkable slopes (0.93 ± 0.14 K -1 , and
0.275 ± 0.044 K -1 , respectively). Binding of the L-isoleucine tRNA ligase to L-isoleucine
and L-valine is not accompanied by any conformational changes of the large (120 kD),
multidomain protein, which is known from the crystal structures of the native enzyme and

its complexes. 286 The heat capacity changes for binding of various substrates to the enzyme
are approximately constant, though the range of the corresponding free energy changes
encompasses almost 20 kJ/mol. 287 Drug - DNA intercalation is related partially to the
binding-induced changes in non-polar and polar solvent-accessible surface areas, and
consequently the binding free energy is proportional to the hydrophobic effect (slight
dependence). 120,121,288 Specific interactions of DNA with repressors and RNA polymerase,
driven by hydrophobicity of the specific sites, are characterized by the wide-spread protein
conformational changes and large negative heat capacity changes, while non-specific
interactions, driven only by the counterion release, are accomplished with
117,196,289-293
130
o
Δ C p = 0. 112-
However, no systematic literature data for the ΔG°-dependent positive heat capacity
changes are available. Searching for origins of the positive
o
Δ C p and for the causal
explanation of the apparent linear relationship in terms of statistical physics will be the
subject of further investigations.
4.4.11. Discussion with Other Authors
The results described herein are contradictory to the first conclusions drawn from
studies of 7-methylGTP and 7-methylGpppG binding to eIF4E from human
erythrocytes 156 . Although the human 49 and murine 50 proteins are almost identical (98%
homology for the full length proteins, and 100% for truncated (28-217) eIF4E), those
results suggested that the eIF4E − cap binding was entropy-driven and enthalpy-opposed,
with the constant thermodynamic parameters in the 278-308 K temperature range (ΔS° =
+219 ± 11 J⋅mol -1 K -1 , ΔH° = +33.9 ± 1.7 kJ⋅mol -1 for 7-methylGpppG 156 ). However, the
human protein was purified by means of the cap-affinity chromatography, what could
result in up to 60% of the unremovable cap analogue bound to eIF4E. 1,208 This purification
method yielded the apparent association constants up to 300-fold lower 5 then those
measured for eIF4E purified without contact with cap 1 (see Table 4-2, p. 61). Moreover,
neither the decreasing temperature-dependence of the human eIF4E activity nor
fluorescence of the cap analogues were taken into account. The authors also reported the
fluorescence intensity of eIF4E and the analogues as invariant over the temperature range
studied, which is impossible. Taken together, these main reasons could lead to
misinterpretation of the experimental data.

The same reservation but even to much greater extent concerns the work of Shen et
al. 294 Apparently higher association constants for dinucleotides than that for 7-methylGTP
results simply from the inner filter effect, as no corrections were applied in spite of the
huge concentration of eIF4E (10 μM) and the cap (22 μM). Thermodynamic parameters
derived in such manner are fortuitous and do not allow a reasonable analysis.
Recently, the human eIF4E − 7-methylGpppG binding affinity was fluorometrically
studied in the context of cell growth suppression, and the association constant of 0.83 ±
0.14 μM -1 was obtained with use of some corrections (at 296.2 K, pH 7.5, 300 mM
KCl). 295 The corresponding Kas value reported herein for truncated murine eIF4E (28-217)
is 5.13 ± 0.81 μM -1 (at 297.6 K, pH 7.2, 100 mM KCl, Table 4-7, p. 101). These results are
in an excellent agreement, since elevation of KCl concentration from 100 to 300 mM
causes a significant, ~ 6-fold decrease of the association constant due to screening of the
electrostatic attraction between the basic amino acids in the cap-binding site of eIF4E and
the phosphate chain of 7-methylGpppG (see 4.2.3.1., p. 74).
4.4.12. Conclusions
The positive heat capacity change at constant pressure appears to be a characteristic
feature of eIF4E binding to mRNA 5' cap. The chemical cap analogues of the highest
specificity exhibit the heat capacity change proportionally shifted toward less positive or
undetectable values. Data obtained from two independent methods, fluorescence
quenching and isothermal titration calorimetry provided excellently accordant
thermodynamic parameters. The microcalorimetric results additionally yielded a
quantitative confirmation of partial protonation of the ligand, which is necessary to form
one of the crucial hydrogen bonds in the protein binding centre.
Isothermal enthalpy-entropy compensation among the cap analogues of different
specificity for eIF4E at 293 K points to several thermodynamic features: the dominating
enthalpic character for the whole congener series, a great instability of the apo-form of
eIF4E, and to conformational rearrangement of the whole protein upon the complex
formation, leading to the final, stable, cap-bound state.
The exceptionally large positive
131
o Δ Cp
of the binding can be attributed to
simultaneous interplay of various intermolecular processes: the extensive additional
hydration of the eIF4E hydrophobic dorsal surface, dehydration of the ionized groups,

including the 7-methylguanosine cation, and the burial of polar groups of the interacting
molecules, as well as to the thermodynamically coupled differential ligand protonation and
the potassium cation release from the ligand. The negative contribution cancelling the
positive one for the most tightly binding analogues can arise from the global protein
conformational change that stiffen the complex structure. The induced shift in the self-
stacking equilibrium of the dinucleotide cap analogues gives a negligible negative
o
p
contribution to the overall Δ C .
Because of the non-zero heat capacity changes, the interactions of eIF4E with natural
dinucleotide cap analogues are both enthalpy- and entropy-driven over the range of
biological temperatures, and stability of the eIF4E − cap complex is almost temperature-
independent.
The general description of the cap – eIF4E association is intricate due to strict
synergy of charge-related and hydrophobic interactions within the binding site and on the
solvent accessible molecular surface of the complex. These properties make this molecular
system unique in the thermodynamic sense.
132

5. Summary
The thesis was aimed at revealing the molecular mechanism of recognition of mRNA
5' cap structure by eIF4E, previously limited to a static view from the crystal structures.
Chemically modified cap-analogues proved to be a valuable tool to probe contribution of
various types of molecular interaction to the eIF4E-cap complex stability via emission
spectroscopy measurements. A fast and accurate method of synchronized fluorescence
titration has been designed to obtain reliable equilibrium association constant values,
which are not only relative to one another, but for the first time have an absolute meaning,
and thus can be interpreted in terms of the free energy of binding. The mechanism of the
complex formation at physiological pH can be now presented as an interplay of several
intermolecular processes (Scheme 5-1). Recognition of the 5' cap structure by eIF4E
binding site begins with electrostatic attraction of the cap phosphate chain, from which one
potassium cation is being removed. In the initial encounter complex ([eIF4E•cap]*) the
cap is first anchored by the phosphate chain and next, cooperative cation - π sandwich
stacking together with hydrogen bonding of 7-methylguanine occurs. Differential
protonation at the N(1) position of 7-methylguanine is necessary to enhance stacking,
which in turn conditions formation of three hydrogen bonds. The second step of
association is accompanied by preferential hydration involving ~ 65 water molecules.
Some of them are trapped inside the cap-binding slot, while several tens supply the first-
layer hydration shell of the complex. This extensive water reorganization implies a
significant conformational change of the whole protein. The latter conclusion comes
independently of several other sets of experimental results: isothermal enthalpy-entropy
compensation, deaggregation of eIF4E forced by association with cap, cooperativity of
cap-binding and eIF4G/4E-BP1-binding sites.
- 1⋅ K + + 0.5 ⋅ H +
eIF4E + cap [eIF4E cap]* eIF4E cap
133
+ 65 ⋅ H2O
Scheme 5-1. Proposed model of two-step association of eIF4E with the mRNA 5' cap

The appropriate equilibrium equation for eIF4E-cap binding has the following form:
K
t
[ eIF4E
• cap][
K ]
=
65 +
[ eIF4E]
[ cap]
[ H O]
[ H ]
0
0
2
+
0.
5
(without terms for buffer; index "0" denotes equilibrium concentrations of free species).
Comparison of the eIF4E-binding affinity of natural vs structurally modified cap
analogues together with the structures of the eIF4E-cap complexes makes a foundation to
rational design of new cap analogues with better inhibitory properties than those
synthesized hitherto. Quantitative analysis of the binding free energy shows that the cap
triphosphate chain contributes in one half to it. Since the negative charge at the cap
phosphate chain is so crucial for the efficient binding to eIF4E, it should be indispensably
included in the designed agents. Hence, thinking about inhibition of the eIF4E cellular
activity by 5' cap analogues must involve an active transport of the charged species through
the cellular bilayer phospholipid membrane. One hopeful analogue is the p-Ch-bz7GTP,
which strongly binds to eIF4E with the affinity of 10 7 – 10 8 M -1 . The equilibrium binding
constant is relatively weakly temperature-dependent, since the association is both enthalpy-
and entropy-driven. This artificial cap analogue should not be easily metabolized or
trapped by other proteins, and thus it can be expected that its half lifetime could be long
enough to render significant translation inhibition. This hope is supported by the
observation that the overall inhibition constant (KI) of p-Ch-bz7GTP determined from
biological in vitro assay in a rabbit reticulocyte lizate 68 is shifted toward lower values in
comparison with that resulting from the average correlation for all cap analogues (Fig. 4-
11, p. 63).
The thermodynamic studies revealed unique, large and positive heat capacity
changes that accompany the eIF4E – cap interactions. The binding of natural dinucleotide
cap analogues are both enthalpy- and entropy-driven over the range of biological
temperatures, and stability of the eIF4E − cap complex is almost temperature-independent.
These findings are suitable for further development of quantitative interpretation of
intermolecular recognition specificity, and are also a kind of challenge for theoretical
interpretation.
The author hopes that the presented thesis contribute to more profound insights into
how eIF4E interacts with other components of the cytoplasmic machinery responsible for
cap-dependent translation initiation.
134

