Protein A - esonn

Advanced biophysics for micro-system design 

Learning from nature the future of nanobiotechnology 

Ruud Hovius & Joachim Piguet 

Laboratoire de Chmie Physique des Polymères et Membranes 

Ecole Polytechnique Fédérale 

Lausanne 

Ruud.Hovius@epfl.ch 

Joachim.Piguet@epfl.ch 

1

Structure of course 

Part I - Molecules and Labeling 

• Introduction 

÷ Molecular interactions 

÷ Molecular machines 

÷ Fluorescence 

• Labeling proteins 

÷ During biosynthetic 

Green fluorescent protein 

Non-natural amino acids 

÷ After biosynthesis 

Enzyme or substrate fusion 

Binding partners 

Chemical reactivity 

÷ Covalent vs reversible 

Part II - Single molecule imaging and 

spectroscopy 

• Single molecule detection 

÷ Theory 

÷ Experiment 

• Wide-field imaging 

÷ Molecular motions 

÷ Interactions & catalysis 

• Confocal detection 

÷ Fluctuation analysis 

÷ Multi-parameter fluorescence 

• Nano-devices 

2

What does one see in a living cell 

What changes 

• Activity 

• Structure 

• Location 

• Concentration 

• Diffusion 

• Transport 

• Interactions 

3

Molecular interactions - Biological networks 

• The cell is extremely complex 

many intertwined signaling and metabolic multi-dimensional networks 

a strong spatial-temporal organization 

4

Cells - Very complicated reaction conditions 

• Highly heterogeneous - compartments with very different contents 

• Transport and separation 

• Size-dependent effects on diffusion, kinetics and thermodynamics 

• Non-equilibrium state 

• Many components - 5’000 different gene products 

- many proteins have similar functions 

=> Systems biology : 

Description in space (x,y,z) and time (t) of a cell, an organism or even biotope 

Goodsell, Nat Chem Biol 3 (2007) p.681 

5

Cells - Highly crowded 

6

Molecular interactions - Reductionist’s approach 


⇒ Simplification, e.g. 

⇒ Isolated membranes 

⇒ Purified proteins 

⇒ Bio-inspired systems 

7

Biological networks - Molecular interactions 

• Interacting neurons 

• Synaptic organization 

Signaling events within a cell 

⇒ Right place 

⇒ Right time 

⇒ Proper arrangement 

⇒ Specificity & right partners 

Hammond “Cell & Mol Neurobiology” 

Kim, Nat Rev Neurosci 5 (2004) 772 

through reversible and adjustable 

molecular interactions. 

8

Proteins - Modular assembly of functional domains 

• Proteins must be multi-functional 

- specific interaction 

- regulation of interaction 

- effector 

• Proteins are in general not monolithic globules 

but rather have a beads-on-a-string multi-domain structures, 

where each domain has a specific function 

• Domains: small (70-140 amino acids) autonomously folding sequences 

specific function e.g. catalysis or binding 

> 1’000 type of domains present in human genome 

• Linear combination of a few domains yields an infinite variation of proteins 

with each unique properties 

“Modular Protein Domains” Cesareni e.a. (Eds) 2005 Wiley 

9

Reversible & adjustable protein interactions 

M-M Zhou, Bhattacharyya Ann Rev Bc 2006 

10


Example 1: The SH2 domain family 

- binds specifically to the protein sequence: ….-pY-x-x-hydrophobic-… 

where pY is a tyrosine phosphorylated by the action of a specific kinase: 

- The domains have a conserved structure and 

- The ligands are positioned similarly. 

- Specificity is detemined by the 3rd amino acid 

after -pY- determines specificity 

11


Growth factor receptors are also called receptor tyrosine kinases. They are present 

on the surface of cells as monomers. 

Upon ligand binding the receptors dimerize and trans-phosphorylate eachother’s 

tyrosines => activation of cellular signalling 

12


Example 2: The PH domain family 

- the human genome encodes about 250 PH domains 

some bind to PIP 2 , 

an abundant lipid in the plasma membrane of cells 

13

Imaging PIP 2 in vivo using PH-GFP chimeras 

• PIP 2 

is rapidly broken down upon activation of e.g. the angiotensin receptor 

• A fusion protein PH_GFP, composed of a green fluorescent protein (GFP) and a 

PH domain binds to PIP 2 

in the plasma membrane 

• Angiotensin addition leads to PIP 2 

breakdown 

=> PH-GFP can not bind to the membrane anymore 

PI(4,5)P 2 

specific domain: 