6. References
1. Niedzwiecka,A., J.Marcotrigiano, J.Stepinski, M.Jankowska-Anyszka, A.Wyslouch-Cieszynska, M.Dadlez,
A.C.Gingras, P.Mak, E.Darzynkiewicz, N.Sonenberg, S.K.Burley, and R.Stolarski. (2002). Biophysical
studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G
and 4E-BP1 proteins. J. Mol. Biol., 319, 615-635.
2. Niedzwiecka,A., J.Stepinski, E.Darzynkiewicz, N.Sonenberg, and R.Stolarski. (2002). Positive heat capacity
change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap. Biochemistry, 41, 12140-
12148.
3. Gingras,A.C., B.Raught, S.P.Gygi, A.Niedzwiecka, M.Miron, S.K.Burley, R.D.Polakiewicz, A.Wyslouch-
Cieszynska, R.Aebersold, and N.Sonenberg. (2001). Hierarchical phosphorylation of the translation inhibitor
4E-BP1. Genes Dev., 15, 2852-2864.
4. Blachut-Okrasinska,E., E.Bojarska, A.Niedzwiecka, L.Chlebicka, E.Darzynkiewicz, R.Stolarski, J.Stepinski, and
J.M.Antosiewicz. (2000). Stopped-flow and Brownian dynamics studies of electrostatic effects in the kinetics
of binding of 7-methyl-GpppG to the protein eIF4E. Eur. Biophys. J., 29, 487-498.
5. Wieczorek,Z., A.Niedzwiecka-Kornas, L.Chlebicka, M.Jankowska, K.Kiraga, J.Stepinski, M.Dadlez, R.Drabent,
E.Darzynkiewicz, and R.Stolarski. (1999). Fluorescence studies on association of human translation initiation
factor eIF4E with mRNA cap-analogues. Z. Naturforsch. [C. ], 54, 278-284.
6. Niedzwiecka-Kornas,A., L.Chlebicka, J.Stepinski, M.Jankowska-Anyszka, Z.Wieczorek, E.Darzynkiewicz,
R.E.Rhoads, and R.Stolarski. (1999). Spectroscopic studies on association of mRNA cap-analogues with
human translation factor eIF4E. From modelling of interactions to inhibitory properties. Collect. Symp. Ser.,
2, 214-218.
7. Niedzwiecka-Kornas,A., R.Przedmojski, L.Balaspiri, Z.Wieczorek, J.Stepinski, M.Jankowska, H.Lonnberg,
E.Darzynkiewicz, and R.Stolarski. (1999). Studies on association of mRNA cap-analogues with a synthetic
dodecapeptide DGIEPMWEDEKN. Nucleosides & Nucleotides, 18, 1105-1106.
8. Crick,F. (1970). Central dogma of molecular biology. Nature, 227, 561-563.
9. Raven, P. H. and Johnson, G. B. (1996). Biology. WCB Publishers.
10. Proudfoot,N.J., A.Furger, and M.J.Dye. (2002). Integrating mRNA processing with transcription. Cell, 108, 501-
512.
11. Creighton, T. E. (1993). Proteins: structures and molecular properties. W. H. Freeman and Co., New York.
12. Sonenberg,N., M.A.Morgan, W.C.Merrick, and A.J.Shatkin. (1978). A polypeptide in eukaryotic initiation factors
that crosslinks specifically to the 5'-terminal cap in mRNA. Proc. Natl. Acad. Sci. U. S. A, 75, 4843-4847.
13. Gingras,A.C., B.Raught, and N.Sonenberg. (1999). eIF4 initiation factors: effectors of mRNA recruitment to
ribosomes and regulators of translation. Annu. Rev. Biochem., 68, 913-963.
14. Muthukrishnan,S., G.W.Both, Y.Furuichi, and A.J.Shatkin. (1975). 5'-Terminal 7-methylguanosine in eukaryotic
mRNA is required for translation. Nature, 255, 33-37.
15. Hershey, J. W. and Merrick, W. C. (2000). The pathway and mechanism of initiation of protein synthesis.
Sonenberg, N, Hershey, J. W., and Mathews, M. B. [39], 33-88, CSHL Press, Cold Spring Harbor, NY.
Translational control of gene expression.
16. Konarska,M.M., R.A.Padgett, and P.A.Sharp. (1984). Recognition of cap structure in splicing in vitro of mRNA
precursors. Cell, 38, 731-736.
17. Izaurralde,E., J.Lewis, C.McGuigan, M.Jankowska, E.Darzynkiewicz, and I.W.Mattaj. (1994). A nuclear cap
binding protein complex involved in pre-mRNA splicing. Cell, 78, 657-668.
135

18. Izaurralde,E., J.Lewis, C.Gamberi, A.Jarmolowski, C.McGuigan, and I.W.Mattaj. (1995). A cap-binding protein
complex mediating U snRNA export. Nature, 376, 709-712.
19. Izaurralde,E., J.Stepinski, E.Darzynkiewicz, and I.W.Mattaj. (1992). A cap binding protein that may mediate
nuclear export of RNA polymerase II-transcribed RNAs. J. Cell Biol., 118, 1287-1295.
20. Lewis,J.D. and E.Izaurralde. (1997). The role of the cap structure in RNA processing and nuclear export. Eur. J.
Biochem., 247, 461-469.
21. Sonenberg,N. (1988). Cap-binding proteins of eukaryotic messenger RNA: functions in initiation and control of
translation. Prog. Nucleic Acid Res. Mol. Biol., 35, 173-207.
22. Mattaj,I.W. (1986). Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding.
Cell, 46, 905-911.
23. Hamm,J., E.Darzynkiewicz, S.M.Tahara, and I.W.Mattaj. (1990). The trimethylguanosine cap structure of U1
snRNA is a component of a bipartite nuclear targeting signal. Cell, 62, 569-577.
24. Gorlich,D. and I.W.Mattaj. (1996). Nucleocytoplasmic transport. Science, 271, 1513-1518.
25. Sharp,P.A. (1994). Split genes and RNA splicing. Cell, 77, 805-815.
26. Eschenfeldt,W.H., B.G.Cohen, and R.E.Rhoads. (1983). Structure of the 5' terminus of hen oviduct lysozyme
messenger ribonucleic acid. J. Biol. Chem., 258, 13076-13081.
27. Maroney,P.A., J.A.Denker, E.Darzynkiewicz, R.Laneve, and T.W.Nilsen. (1995). Most mRNAs in the nematode
Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency. RNA., 1,
714-723.
28. Vandenberghe,A.E., T.H.Meedel, and K.E.Hastings. (2001). mRNA 5'-leader trans-splicing in the chordates.
Genes Dev., 15, 294-303.
29. Van Doren,K. and D.Hirsh. (1990). mRNAs that mature through trans-splicing in Caenorhabditis elegans have a
trimethylguanosine cap at their 5' termini. Mol. Cell Biol., 10, 1769-1772.
30. Sachs,A.B., P.Sarnow, and M.W.Hentze. (1997). Starting at the beginning, middle, and end: translation initiation
in eukaryotes. Cell, 89, 831-838.
31. Sonenberg,N. (1996). mRNA 5' cap-binding protein eIF4E and control of cell growth. In Translational Control
(J.W.B.Hershey, M.B.Mathews, and N.Sonenberg, editors), pp.245-69, Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York.
32. Pause,A., N.Methot, Y.Svitkin, W.C.Merrick, and N.Sonenberg. (1994). Dominant negative mutants of
mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and capindependent
initiation of translation. EMBO J., 13, 1205-1215.
33. Hentze,M.W. (1997). eIF4G: a multipurpose ribosome adapter? Science, 275, 500-501.
34. Imataka,H., A.Gradi, and N.Sonenberg. (1998). A newly identified N-terminal amino acid sequence of human
eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation. EMBO J., 17, 7480-
7489.
35. Gradi,A., H.Imataka, Y.V.Svitkin, E.Rom, B.Raught, S.Morino, and N.Sonenberg. (1998). A novel functional
human eukaryotic translation initiation factor 4G. Mol. Cell Biol., 18, 334-342.
36. Wei,C.C., M.L.Balasta, J.Ren, and D.J.Goss. (1998). Wheat germ poly(A) binding protein enhances the binding
affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogues. Biochemistry, 37, 1910-1916.
37. Lejbkowicz,F., C.Goyer, A.Darveau, S.Neron, R.Lemieux, and N.Sonenberg. (1992). A fraction of the mRNA 5'
cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus. Proc. Natl. Acad. Sci. U. S. A, 89,
9612-9616.
136