- [PI(4,5)P 2 

] high in membrane 

- Angiotensin promotes breakdown 

Balla, TiPS 2000 

14

Domain combination - Variations on a theme 

“Combinatorial” protein design to create proteins with new properties 

15

Molecular interactions exploiting domains 

• Organizing molecules 

• Scaffolds allow regulation and integration of signals and 

organization of signaling molecules in space and time 

• Coincidence of events is important for both control and out-put 

16

Scaffolds - regulation and integration 

17

Modified scaffolds => modified output 

Comparison of pheromone and osmo-sensing in yeast 

organisation by different scaffolds => different cellular response 

INPUT 

Chimeric scaffold 

OUTPUT 

18

Protein interactions: Genome wide approaches 

• Classical methods to determine protein-protein interactions: 

e.g. 2-hybrid, GST-pull down, phage display 

⇒ protein interaction maps 

⇒“simplistic” view of proteins 

19


• Domains: 

- approx 1’000 types of domains in genome 

÷ for about 750 types, 1 or more structures known 

- about 10’000 types of domain-domain interactions are predicted 

÷ 20% have been structurally characterized 

=> Use functional and structural information to predict 

- interactions between domains, and thus between proteins 

- identify interactions that 

÷ can happen simultaneously 

÷ are sterically overlapping, and thus excluding 

- estimate by modeling 

÷ affinities 

÷ rate constants 

20


21


• An example for the protein Ras: 

22

Interacting molecules - Nanoscopic building blocks 

Constructing supra-molecular functional entities using nature’s components. 

=> in solution or on surfaces 

Commonly used constructing materials: 

Streptavidin 

Biotin 

Immunoglobulins Antigens Protein A or G 

Oligonucleotides 

23

Interacting molecules: Streptavidin 

• Tetrameric protein from Streptomyces avidinii, about 65 kDa (approx 5x5x5 nm) 

• each monomer binds very tightly to biotin : K d 

~ 10 -14 M or ΔG ~ 32 kT. 

Biotin, also known as vitamin H or B7 

O 

H 

HN 

NH 

H 

S 

COOH 

• Avidin is a glycoprotein from bird eggs with comparable structure and properties. 

It prevents biotin absorption in the gastrointestinal tract. 

• Biotin can easily be linked chemically to many substances. 

24

Interacting molecules: Monovalent streptavidin 

Streptavidin is a tetrameric protein 

- Danger of cross-linking biotinylated molecules 

=> Design a “Monovalent streptavidin” using 

“dead” subunits carrying mutations N23A,S27D &S45A => K a = 10 3 M -1 

1”alive” + 3 “dead” => K d 

~ 2.10 -13 M 

Howarth, Nature Meth 3 (2006) 267 

25

Interacting molecules: Streptavidin 

How to bind proteins to streptavidin 

=> biotinylation either chemical of biosynthetically 

=> fusion with a Streptavidin-tag 

Analysis of peptide libraries identified peptide with good affinities for streptavidin 

• Strep-tag WSHPQFEK-COOH K d 

= 72 µM 

K d 

= 1 µM to engineered streptavidin 

Voss & Skerra, Protein Eng 10 (1997) 975 

• SBP-tag : 

K d 

= 2.5 nM 

MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP 

Keefe, Prot Exp Purif 23 (2001) 440 

26

Interacting molecules: Immunoglobins 

A binding partner to almost anything, the immune system uses immunoglobins, 

also called antibodies: 

• the antigen is bound to the variable regions 

• genetic recombination yield infinite variations 

Antibody of IgG class 

• Each domain is approx 50 kDa and 5 nm long. 

• Dissociation constants are nM for good antibodies. 

27

Interacting molecules: Immunoglobins 

Potential disadvantages: 

• large size (150 kDa) steric problems 

• 2 binding sites 

cross-linking 

Solution: make it smaller!! 

=> Enzymatic treatment => Genetic engineering 

F ab 

- fragments 

scFV-fragments 

28

Interacting molecules: Antibody-binders 

• Bacterial have developed antibody-binding proteins as a means of defense 

Protein A is a cell wall protein found in Staphylococcus aureus, 

4 binding sites for heavy chain on F c 

with a K d 

= 10 -8 M 

Protein G is a cell wall protein from G. streptococci, 

2 F c 

binding sites 

Protein L is from Peptostreptococcus magnus, 

binds to Ig’s with a particular kappa-light chain 

These proteins are often used to coat surfaces for further antibody Y 

immobilization. 