38. Strudwick,S. and K.L.Borden. (2002). The emerging roles of translation factor eIF4E in the nucleus.
Differentiation, 70, 10-22.
39. Dostie,J., F.Lejbkowicz, and N.Sonenberg. (2000). Nuclear eukaryotic initiation factor 4E (eIF4E) colocalizes
with splicing factors in speckles. J. Cell Biol., 148, 239-247.
40. Dostie,J., M.Ferraiuolo, A.Pause, S.A.Adam, and N.Sonenberg. (2000). A novel shuttling protein, 4E-T, mediates
the nuclear import of the mRNA 5' cap-binding protein, eIF4E. EMBO J., 19, 3142-3156.
41. Duncan,R., S.C.Milburn, and J.W.Hershey. (1987). Regulated phosphorylation and low abundance of HeLa cell
initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. J. Biol. Chem.,
262, 380-388.
42. Raught,B. and A.C.Gingras. (1999). eIF4E activity is regulated at multiple levels. Int. J. Biochem. Cell Biol., 31,
43-57.
43. Jones,R.M., J.Branda, K.A.Johnston, M.Polymenis, M.Gadd, A.Rustgi, L.Callanan, and E.V.Schmidt. (1996). An
essential E box in the promoter of the gene encoding the mRNA cap- binding protein (eukaryotic initiation
factor 4E) is a target for activation by c-myc. Mol. Cell Biol., 16, 4754-4764.
44. Guruprasad,K., B.V.Reddy, and M.W.Pandit. (1990). Correlation between stability of a protein and its dipeptide
composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein
Eng, 4, 155-161.
45. Altmann,M., N.Schmitz, C.Berset, and H.Trachsel. (1997). A novel inhibitor of cap-dependent translation
initiation in yeast: p20 competes with eIF4G for binding to eIF4E. EMBO J., 16, 1114-1121.
46. Kabsch,W. and C.Sander. (1983). Dictionary of protein secondary structure: pattern recognition of hydrogenbonded
and geometrical features. Biopolymers, 22, 2577-2637.
47. Rost,B. and C.Sander. (1994). Conservation and prediction of solvent accessibility in protein families. Proteins,
20, 216-226.
48. Tomoo,K., X.Shen, K.Okabe, Y.Nozoe, S.Fukuhara, S.Morino, T.Ishida, T.Taniguchi, H.Hasegawa,
A.Terashima, M.Sasaki, Y.Katsuya, K.Kitamura, H.Miyoshi, M.Ishikawa, and K.Miura. (2002). Crystal
structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-
5',5'-triphosphate (m(7)GpppA)- bound human full-length eukaryotic initiation factor 4E: biological
importance of the C-terminal flexible region. Biochem. J., 362, 539-544.
49. Rychlik,W., L.L.Domier, P.R.Gardner, G.M.Hellmann, and R.E.Rhoads. (1987). Amino acid sequence of the
mRNA cap-binding protein from human tissues. Proc. Natl. Acad. Sci. U. S. A, 84, 945-949.
50. Altmann,M., P.P.Muller, J.Pelletier, N.Sonenberg, and H.Trachsel. (1989). A mammalian translation initiation
factor can substitute for its yeast homologue in vivo. J. Biol. Chem., 264, 12145-12147.
51. Rychlik,W. and R.E.Rhoads. (1992). Nucleotide sequence of rabbit eIF-4E cDNA. Nucleic Acids Res., 20, 6415.
52. Wakiyama,M., M.Saigoh, K.Shiokawa, and K.Miura. (1995). mRNA encoding the translation initiation factor
eIF-4E is expressed early in Xenopus embryogenesis. FEBS Lett., 360, 191-193.
53. Dyer,J.R., A.M.Pepio, S.K.Yanow, and W.S.Sossin. (1998). Phosphorylation of eIF4E at a conserved serine in
Aplysia. J. Biol. Chem., 273, 29469-29474.
54. Hernandez,G. and J.M.Sierra. (1995). Translation initiation factor eIF-4E from Drosophila: cDNA sequence and
expression of the gene. Biochim. Biophys. Acta, 1261, 427-431.
55. Jankowska-Anyszka,M., B.J.Lamphear, E.J.Aamodt, T.Harrington, E.Darzynkiewicz, R.Stolarski, and
R.E.Rhoads. (1998). Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis
elegans can distinguish between mono- and trimethylated mRNA cap structures. J. Biol. Chem., 273, 10538-
10542.
56. Manjunath,S., A.J.Williams, and J.Bailey-Serres. (1999). Oxygen deprivation stimulates Ca2+-mediated
phosphorylation of mRNA cap- binding protein eIF4E in maize roots. Plant J., 19, 21-30.
137

57. Aliyeva,E., A.M.Metz, and K.S.Browning. (1996). Sequences of two expressed sequence tags (EST) from rice
encoding different cap-binding proteins. Gene, 180, 221-223.
58. Metz,A.M., R.T.Timmer, and K.S.Browning. (1992). Isolation and sequence of a cDNA encoding the cap binding
protein of wheat eukaryotic protein synthesis initiation factor 4F. Nucleic Acids Res., 20, 4096.
59. Rodriguez,C.M., M.A.Freire, C.Camilleri, and C.Robaglia. (1998). The Arabidopsis thaliana cDNAs coding for
eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially
expressed during plant development. Plant J., 13, 465-473.
60. Ono N. and Sudoh M. (2003). Candida glabrata eIF4E gene. EMBL/GenBank/DDBJ databases,
61. Altmann,M., C.Handschin, and H.Trachsel. (1987). mRNA cap-binding protein: cloning of the gene encoding
protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae. Mol. Cell Biol., 7, 998-1003.
62. Ptushkina,M., I.Fierro-Monti, J.van den Heuvel, S.Vasilescu, R.Birkenhager, K.Mita, and J.E.McCarthy. (1996).
Schizosaccharomyces pombe has a novel eukaryotic initiation factor 4F complex containing a cap-binding
protein with the human eIF4E C- terminal motif KSGST. J. Biol. Chem., 271, 32818-32824.
63. Thompson,J.D., D.G.Higgins, and T.J.Gibson. (1994). CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res., 22, 4673-4680.
64. Risler,J.L., M.O.Delorme, H.Delacroix, and A.Henaut. (1988). Amino acid substitutions in structurally related
proteins. A pattern recognition approach. Determination of a new and efficient scoring matrix. J. Mol. Biol.,
204, 1019-1029.
65. Joshi,B., A.L.Cai, B.D.Keiper, W.B.Minich, R.Mendez, C.M.Beach, J.Stepinski, R.Stolarski, E.Darzynkiewicz,
and R.E.Rhoads. (1995). Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209. J.
Biol. Chem., 270, 14597-14603.
66. Miyamoto,S., S.R.Kimball, and B.Safer. (2000). Signal transduction pathways that contribute to increased protein
synthesis during T-cell activation. Biochim. Biophys. Acta, 1494, 28-42.
67. Pause,A., G.J.Belsham, A.C.Gingras, O.Donze, T.A.Lin, J.C.Lawrence, Jr., and N.Sonenberg. (1994). Insulindependent
stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature, 371,
762-767.
68. Whalen,S.G., A.C.Gingras, L.Amankwa, S.Mader, P.E.Branton, R.Aebersold, and N.Sonenberg. (1996).
Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4Ebinding
proteins. J. Biol. Chem., 271, 11831-11837.
69. Duncan,R.F., H.Peterson, C.H.Hagedorn, and A.Sevanian. (2003). Oxidative stress increases eukaryotic initiation
factor 4E phosphorylation in vascular cells. Biochem. J., 369, 213-225.
70. Minich,W.B., M.L.Balasta, D.J.Goss, and R.E.Rhoads. (1994). Chromatographic resolution of in vivo
phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity
of the phosphorylated form. Proc. Natl. Acad. Sci. U. S. A, 91, 7668-7672.
71. Morley,S.J. and S.Naegele. (2002). Phosphorylation of eukaryotic initiation factor (eIF) 4E is not required for de
novo protein synthesis following recovery from hypertonic stress in human kidney cells. J. Biol. Chem., 277,
32855-32859.
72. Scheper,G.C. and C.G.Proud. (2002). Does phosphorylation of the cap-binding protein eIF4E play a role in
translation initiation? Eur. J. Biochem., 269, 5350-5359.
73. Scheper,G.C., B.van Kollenburg, J.Hu, Y.Luo, D.J.Goss, and C.G.Proud. (2002). Phosphorylation of Eukaryotic
Initiation Factor 4E Markedly Reduces Its Affinity for Capped mRNA. J. Biol. Chem., 277, 3303-3309.
74. Zuberek,J., A.Wyslouch-Cieszynska, A.Niedzwiecka, M.Dadlez, J.Stepinski, W.Augustyniak, A.C.Gingras,
Z.Zhang, S.K.Burley, N.Sonenberg, R.Stolarski, and E.Darzynkiewicz. (2003). Phosphorylation of eIF4E
attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: Intein-mediated protein
ligation strategy to obtain phosphorylated protein. RNA, 9, 52-61.
138

75. Marcotrigiano,J., A.C.Gingras, N.Sonenberg, and S.K.Burley. (1997). Cocrystal structure of the messenger RNA
5' cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell, 89, 951-961.
76. Matsuo,H., H.Li, A.M.McGuire, C.M.Fletcher, A.C.Gingras, N.Sonenberg, and G.Wagner. (1997). Structure of
translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nat. Struct. Biol., 4, 717-
724.
77. Mathews, M. B., Sonenberg, N, and Hershey, J. W. (2000). Origins and principles of translational control.
Sonenberg, N, Hershey, J. W., and Mathews, M. B. [39], 1-31, CSHL Press, Cold Spring Harbor, NY.
Translational control of gene expression.
78. Tang,S.J., G.Reis, H.Kang, A.C.Gingras, N.Sonenberg, and E.M.Schuman. (2002). A rapamycin-sensitive
signaling pathway contributes to long-term synaptic plasticity in the hippocampus. Proc. Natl. Acad. Sci. U. S.
A, 99, 467-472.
79. Martin,K.C., A.Casadio, H.Zhu, E.Yaping, J.C.Rose, M.Chen, C.H.Bailey, and E.R.Kandel. (1997). Synapsespecific,
long-term facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in
memory storage. Cell, 91, 927-938.
80. Casadio,A., K.C.Martin, M.Giustetto, H.Zhu, M.Chen, D.Bartsch, C.H.Bailey, and E.R.Kandel. (1999). A
transient, neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by
local protein synthesis. Cell, 99 , 221-237.
81. Vries,R.G., A.Flynn, J.C.Patel, X.Wang, R.M.Denton, and C.G.Proud. (1997). Heat shock increases the
association of binding protein-1 with initiation factor 4E. J. Biol. Chem., 272, 32779-32784.
82. Gingras,A.C., Y.Svitkin, G.J.Belsham, A.Pause, and N.Sonenberg. (1996). Activation of the translational
suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus. Proc. Natl. Acad. Sci.
U. S. A, 93, 5578-5583.
83. Ohlmann,T., D.Prevot, D.Decimo, F.Roux, J.Garin, S.J.Morley, and J.L.Darlix. (2002). In vitro cleavage of
eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate
system. J. Mol. Biol., 318, 9-20.
84. De Benedetti,A. and A.L.Harris. (1999). eIF4E expression in tumors: its possible role in progression of
malignancies. Int. J. Biochem. Cell Biol., 31, 59-72.
85. Sorrells,D.L., G.E.Ghali, C.Meschonat, R.J.DeFatta, D.Black, L.Liu, A.De Benedetti, C.A.Nathan, and B.D.Li.
(1999). Competitive PCR to detect eIF4E gene amplification in head and neck cancer. Head Neck, 21, 60-65.
86. DeFatta,R.J., E.A.Turbat-Herrera, B.D.Li, W.Anderson, and A.De Benedetti. (1999). Elevated expression of
eIF4E in confined early breast cancer lesions: possible role of hypoxia. Int. J. Cancer, 80, 516-522.
87. Hershey, J. W. and Miyamoto, S. (2000). Translational control and cancer. Sonenberg, N, Hershey, J. W., and
Mathews, M. B. [39], 637-654, CSHL Press, Cold Spring Harbor, NY. Translational control of gene
expression.
88. Haghighat,A., S.Mader, A.Pause, and N.Sonenberg. (1995). Repression of cap-dependent translation by 4Ebinding
protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J., 14, 5701-
5709.
89. Mader,S., H.Lee, A.Pause, and N.Sonenberg. (1995). The translation initiation factor eIF-4E binds to a common
motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol.
Cell Biol., 15, 4990-4997.
90. Poulin,F., A.C.Gingras, H.Olsen, S.Chevalier, and N.Sonenberg. (1998). 4E-BP3, a new member of the
eukaryotic initiation factor 4E-binding protein family. J. Biol. Chem., 273, 14002-14007.
91. Marcotrigiano,J., A.C.Gingras, N.Sonenberg, and S.K.Burley. (1999). Cap-dependent translation initiation in
eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell, 3, 707-716.
139

92. Gingras,A.C., S.P.Gygi, B.Raught, R.D.Polakiewicz, R.T.Abraham, M.F.Hoekstra, R.Aebersold, and
N.Sonenberg. (1999). Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev., 13,
1422-1437.
93. Mothe-Satney,I., G.J.Brunn, L.P.McMahon, C.T.Capaldo, R.T.Abraham, and J.C.Lawrence, Jr. (2000).
Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by
phospho-specific antibodies. J. Biol. Chem., 275 , 33836-33843.
94. Karim,M.M., J.M.Hughes, J.Warwicker, G.C.Scheper, C.G.Proud, and J.E.McCarthy. (2001). A quantitative
molecular model for modulation of mammalian translation by the eIF4E-binding protein 1. J. Biol. Chem.,
95. Mothe-Satney,I., D.Yang, P.Fadden, T.A.Haystead, and J.C.Lawrence, Jr. (2000). Multiple mechanisms control
phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression. Mol. Cell Biol., 20, 3558-
3567.
96. Gingras,A.C., B.Raught, and N.Sonenberg. (2001). Regulation of translation initiation by FRAP/mTOR. Genes
Dev., 15, 807-826.
97. Chung,J., R.E.Bachelder, E.A.Lipscomb, L.M.Shaw, and A.M.Mercurio. (2002). Integrin (alpha 6 beta 4)
regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells. J. Cell Biol.,
158, 165-174.
98. Nathan,C.A., L.Liu, B.D.Li, F.W.Abreo, I.Nandy, and A.De Benedetti. (1997). Detection of the proto-oncogene
eIF4E in surgical margins may predict recurrence in head and neck cancer. Oncogene, 15, 579-584.
99. Cohen,N., M.Sharma, A.Kentsis, J.M.Perez, S.Strudwick, and K.L.Borden. (2001). PML RING suppresses
oncogenic transformation by reducing the affinity of eIF4E for mRNA. EMBO J., 20, 4547-4559.
100. Nathan,C.A., P.Carter, L.Liu, B.D.Li, F.Abreo, A.Tudor, S.G.Zimmer, and A.De Benedetti. (1997). Elevated
expression of eIF4E and FGF-2 isoforms during vascularization of breast carcinomas. Oncogene, 15, 1087-
1094.
101. Gradi,A., Y.V.Svitkin, H.Imataka, and N.Sonenberg. (1998). Proteolysis of human eukaryotic translation
initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus
infection. Proc. Natl. Acad. Sci. U. S. A, 95, 11089-11094.
102. Svitkin,Y.V., A.Gradi, H.Imataka, S.Morino, and N.Sonenberg. (1999). Eukaryotic initiation factor 4GII
(eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human
rhinovirus infection. J. Virol., 73, 3467-3472.
103. Morley,S.J., L.McKendrick, and M.Bushell. (1998). Cleavage of translation initiation factor 4G (eIF4G) during
anti-Fas IgM-induced apoptosis does not require signalling through the p38 mitogen-activated protein (MAP)
kinase. FEBS Lett., 438, 41-48.
104. Bushell,M., L.McKendrick, R.U.Janicke, M.J.Clemens, and S.J.Morley. (1999). Caspase-3 is necessary and
sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis. FEBS Lett., 451,
332-336.
105. Thomas,G. and M.N.Hall. (1997). TOR signalling and control of cell growth. Curr. Opin. Cell Biol., 9, 782-787.
106. Huang,S. and P.J.Houghton. (2002). Inhibitors of mammalian target of rapamycin as novel antitumor agents: from
bench to clinic. Curr. Opin. Investig. Drugs, 3, 295-304.
107. Schmelzle,T. and M.N.Hall. (2000). TOR, a central controller of cell growth. Cell, 103, 253-262.
108. Huang,S. and P.J.Houghton. (2001). Resistance to rapamycin: a novel anticancer drug. Cancer Metastasis Rev.,
20, 69-78.
109. Huang,S. and P.J.Houghton. (2001). Mechanisms of resistance to rapamycins. Drug Resist. Updat., 4, 378-391.
110. DeFatta,R.J., Y.Li, and A.De Benedetti. (2002). Selective killing of cancer cells based on translational control of a
suicide gene. Cancer Gene Ther., 9, 573-578.
140

111. Patikoglou,G. and S.K.Burley. (1997). Eukaryotic transcription factor-DNA complexes. Annu. Rev. Biophys.
Biomol. Struct., 26, 289-325.
112. DeHaseth,P.L., T.M.Lohman, and M.T.Record, Jr. (1977). Nonspecific interaction of lac repressor with DNA: an
association reaction driven by counterion release. Biochemistry, 16, 4783-4790.
113. Record,M.T., Jr., P.L.DeHaseth, and T.M.Lohman. (1977). Interpretation of monovalent and divalent cation
effects on the lac repressor-operator interaction. Biochemistry, 16, 4791-4796.
114. Record,M.T., Jr., C.F.Anderson, and T.M.Lohman. (1978). Thermodynamic analysis of ion effects on the binding
and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening,
and ion effects on water activity. Q. Rev. Biophys., 11, 103-178.
115. Lohman,T.M., P.L.DeHaseth, and M.T.Record, Jr. (1978). Analysis of ion concentration effects of the kinetics of
protein- nucleic acid interactions. Application to lac repressor-operator interactions. Biophys. Chem., 8, 281-
294.
116. Roe,J.H. and M.T.Record, Jr. (1985). Regulation of the kinetics of the interaction of Escherichia coli RNA
polymerase with the lambda PR promoter by salt concentration. Biochemistry, 24, 4721-4726.
117. Ha,J.H., M.W.Capp, M.D.Hohenwalter, M.Baskerville, and M.T.Record, Jr. (1992). Thermodynamic
stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac
repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions. J.
Mol. Biol., 228, 252-264.
118. Record,M.T., Jr., J.H.Ha, and M.A.Fisher. (1991). Analysis of equilibrium and kinetic measurements to determine
thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes
between proteins and helical DNA. Methods Enzymol., 208, 291-343.
119. Parsegian,V.A., R.P.Rand, and D.C.Rau. (1995). Macromolecules and water: probing with osmotic stress.
Methods Enzymol., 259, 43-94.
120. Chaires,J.B., S.Satyanarayana, D.Suh, I.Fokt, T.Przewloka, and W.Priebe. (1996). Parsing the free energy of
anthracycline antibiotic binding to DNA. Biochemistry, 35, 2047-2053.
121. Qu,X. and J.B.Chaires. (2001). Hydration Changes for DNA Intercalation Reactions. J. Am. Chem. Soc., 123, 1-7.
122. Eftink,M.R., A.C.Anusiem, and R.L.Biltonen. (1983). Enthalpy-entropy compensation and heat capacity changes
for protein- ligand interactions: general thermodynamic models and data for the binding of nucleotides to
ribonuclease A. Biochemistry, 22, 3884-3896.
123. Luther,M.A., G.Z.Cai, and J.C.Lee. (1986). Thermodynamics of dimer and tetramer formations in rabbit muscle
phosphofructokinase. Biochemistry, 25, 7931-7937.
124. Baker,B.M. and K.P.Murphy. (1996). Evaluation of linked protonation effects in protein binding reactions using
isothermal titration calorimetry. Biophys. J., 71, 2049-2055.
125. Guminski, K. (1974). Termodynamika. PWN, Warszawa.
126. Parsegian,V.A., R.P.Rand, and D.C.Rau. (2000). Osmotic stress, crowding, preferential hydration, and binding: A
comparison of perspectives. Proc. Natl. Acad. Sci. U. S. A, 97, 3987-3992.
127. McNaught, A. D. and Wilkinson, A. (1997). Compendium of Chemical Terminology. Blackwell Science.
128. Bloomfield,V.A. (2001). Thermodynamics and Statistical Thermodynamics. In Biophysics Textbook online
(E.Freire, editor), Biophysical Society & IUPAB,
129. Dougherty,D.A. (1996). Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and
Trp. Science, 271, 163-168.
130. Makhatadze,G.I. and P.L.Privalov. (1995). Energetics of protein structure. Adv. Protein Chem., 47, 307-425.
141