Y Y Y Y 

29

Interacting molecules: Oligonucleotides 

Oligonucleotides are polymers made of the 4 (ribo)nucleobases : A C G T 

Complementary antiparallel strands hybridize through base pairing and sequence 

recognition 

30


Complementary strands are kept together with many weak H-bonds 

=> temperature sensitive 

DNA double strands dissociate at a ginven temperature: the melting temperature T m 

GATC 

|||| melts at 12 °C 

CTAG 

31


Base pairing & sequence recognition can be used e.g. to construct 

complicated structures: 

165x165 nm 

Rothemund, Nature 440 (2006) 297 

Scale: 1 to 2.10 14 

32


Alternative new proteins: See reviews 

• 

• 

Advantages are: 

• small 

• easier to optimize against any target 

• can be expressed in large amounts 

33


Organic molecules specifically recognizing oxide surfaces: 

=> Selective Molecular Assembly Patterning (SMAP) 

e.g. - alkane phosphates (DDP) => Nb 2 

O 5 

or Ta 2 

O 5 

- Poly-l-lysine-PEG (PLL-PEG) => SiO 2 

N. Spencer, ETH-Zürich 

34

Interacting molecules: LEGO for the advanced 

35

Interacting molecules: LEGO for the advanced 


36

Molecular machines 

⇒ Linear motors 

⇒ Rotational motors 

⇒ Energy conversion 

⇒ Protein factories 

⇒ Transport systems 

⇒ Channels 

Motors walk on filament 

Kinesin on microtuble 

Filament slide on immobilized motors 

Myosin on Actin 

Typical speed: 

Step size: 

about 1 µm/s 

about 10 nm 

37







⇒ Channels 

Bacterial flagella 

Energy conversion: Electro-chemical >> mechanical 

Number of H + per revolution: ~ 1000 (~ 6000 kT) 

Maximum rotation rate: 300 Hz (H + ) or 1700 Hz (Na + ) 

Maximal speed: 

1 µm/s 

Diameter: 

50 nm 

38







⇒ Channels 

ATPase uses H +- gradient to produce ATP: 3-5 H + needed per ATP 

Energy conversion: Electro-chemical >> mechanical >> chemical 

Dimensions: 

Diameter about 10 nm 

Rotations: 

> 3000 rpm => >10’000 ATP/min 

39







⇒ Channels 

Energy conversion: 

Speed: 

Chemical => chemical 

Few residues per second 

40







⇒ Channels 

In vitro 

Energy conversion: 

Speed: 

In vivo 

Chemical => motion 

µm/s 

41







⇒ Channels 

closed ⇔ open 

Kuo, Structure 13 (2005) 1463 

Voltage-gated potassium channels 

Permeability: 

10 6 ions/s 

Selectivity: 

only potassium 

Conversion: 

potential => current 

42

Biomolecular nanotechnology => Nanomachines 

÷ Molecular interactions 

÷ Molecular machines 

=> Nanobiomachines 

NASA/Rutgers 

Arizona 

43

Biomolecular nanotechnology => Nanomachines 

Nanobiomachines: A recent example of a miniaturized flee circus 

Hiratsuka, PNAS 103 (2006) 13618 

44

Where to study the biological objects 

• In vitro 

+ Purified, well controlled system 

+ Easy to label 

- Out of cellular matrix/context 

• In vivo 

+ In the cellular context 

+ Can be labelled biosynthetically 

- Hard to access 

- Very complex 

- Difficult to control 

45

Method of choice: Fluorescence 

• Non-invasive => in vivo 

• Sensitive => down to a single molecule 

• On line => direct info from ns to days 

• High information content 

– Location, movement & distance 

– Microenvironment 

– Molecular interaction 

• Many parameters 

– Intensity 

– Spectral shape 

– Lifetime 

– Anisotropy 

– Position 

46

Method of choice: Fluorescence 

What is needed 

• Molecular and cellular biology 

Design, engineering, expression, purification of desired molecules 

• Instrumentation and methodology 

Acquisition and data treatment, interpretation 

• Labeling 

Specific introduction of non-perturbing probes 

47

Labeling is needed for identification 

=> Discriminate from matrix and between molecules of interest 

- Cells are full of fluorescent molecules 

=> Detect the parameter of interest 

Ideal: 

• Specific labeling of molecular of interest within a living cell 

• Stoichiometric 

• Non-perturbing 

• Bright and stable labels 

48

What kind of molecules to label 

• Sugars Glycomics 

– Cell recognition 

– Protection 

• Lipids Lipidomics 

– Membrane barrier 

– Signaling 

• Nucleic acids Genomics 

– Genetic information 

– Catalysis 

• Proteins Proteomics 

– Catalysis 

– Machines 

• Small molecules Metabolomics 

Pharmacolomics 

49

A classical fluorophore: 

Fluorescein 

Some basics on fluorescence 

• Wavelength of excitation < emission => Stokes’ shift 

• Lifetime τ of the excited state ns - ms 

• Extinction coefficient: probability of absorbance 10 4 - 10 6 M -1 cm -1 

• Quantum yield q : photons emitted/absorbed 0 - 1 

• Total number of photons before photo-damage 10 4 - 10 6 

• Rate of emission ≤ 2.5 10 6 s -1 

50

Photoselection and anisotropy of fluorescence 

Steady-state anisotropy r is defined by: 

with the rotational correlation time 

=> r depends also on mass. 