131. Sturtevant,J.M. (1977). Heat capacity and entropy changes in processes involving proteins. Proc. Natl. Acad. Sci.
U. S. A, 74, 2236-2240.
132. Murphy,K.P. and S.J.Gill. (1991). Solid model compounds and the thermodynamics of protein unfolding. J. Mol.
Biol., 222, 699-709.
133. Murphy,K.P. and E.Freire. (1992). Thermodynamics of structural stability and cooperative folding behavior in
proteins. Adv. Protein Chem., 43, 313-361.
134. Spolar,R.S., J.R.Livingstone, and M.T.Record, Jr. (1992). Use of liquid hydrocarbon and amide transfer data to
estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar
surface from water. Biochemistry, 31, 3947-3955.
135. (1994). The encyclopedia of molecular biology. Kendrew, J. and Lawrence, E. Blackwell Science, Oxford.
136. Stryer, L. (1995). Biochemistry. W. H. Freeman & Co., New York.
137. Fersht, A. R. (1999). Structure and mechanism in protein science. W. H. Freeman & Co., New York.
138. Horovitz,A., L.Serrano, B.Avron, M.Bycroft, and A.R.Fersht. (1990). Strength and co-operativity of contributions
of surface salt bridges to protein stability. J. Mol. Biol., 216, 1031-1044.
139. Kuntz,I.D., K.Chen, K.A.Sharp, and P.A.Kollman. (1999). The maximal affinity of ligands. Proc. Natl. Acad. Sci.
U. S. A, 96, 9997-10002.
140. Serrano,L., J.T.Kellis, Jr., P.Cann, A.Matouschek, and A.R.Fersht. (1992). The folding of an enzyme. II.
Substructure of barnase and the contribution of different interactions to protein stability. J. Mol. Biol., 224,
783-804.
141. Shirley,B.A., P.Stanssens, U.Hahn, and C.N.Pace. (1992). Contribution of hydrogen bonding to the
conformational stability of ribonuclease T1. Biochemistry, 31, 725-732.
142. Hu,C.Q., J.M.Sturtevant, J.A.Thomson, R.E.Erickson, and C.N.Pace. (1992). Thermodynamics of ribonuclease
T1 denaturation. Biochemistry, 31, 4876-4882.
143. Pace,C.N., B.A.Shirley, M.McNutt, and K.Gajiwala. (1996). Forces contributing to the conformational stability of
proteins. FASEB J., 10, 75-83.
144. Sneddon,S.F., D.J.Tobias, and C.L.Brooks, III. (1989). Thermodynamics of amide hydrogen bond formation in
polar and apolar solvents. J. Mol. Biol., 209, 817-820.
145. Pace,C.N. (1992). Contribution of the hydrophobic effect to globular protein stability. J. Mol. Biol., 226, 29-35.
146. McGaughey,G.B., M.Gagne, and A.K.Rappe. (1998). pi-Stacking interactions. Alive and well in proteins. J. Biol.
Chem., 273, 15458-15463.
147. Serrano,L., M.Bycroft, and A.R.Fersht. (1991). Aromatic-aromatic interactions and protein stability. Investigation
by double-mutant cycles. J. Mol. Biol., 218, 465-475.
148. Mecozzi,S., A.P.West, Jr., and D.A.Dougherty. (1996). Cation-pi interactions in aromatics of biological and
medicinal interest: electrostatic potential surfaces as a useful qualitative guide. Proc. Natl. Acad. Sci. U. S. A,
93, 10566-10571.
149. Gallivan,J.P. and D.A.Dougherty. (1999). Cation-pi interactions in structural biology. Proc. Natl. Acad. Sci. U. S.
A, 96, 9459-9464.
150. Jayaram,B., K.J.McConnell, S.B.Dixit, and D.L.Beveridge. (1999). Free energy analysis of protein-DNA binding:
the EcoRI endonuclease-DNA complex. J. Comput. Phys., 151, 333-357.
151. Haq,I., J.E.Ladbury, B.Z.Chowdhry, T.C.Jenkins, and J.B.Chaires. (1997). Specific binding of hoechst 33258 to
the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies. J. Mol. Biol., 271, 244-257.
142

152. Boresch,S., G.Archontis, and M.Karplus. (1994). Free energy simulations: the meaning of the individual
contributions from a component analysis. Proteins, 20, 25-33.
153. Mark,A.E. and W.F.van Gunsteren. (1994). Decomposition of the free energy of a system in terms of specific
interactions. Implications for theoretical and experimental studies. J. Mol. Biol., 240, 167-176.
154. Brady,G.P., A.Szabo, and K.A.Sharp. (1996). On the decomposition of free energies. J. Mol. Biol., 263, 123-125.
155. Rhoads,R.E., G.M.Hellmann, P.Remy, and J.P.Ebel. (1983). Translational recognition of messenger ribonucleic
acid caps as a function of pH. Biochemistry, 22, 6084-6088.
156. Carberry,S.E., R.E.Rhoads, and D.J.Goss. (1989). A spectroscopic study of the binding of m7GTP and m7GpppG
to human protein synthesis initiation factor 4E. Biochemistry, 28, 8078-8083.
157. Cai,A., M.Jankowska-Anyszka, A.Centers, L.Chlebicka, J.Stepinski, R.Stolarski, E.Darzynkiewicz, and
R.E.Rhoads. (1999). Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.
Biochemistry, 38, 8538-8547.
158. McCubbin,W.D., I.Edery, M.Altmann, N.Sonenberg, and C.M.Kay. (1988). Circular dichroism and fluorescence
studies on protein synthesis initiation factor eIF-4E and two mutant forms from the yeast Saccharomyces
cerevisiae. J. Biol. Chem., 263, 17663-17671.
159. Carberry,S.E., E.Darzynkiewicz, J.Stepinski, S.M.Tahara, R.E.Rhoads, and D.J.Goss. (1990). A spectroscopic
study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E.
Biochemistry, 29, 3337-3341.
160. Goss,D.J., S.E.Carberry, T.E.Dever, W.C.Merrick, and R.E.Rhoads. (1990). Fluorescence study of the binding of
m7GpppG and rabbit globin mRNA to protein synthesis initiation factors 4A, 4E, and 4F. Biochemistry, 29,
5008-5012.
161. Ueda,H., H.Maruyama, M.Doi, M.Inoue, T.Ishida, H.Morioka, T.Tanaka, S.Nishikawa, and S.Uesugi. (1991).
Expression of a synthetic gene for human cap binding protein (human IF- 4E) in Escherichia coli and
fluorescence studies on interaction with mRNA cap structure analogues. J. Biochem. (Tokyo), 109, 882-889.
162. Morino,S., M.Yasui, M.Doi, M.Inoue, T.Ishida, H.Ueda, and S.Uesugi. (1994). Direct expression of a synthetic
gene in Escherichia coli: purification and physicochemical properties of human initiation factor 4E. J.
Biochem. (Tokyo), 116, 687-693.
163. Hagedorn,C.H., T.Spivak-Kroizman, D.E.Friedland, D.J.Goss, and Y.Xie. (1997). Expression of functional eIF-
4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins. Protein
Expr. Purif., 9, 53-60.
164. Hsu,P.C., M.R.Hodel, J.W.Thomas, L.J.Taylor, C.H.Hagedorn, and A.E.Hodel. (2000). Structural requirements
for the specific recognition of an m7G mRNA cap. Biochemistry, 39, 13730-13736.
165. Burley, S. K. (1998). Personal communication.
166. Ptushkina,M., T.von der Haar, M.M.Karim, J.M.Hughes, and J.E.McCarthy. (1999). Repressor binding to a dorsal
regulatory site traps human eIF4E in a high cap-affinity state. EMBO J., 18, 4068-4075.
167. Ptushkina,M., T.von der Haar, S.Vasilescu, R.Frank, R.Birkenhager, and J.E.McCarthy. (1998). Cooperative
modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.
EMBO J., 17, 4798-4808.
168. von der Haar,T., P.D.Ball, and J.E.McCarthy. (2000). Stabilization of eukaryotic initiation factor 4E binding to
the mRNA 5'- Cap by domains of eIF4G. J. Biol. Chem., 275, 30551-30555.
169. Darzynkiewicz,E., I.Ekiel, S.M.Tahara, L.S.Seliger, and A.J.Shatkin. (1985). Chemical synthesis and
characterization of 7-methylguanosine cap analogues. Biochemistry, 24, 1701-1707.
170. Jankowska,M., J.Stepinski, R.Stolarski, A.Temeriusz, and E.Darzynkiewicz. (1993). Synthesis and properties of
new NH 2 and N7 substituted GMP and GTP 5'-mRNA cap analogues. Collect. Czech. Chem. Commun., 58,
138-141.
143

171. Darzynkiewicz,E., J.Stepinski, I.Ekiel, Y.Jin, D.Haber, T.Sijuwade, and S.M.Tahara. (1988). Beta-globin mRNAs
capped with m7G, m2.7(2)G or m2.2.7(3)G differ in intrinsic translation efficiency. Nucleic Acids Res., 16,
8953-8962.
172. Stepinski,J., M.Bretner, M.Jankowska, K.Felczak, R.Stolarski, Z.Wieczorek, A.L.Cai, R.E.Rhoads, A.Temeriusz,
D.Haber, and E.Darzynkiewicz. (1995). Synthesis and properties of P 1 ,P 2 -, P 1 ,P 3 - and P 1 ,P 4 - dinucleotide di-,
tri- and tetraphosphate mRNA 5'-cap analogues. Nucleosides & Nucleotides, 14, 717-721.
173. Jankowska,M., J.Stepinski, R.Stolarski, Z.Wieczorek, A.Temeriusz, D.Haber, and E.Darzynkiewicz. (1996). H-1
NMR and fluorescence studies of new mRNA 5'-cap analogues. Collect. Czech. Chem. Commun., 61, 197-
202.
174. Darzynkiewicz,E., J.Stepinski, S.M.Tahara, R.Stolarski, I.Ekiel, D.Haber, K.Neuvonen, P.Lehikoinen, I.Labadi,
and H.Lonnberg. (1990). Synthesis, conformation and hydrolytic stability of P 1 ,P 3 -dinucleoside triphosphates
related to mRNA 5'-cap, and comparative kinetic studies on their nucleoside and nucleoside monophosphate
analogues. Nucleosides & Nucleotides, 9, 599-618.
175. Ruszczynska, K. and Stolarski, R. (2000). Personal Communication.
176. Schnolzer,M., P.Alewood, A.Jones, D.Alewood, and S.B.Kent. (1992). In situ neutralization in Boc-chemistry
solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res., 40,
180-193.
177. Wieczorek,Z., J.Stepinski, M.Jankowska, and H.Lonnberg. (1995). Fluorescence and absorption spectroscopic
properties of RNA 5'-cap analogues derived from 7-methyl-, N2,7-dimethyl- and N2,N2,7-trimethylguanosines.
J. Photochem. Photobiol. B, 28, 57-63.
178. Dawson, R. M., Elliott, D. C., Elliott, W. H., and Jones, K. M. (1969). Data for biochemical research. 2, 174-175,
Clarendon Press, Oxford.
179. Edery,I., M.Altmann, and N.Sonenberg. (1988). High-level synthesis in Escherichia coli of functional cap-binding
eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin. Gene, 74, 517-
525.
180. Stern,B.D., M.Wilson, and R.Jagus. (1993). Use of nonreducing SDS-PAGE for monitoring renaturation of
recombinant protein synthesis initiation factor, eIF-4 alpha. Protein Expr. Purif., 4, 320-327.
181. Webb,N.R., R.V.Chari, G.DePillis, J.W.Kozarich, and R.E.Rhoads. (1984). Purification of the messenger RNA
cap-binding protein using a new affinity medium. Biochemistry, 23, 177-181.
182. Lakowicz, J. R. (1999). Principles of fluorescence spectroscopy. Kluwer Academic/Plenum Publishers, New
York.
183. Parker, C. A. (1968). Photoluminescence of Solutions. Elsevier Publishing Company, Amsterdam.
184. Callis,P.R. (1997). 1La and 1Lb transitions of tryptophan: applications of theory and experimental observations to
fluorescence of proteins. Methods Enzymol., 278, 113-150.
185. Antosiewicz, J. (2003). Personal communication.
186. Wieczorek,Z., J.Stepinski, E.Darzynkiewicz, and H.Lonnberg. (1993). Association of nucleosides and their 5'monophosphates
with a tryptophan containing tripeptide, Trp-Leu-Glu: the source of an overestimation by
fluorescence spectroscopy. Biophys. Chem., 47, 233-240.
187. Eadie,G.S. (1942). J. Biol. Chem., 146, 85-93.
188. Hofstee,B.H.J. (1959). Nature, Lond., 184, 1296.
189. (2001). Database of osmotic pressures.
190. Marcus, Y. (1997). Ion Properties. Marcel Dekker, Inc., New York.
191. Robinson, R. A. and Stokes, R. H. (1970). Electrolyte Solutions. Butterworths, London.
144

192. Davies, C. W. (1962). Ion Association. Butterworths, London.
193. Wyman,J.Jr. (1964). Linked functions and reciprocal effects in hemoglobin: a second look. Adv. Protein Chem.,
223-286.
194. Baldwin,R.L. (1986). Temperature dependence of the hydrophobic interaction in protein folding. Proc. Natl.
Acad. Sci. U. S. A, 83, 8069-8072.
195. Becktel,W.J. and J.A.Schellman. (1987). Protein stability curves. Biopolymers, 26, 1859-1877.
196. Ha,J.H., R.S.Spolar, and M.T.Record, Jr. (1989). Role of the hydrophobic effect in stability of site-specific
protein- DNA complexes. J. Mol. Biol., 209, 801-816.
197. Beyer, W. H. (1987). CRC Standard mathematical tables. 28th, 536, CRC Press, Boca Raton, FL.
198. Wieczorek,Z., K.Zdanowski, L.Chlebicka, J.Stepinski, M.Jankowska, B.Kierdaszuk, A.Temeriusz,
E.Darzynkiewicz, and R.Stolarski. (1997). Fluorescence and NMR studies of intramolecular stacking of
mRNA cap- analogues. Biochim. Biophys. Acta, 1354 , 145-152.
199. Sharp,K. (2001). Entropy-enthalpy compensation: fact or artifact? Protein Sci., 10, 661-667.
200. Ladbury,J.E. and B.Z.Chowdhry. (1996). Sensing the heat: the application of isothermal titration calorimetry to
thermodynamic studies of biomolecular interactions. Chem. Biol., 3, 791-801.
201. (1991). MicroCal OMEGA Manual. MicroCal, Inc., Northampton, MA, U.S.A.
202. Christensen, J. J., Hansen, L. D., and Izatt, R. M. (1976). Handbook of proton ionization heats and related
thermodynamic quantities. John Wiley & Sons, New York.
203. Harris, R. K. (1983). Nuclear Magnetic Resonance Spectroscopy. A Physicochemical View. Pitman Books Ltd.,
London.
204. Brown,L.R. and B.T.Farmer. (1989). Rotating-frame nuclear Overhauser effect. Methods Enzymol., 176, 199-
216.
205. Hore,P.J. (1983). Solvent suppression in Fourier transform nuclear magnetic resonance. Journal of Magnetic
Resonance, 55, 283-300.
206. Brunner,H. and K.Dransfeld. (1983). Light scattering by macromolecules. In Biophysics (W.Hoppe, W.Lohmann,
H.Markl, and H.Ziegler, editors), pp.93-100, Springer-Verlag, New York.
207. Taylor, J. R. (1982). An introduction to error analysis. University Science Books, Mill Valley, CA.
208. Shibata,S., S.Morino, K.Tomoo, Y.In, and T.Ishida. (1998). Effect of mRNA cap structure on eIF-4E
phosphorylation and cap binding analyses using Ser209-mutated eIF-4Es. Biochem. Biophys. Res. Commun.,
247, 213-216.
209. (1989). Elementy enzymologii . Witwicki, J. and Ardelt, W. 2PWN, Warszawa.
210. Koellner,G., A.Bzowska, B.Wielgus-Kutrowska, M.Luic, T.Steiner, W.Saenger, and J.Stepinski. (2002). Open
and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the
catalytic mechanism. J. Mol. Biol., 315, 351-371.
211. Wielgus-Kutrowska,B., A.Bzowska, J.Tebbe, G.Koellner, and D.Shugar. (2002). Purine nucleoside phosphorylase
from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent
enhancement of enzyme intrinsic fluorescence, and by protective effects of ligands on thermal inactivation of
the enzyme. Biochim. Biophys. Acta, 1597, 320-334.
212. Lodish,H.F. (1974). Model for the regulation of mRNA translation applied to haemoglobin synthesis. Nature,
251, 385-388.
145