51

Photoselection and anisotropy of fluorescence 

• Applications: • Binding of small ligand A to big receptor B: 

A* + B A*B 

• Rotation & orientation of molecules 

e.g. ATPase or flagella 

52


Fluorescence Resonance Energy Transfer (FRET) efficiency E depends 

on: 

- Overlap of emission D and absorption A 

- Distance between D and A 

=> E = 0.5 at the Föster radius R o 

5 Å < R o 

< 80 Å 

53


• Applications: • Molecular interactions: D* + A° D*A° 

• Structural changes: 

D*-A° D* ---A° 

S. Weiss, Science (1999) 

54

Labeling, with what 

The probe should be: 

- Small is beautiful 

- Bright 

- Stable 

- Non-perturbing 

- .. 

The labelling should be/allow: 

- Specific 

- Stoichiometric 

- Inside or on surface of cell 

- Permanent or reversible 

Both should match the lifetime of the 

process of interest 

E. A. Jares, Nat BioTech 21 (2003) 1387 

55

Labeling, with what 

- unspecific 

- unspecific 

- blinking 

+ specific 

• Proteins + (bio)synthetically - large 

+ specific - post-labeling 

56

Labeling with organic dyes 

Very large choice of (reactive) dyes commercially available from e.g. 

Invitrogen (Molecular Probes )(Alexa) 

GE (Amersham) (Cy dye) 

Toronto Research Chemicals 

Chromeon 

Atto-tec (Atto) 

Biotium 

Dyomics 

FluoProbes 

57

Labels: Organic dyes 

Hard to choose form the many dyes available => try many!! 

Important points: 

• Wavelengths of excitation & emission 

• Type of matrix, e.g. living cell or silicon waver 

• Interference with functionality of the molecule to be labeled 

e.g. labelled acetylcholine 

depending on spacer length 

either agonist or antagonist 

Baindur, Drug Development Research 33 (1994) 373 

58

Labels: Organic dyes 

Hard to choose form the many dyes available => try many!! 

Important points: 

• Stability of dye & 

observation time needed 

www.atto-tec.com 

• Hydrophobicity of probe 

Dye molecules contain large conjugated, aromatic systems 

=> aggregation 

=> unspecific binding 

• “Bad ones” Bodipy’s, Dy-655, rhodamines, ATTO647N 

• Nonspecific interactions 

with membranes e.g. Cy5 

with mitochondria e.g. Atto647N 

59

Labels: Quantum dots 

Semiconductor crystals of nm-size show: 

• Size dependant absorption and emission wavelengths 

=> colors can be tuned by varying size 

• Great brightness 

• Narrow emission spectra 

• Highly photostability 

• Broad absorption spectra => allows multiplexing. 

CdSe/CdS 

- Large size & stoichiometry of modification 

Bruchez, Science 281 (1999) 2012 

60

How to get them 

• Home made 

Labels: Quantum dots 

=> quite tedious 

• Commercial sources 

available with e.g. streptavidin or -COOH surface modification 

=> batch to batch variations, 

especially in blinking properties 

Where to get them from 

Invitrogen (Quantum dot) 

Evident 

Biopixels 

61

Labels: Quantum dots in imaging 

• Single AMPA receptor imaging on neurons 

- post-biosynthetic labeling upon bionylation. 

PSD-95-YFP & AMPA receptors with QD605 

1- PSD-95-YFP, then overlaid with DIC image 

2- plus QD605 movie (speeded up 3-fold) 

3- QD605 alone. 

Howarth, PNAS (2005) 1388 

62

Quantum dots for supra-molecular entities 

• Scaffold 

• Multi-functional building block 

Medintz, Nat Materials 2 (2003) 630 

Geissbuehler, Angew. Chem. 44 (2005) 1388 

63

How to label 

Methods: 

Modality: 

• Biosynthetically • Covalent 

- GFP 

- Non-natural amino acids • Reversible 

• Post-biosynthetically 

- Enzymes 

- Small tags 

- Ligands 

• Chemically 

- Reactive functional groups 

64

Reviews 

• General 

Marks & Nolan “Chemical labeling strategies for cell biology” 

Nature Methods 3 (2006) 591 

Prescher & Bertozzi “Chemistry in living systems” 

Nature Chem Biol 1 (2005) 13 

Hovius e.a. “Fluorescent Labelling of Membrane Proteins in Living Cells” 

in "Structural Genomics on Membrane Proteins” CRC Press (2006) 199 

• Fluorescent proteins 

Tsien, R.Y. The green fluorescent protein. 