213. Chu,L.Y. and R.E.Rhoads. (1980). Inhibition of cell-free messenger ribonucleic acid translation by 7methylguanosine
5'-triphosphate: effect of messenger ribonucleic acid concentration. Biochemistry, 19, 184-
191.
214. Rau,M., T.Ohlmann, S.J.Morley, and V.M.Pain. (1996). A reevaluation of the cap-binding protein, eIF4E, as a
rate-limiting factor for initiation of translation in reticulocyte lysate. J. Biol. Chem., 271, 8983-8990.
215. Hiremath,L.S., N.R.Webb, and R.E.Rhoads. (1985). Immunological detection of the messenger RNA cap-binding
protein. J. Biol. Chem., 260, 7843-7849.
216. Haghighat,A. and N.Sonenberg. (1997). eIF4G dramatically enhances the binding of eIF4E to the mRNA 5'-cap
structure. J. Biol. Chem., 272, 21677-21680.
217. Ishida,T., K.Ohnishi, M.Doi, and M.Inoue. (1989). Proton nuclear magnetic resonance study on the aromatic
amino acid- guanine nucleotide system: effect of base methylation on the stacking interaction with tyrosine
and phenylalanine. Chem. Pharm. Bull. (Tokyo), 37, 1-4.
218. Ueda,H., M.Doi, M.Inoue, T.Ishida, T.Tanaka, and S.Uesugi. (1988). A possible recognition mode of mRNA cap
terminal structure by peptide: cooperative stacking and hydrogen-bond pairing interactions between
m7GpppA and Trp-Leu-Glu. Biochem. Biophys. Res. Commun., 154, 199-204.
219. Ueda,H., H.Iyo, M.Doi, M.Inoue, and T.Ishida. (1991). Cooperative stacking and hydrogen bond pairing
interactions of fragment peptide in cap binding protein with mRNA cap structure. Biochim. Biophys. Acta,
1075, 181-186.
220. Ueda,H., H.Iyo, M.Doi, M.Inoue, T.Ishida, H.Morioka, T.Tanaka, S.Nishikawa, and S.Uesugi. (1991).
Combination of Trp and Glu residues for recognition of mRNA cap structure. Analysis of m7G base
recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis. FEBS Lett., 280, 207-210.
221. Hu,G., P.D.Gershon, A.E.Hodel, and F.A.Quiocho. (1999). mRNA cap recognition: dominant role of enhanced
stacking interactions between methylated bases and protein aromatic side chains. Proc. Natl. Acad. Sci. U. S.
A, 96, 7149-7154.
222. Hodel,A.E., P.D.Gershon, X.Shi, and F.A.Quiocho. (1996). The 1.85 A structure of vaccinia protein VP39: a
bifunctional enzyme that participates in the modification of both mRNA ends. Cell, 85, 247-256.
223. Quiocho,F.A., G.Hu, and P.D.Gershon. (2000). Structural basis of mRNA cap recognition by proteins. Curr.
Opin. Struct. Biol., 10, 78-86.
224. Hu,G., A.Oguro, C.Li, P.D.Gershon, and F.A.Quiocho. (2002). The "cap-binding slot" of an mRNA cap-binding
protein: quantitative effects of aromatic side chain choice in the double-stacking sandwich with cap.
Biochemistry, 41, 7677-7687.
225. Martz, E. (2002). Protein Explorer. [1.982 Beta] //www.proteinexplorer.org.
226. Takano,K., J.Funahashi, Y.Yamagata, S.Fujii, and K.Yutani. (1997). Contribution of water molecules in the
interior of a protein to the conformational stability. J. Mol. Biol., 274, 132-142.
227. Lucke,C., S.Huang, M.Rademacher, and H.Ruterjans. (2002). New insights into intracellular lipid binding
proteins: The role of buried water. Protein Sci., 11, 2382-2392.
228. Hempel,R., J.Schmidt-Brauns, M.Gebinoga, F.Wirsching, and A.Schwienhorst. (2001). Cation radius effects on
cell-free translation in rabbit reticulocyte lysate. Biochem. Biophys. Res. Commun., 283, 267-272.
229. Raught,B., A.C.Gingras, and N.Sonenberg. (2000). Regulation of ribosomal recruitment in eukaryotes. In
Translational Control of Gene Expression pp.245-93, Cold Spring Harbor Laboratory Press, New York.
230. Pyronnet,S., H.Imataka, A.C.Gingras, R.Fukunaga, T.Hunter, and N.Sonenberg. (1999). Human eukaryotic
translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E. EMBO J., 18, 270-279.
231. Waskiewicz,A.J., J.C.Johnson, B.Penn, M.Mahalingam, S.R.Kimball, and J.A.Cooper. (1999). Phosphorylation of
the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. Mol. Cell
Biol., 19, 1871-1880.
146

232. Colombo,M.F., D.C.Rau, and V.A.Parsegian. (1992). Protein solvation in allosteric regulation: a water effect on
hemoglobin. Science, 256, 655-659.
233. Colombo,M.F., D.C.Rau, and V.A.Parsegian. (1994). Reevaluation of chloride's regulation of hemoglobin oxygen
uptake: the neglected contribution of protein hydration in allosterism. Proc. Natl. Acad. Sci. U. S. A, 91,
10517-10520.
234. Saenger,W. (1987). Structure and dynamics of water surrounding biomolecules. Annu. Rev. Biophys. Biophys.
Chem., 16, 93-114.
235. Sha,M., Y.Wang, T.Xiang, A.van Heerden, K.S.Browning, and D.J.Goss. (1995). Interaction of wheat germ
protein synthesis initiation factor eIF- (iso)4F and its subunits p28 and p86 with m7GTP and mRNA
analogues. J. Biol. Chem., 270, 29904-29909.
236. Gingras, A. C. (2002). Personal Communication.
237. Niedzwiecka-Kornas, A., Chlebicka, L., Jankowska-Anyszka, M., Stepinski, J., Dadlez, M., Gingras, A. C.,
Marcotrigiano, J., Sonenberg, N., Burley, S. K., Stolarski, R., and Darzynkiewicz, E. (1999). RNA Meeting
Abstract '99.
238. Miyoshi,H., T.Youtani, H.Ide, H.Hori, K.Okamoto, M.Ishikawa, M.Wakiyama, T.Nishino, T.Ishida, and K.Miura.
(1999). Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex
formation. J. Biochem. (Tokyo), 126, 897-904.
239. Fletcher,C.M., A.M.McGuire, A.C.Gingras, H.Li, H.Matsuo, N.Sonenberg, and G.Wagner. (1998). 4E binding
proteins inhibit the translation factor eIF4E without folded structure. Biochemistry, 37, 9-15.
240. Pimentel,G.C. and A.L.McClellan. (1971). Hydrogen bonding. Annu. Rev. Phys. Chem., 22, 347-385.
241. Stolarski,R., A.Sitek, J.Stepinski, M.Jankowska, P.Oksman, A.Temeriusz, E.Darzynkiewicz, H.Lonnberg, and
D.Shugar. (1996). 1H-NMR studies on association of mRNA cap-analogues with tryptophan- containing
peptides. Biochim. Biophys. Acta, 1293, 97-105.
242. Spolar,R.S. and M.T.Record, Jr. (1994). Coupling of local folding to site-specific binding of proteins to DNA.
Science, 263, 777-784.
243. Bhat,T.N., G.A.Bentley, G.Boulot, M.I.Greene, D.Tello, W.Dall'Acqua, H.Souchon, F.P.Schwarz, R.A.Mariuzza,
and R.J.Poljak. (1994). Bound water molecules and conformational stabilization help mediate an antigenantibody
association. Proc. Natl. Acad. Sci. U. S. A, 91, 1089-1093.
244. Naghibi,H., A.Tamura, and J.M.Sturtevant. (1995). Significant discrepancies between van't Hoff and calorimetric
enthalpies. Proc. Natl. Acad. Sci. U. S. A, 92, 5597-5599.
245. Horn,J.R., J.F.Brandts, and K.P.Murphy. (2002). van't Hoff and calorimetric enthalpies II: effects of linked
equilibria. Biochemistry, 41, 7501-7507.
246. Liu,Y. and J.M.Sturtevant. (1995). Significant discrepancies between van't Hoff and calorimetric enthalpies. II.
Protein Sci., 4, 2559-2561.
247. Liu,Y. and J.M.Sturtevant. (1997). Significant discrepancies between van't Hoff and calorimetric enthalpies. III.
Biophys. Chem., 64, 121-126.
248. Chaires,J.B. (1997). Possible origin of differences between van't Hoff and calorimetric enthalpy estimates.
Biophys. Chem., 64, 15-23.
249. Rouzina,I. and V.A.Bloomfield. (1999). Heat capacity effects on the melting of DNA. 1. General aspects.
Biophys. J., 77, 3242-3251.
250. Horn,J.R., D.Russell, E.A.Lewis, and K.P.Murphy. (2001). Van't Hoff and calorimetric enthalpies from
isothermal titration calorimetry: are there significant discrepancies? Biochemistry, 40, 1774-1778.
251. Kavanoor,M. and M.R.Eftink. (1997). Characterization of the role of side-chain interactions in the binding of
ligands to apo trp repressor: pH dependence studies. Biophys. Chem., 66, 43-55.
147

252. Kozlov,A.G. and T.M.Lohman. (1999). Adenine base unstacking dominates the observed enthalpy and heat
capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates.
Biochemistry, 38, 7388-7397.
253. Marcotrigiano, J. (1999). Personal communication.
254. Panniers,R., E.B.Stewart, W.C.Merrick, and E.C.Henshaw. (1985). Mechanism of inhibition of polypeptide chain
initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity. J. Biol.
Chem., 260, 9648-9653.
255. Lamphear,B.J. and R.Panniers. (1991). Heat shock impairs the interaction of cap-binding protein complex with 5'
mRNA cap. J. Biol. Chem., 266, 2789-2794.
256. Morley,S.J. and V.M.Pain. (1995). Translational regulation during activation of porcine peripheral blood
lymphocytes: association and phosphorylation of the alpha and gamma subunits of the initiation factor
complex eIF-4F. Biochem. J., 312 ( Pt 2), 627-635.
257. Sierra,J.M. and J.M.Zapata. (1994). Translational regulation of the heat shock response. Mol. Biol. Rep., 19, 211-
220.
258. Rhoads,R.E. and B.J.Lamphear. (1995). Cap-independent translation of heat shock messenger RNAs. Curr. Top.
Microbiol. Immunol., 203, 131-153.
259. Varani,G. (1997). A cap for all occasions. Structure., 5, 855-858.
260. Huber,J., U.Cronshagen, M.Kadokura, C.Marshallsay, T.Wada, M.Sekine, and R.Luhrmann. (1998). Snurportin1,
an m3G-cap-specific nuclear import receptor with a novel domain structure. EMBO J., 17, 4114-4126.
261. Zorio,D.A., N.N.Cheng, T.Blumenthal, and J.Spieth. (1994). Operons as a common form of chromosomal
organization in C. elegans. Nature, 372, 270-272.
262. Blumenthal,T. and K.Steward. (1997). In C. Elegans II (D.L.Riddle, T.Blumenthal, B.J.Meyer, and J.R.Priess,
editors), pp.117-45, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
263. Stachelska,A., Z.Wieczorek, K.Ruszczynska, R.Stolarski, M.Pietrzak, B.J.Lamphear, R.E.Rhoads,
E.Darzynkiewicz, and M.Jankowska-Anyszka. (2002). Interaction of three Caenorhabditis elegans isoforms of
translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues. Acta Biochim. Pol.,
49, 671-682.
264. Keiper,B.D., B.J.Lamphear, A.M.Deshpande, M.Jankowska-Anyszka, E.J.Aamodt, T.Blumenthal, and
R.E.Rhoads. (2000). Functional characterization of five eIF4E isoforms in Caenorhabditis elegans. J. Biol.
Chem., 275, 10590-10596.
265. Lumry,R. and S.Rajender. (1970). Enthalpy-entropy compensation phenomena in water solutions of proteins and
small molecules: a ubiquitous property of water. Biopolymers, 9, 1125-1227.
266. Krug,R.R., W.G.Hunter, and R.A.Grieger. (1976). Enthalpy-entropy compensation. I. Some fundamental
statistical problems associated with the analysis of van't Hoff and Arrhenius data. J. Phys. Chem., 80, 2335-
2341.
267. Krug,R.R., W.G.Hunter, and R.A.Grieger. (1976). Statistical interpretation of enthalpy-entropy compensation.
Nature, 261, 566-567.
268. Murphy,K.P., V.Bhakuni, D.Xie, and E.Freire. (1992). Molecular basis of co-operativity in protein folding. III.
Structural identification of cooperative folding units and folding intermediates. J. Mol. Biol., 227, 293-306.
269. Spolar,R.S., J.H.Ha, and M.T.Record, Jr. (1989). Hydrophobic effect in protein folding and other noncovalent
processes involving proteins. Proc. Natl. Acad. Sci. U. S. A, 86, 8382-8385.
270. Murphy,K.P., P.L.Privalov, and S.J.Gill. (1990). Common features of protein unfolding and dissolution of
hydrophobic compounds. Science, 247, 559-561.
271. Gomez,J., V.J.Hilser, D.Xie, and E.Freire. (1995). The heat capacity of proteins. Proteins, 22, 404-412.
148

272. Bruzzese,F.J. and P.R.Connelly. (1997). Allosteric properties of inosine monophosphate dehydrogenase revealed
through the thermodynamics of binding of inosine 5'-monophosphate and mycophenolic acid. Temperature
dependent heat capacity of binding as a signature of ligand-coupled conformational equilibria. Biochemistry,
36, 10428-10438.
273. Hileman,R.E., R.N.Jennings, and R.J.Linhardt. (1998). Thermodynamic analysis of the heparin interaction with a
basic cyclic peptide using isothermal titration calorimetry. Biochemistry, 37, 15231-15237.
274. Matulis,D., I.Rouzina, and V.A.Bloomfield. (2000). Thermodynamics of DNA binding and condensation:
isothermal titration calorimetry and electrostatic mechanism. J. Mol. Biol., 296, 1053-1063.
275. Gill,D.S., D.J.Roush, K.A.Shick, and R.C.Willson. (1995). Microcalorimetric characterization of the anionexchange
adsorption of recombinant cytochrome b5 and its surface-charge mutants. J. Chromatogr. A, 715,
81-93.
276. Oda,M., K.Furukawa, K.Ogata, A.Sarai, and H.Nakamura. (1998). Thermodynamics of specific and non-specific
DNA binding by the c-Myb DNA-binding domain. J. Mol. Biol., 276, 571-590.
277. Madan,B. and K.A.Sharp. (2001). Hydration heat capacity of nucleic acid constituents determined from the
random network model. Biophys. J., 81, 1881-1887.
278. Gallagher,K. and K.Sharp. (1998). Electrostatic contributions to heat capacity changes of DNA-ligand binding.
Biophys. J., 75, 769-776.
279. Ma,J.C. and D.A.Dougherty. (1997). The cation-pi interaction. Chemical Reviews, 97, 1303-1324.
280. Mecozzi,S., A.P.West, and D.A.Dougherty. (1996). Cation-pi interactions in simple aromatics: Electrostatics
provide a predictive tool. Journal of the American Chemical Society, 118, 2307-2308.
281. Przedmojski, R. (1998). Masters thesis.
282. Filimonov,V.V. and P.L.Privalov. (1978). Thermodynamics of base interaction in (A)n and (A.U)n. J. Mol. Biol.,
122, 465-470.
283. Breslauer,K.J. and J.M.Sturtevant. (1977). A calorimetric investigation of single stranded base stacking in the
ribo-oligonucleotide A7. Biophys. Chem., 7, 205-209.
284. Berg,O.G., R.B.Winter, and P.H.von Hippel. (1981). Diffusion-driven mechanisms of protein translocation on
nucleic acids. 1. Models and theory. Biochemistry, 20, 6929-6948.
285. Griffon,N. and E.Di Stasio. (2001). Thermodynamics of Na+ binding to coagulation serine proteases. Biophys.
Chem., 90, 89-96.
286. Nureki,O., D.G.Vassylyev, M.Tateno, A.Shimada, T.Nakama, S.Fukai, M.Konno, T.L.Hendrickson, P.Schimmel,
and S.Yokoyama. (1998). Enzyme structure with two catalytic sites for double-sieve selection of substrate.
Science, 280, 578-582.
287. Hinz,H.J., K.Weber, J.Flossdorf, and M.R.Kula. (1976). Thermodynamic studies on the specificity of Lisoleucine-tRNA
ligase of Escherichia coli MRE 600. Calorimetric investigations on binding of amino acids
and isoleucinol to the enzyme. Eur. J. Biochem., 71, 437-442.
288. Ren,J., T.C.Jenkins, and J.B.Chaires. (2000). Energetics of DNA intercalation reactions. Biochemistry, 39, 8439-
8447.
289. DeHaseth,P.L., T.M.Lohman, R.R.Burgess, and M.T.Record, Jr. (1978). Nonspecific interactions of Escherichia
coli RNA polymerase with native and denatured DNA: differences in the binding behavior of core and
holoenzyme. Biochemistry, 17, 1612-1622.
290. Strauss,H.S., R.R.Burgess, and M.T.Record, Jr. (1980). Binding of Escherichia coli ribonucleic acid polymerase
holoenzyme to a bacteriophage T7 promoter-containing fragment: evaluation of promoter binding constants as
a function of solution conditions. Biochemistry, 19, 3504-3515.
149

291. Takeda,Y., P.D.Ross, and C.P.Mudd. (1992). Thermodynamics of Cro protein-DNA interactions. Proc. Natl.
Acad. Sci. U. S. A, 89, 8180-8184.
292. Jin,L., J.Yang, and J.Carey. (1993). Thermodynamics of ligand binding to trp repressor. Biochemistry, 32, 7302-
7309.
293. Ladbury,J.E., J.G.Wright, J.M.Sturtevant, and P.B.Sigler. (1994). A thermodynamic study of the trp repressoroperator
interaction. J. Mol. Biol., 238, 669-681.
294. Shen,X., K.Tomoo, S.Uchiyama, Y.Kobayashi, and T.Ishida. (2001). Structural and thermodynamic behavior of
eukaryotic initiation factor 4E in supramolecular formation with 4E-binding protein 1 and mRNA cap
analogue, studied by spectroscopic methods. Chem. Pharm. Bull. (Tokyo), 49, 1299-1303.
295. Kentsis,A., E.C.Dwyer, J.M.Perez, M.Sharma, A.Chen, Z.Q.Pan, and K.L.Borden. (2001). The RING Domains of
the Promyelocytic Leukemia Protein PML and the Arenaviral Protein Z Repress Translation by Directly
Inhibiting Translation Initiation Factor eIF4E. J. Mol. Biol., 312, 609-623.
150

151