Annu. Rev. Biochem., 67, 509–544,1998. 

Zimmer, M. Green fluorescent protein (GFP): applications, structure, and related 

photophysical behavior. 

Chem. Rev., 102, 759–781,2002. 

• Nonnatural amino acidsand suppressor tRNA 

Wang, Xie & Schultz “Expanding the genetic code” 

Annu Rev Biophys Biomol Struct 35 (2006) 225 

65

Biosynthetic labeling 

• Labeling during biosynthesis of the protein 

Advantages: 

- in the cell 

- can be targeted to compartment of wish 

- on the protein of wish 

- site-specific 

Disadvantages: 

- limited choice of labels 

- limited quality 

- compatibility with protein 

- incomplete targeting 

66

Biosynthetic labeling: Fluorescent proteins 

Green Fluorescent Protein from the 

jellyfish Aequorea victoria 

• 26 kDa protein with a β-barrel structure 

• Chromophore within barrel => very stable!! 

R. Y. Tsien 

M. Zimmer, Chem Rev (2002) 

67


• Many color variants either by mutagenesis or from other species 

=> Very differing spectroscopic properties !! 

Ex (nm) Em (nm) Abs coeff (M - Q τ (ns) r o φ (ns) brillance 

1 

cm -1 ) 

eCFP 434 477 26'000 0.4 1-1.5 (50 %) 

0.2 

3.3-4.0 

eBFP 380 440 31'000 0.18 3 0.11 Bleaches fast 

eYFP 514 527 84'000 0.61 3.7 1.0 Bleaches fast ! 

Long dark states 

eGFP 489 508 55'000 0.6 2.5 0.33 24 0.64 pH sensitive 

DsRed 558 583 22'5 - 27'000 0.25-0.4 4 ±0.15 Aggregates 

far reds ±590 ±640 

A. Miyawaki, Nat. Neurosci Sept 2003, S1 

68


• Fusion to protein of interest 

N- or C-terminal, internal 

• Can be targetted to all organelles 

Clontech 

• Not (too) toxic 

R. Y. Tsien 

69


Combination of several spectral variants facilitates multi-color-imaging 

up to 6 colors in parallel 

• E.g. using an LSM510 equipped with a 32 channel META detector 

and excitation at 458 nm. 

T. Kogure, Nat Biotech 24 (2006) 577 

70


Timer, a mutant red fluorescent protein, which slowly changes color. 

In vitro 

In vivo: Following protein expression 

and transport in time 

A. Terskikh, Science 290 (2000) 1585 

71

Fluorescent proteins : Photoactivation 

• GFP Mutant T203H: Irradiation at 413nm => inversed absorbance spectrum 

=> has a 100-fold contrast upon excitation at 480 nm!! 

• In vitro: Immobilized GFP 

Excitation at 488 nm 

Activation at 413 

=> Photo-lithography 

G. H. Patterson, Science 297 (2002) 1873 

72

Fluorescent proteins : Switching 

• mCherry mutant can be switched on and off repetitively!! 

Experiment: live E. coli’s expressing the proteins & using a Hg-lamp. 

Stiel (2008) Biophys J 2989 

73


Molecular interactions or conformational changes can be detected by FRET. 

Combining fluorescent proteins one can fine-tune the distance dependence 

of FRET to the system under investigation. 

G. H. Patterson, Anal Bc 284 (2002) 438 

74


Molecular interactions or conformational changes can be detected by FRET. 

e.g. a sensor of tyrosine phosphorylation: 

Sato, Nat Biotech (2002) 278 

75

Fluorescent proteins: Transducers in sensors 

Variations on a theme to detect molecular interactions, changes of 

conformation or of analyte concentrations. 

J. Zhang, Nat Rev MolCelBiol 3 (2002) 906 

76


Fluorescent protein based sensors have been used to measure: 

[Ca 2+ ] A. Miyawaki. Nature 388 (1997) 882 

[H + ] J. Llopis, PNAS 95 (1998) 6803 

[Cl - ] S. Jayaraman, JBC 275 (2000) 6047 

cAMP M. Zaccolo, Nature Cell Biology 2 (2000) 25 

[Zn 2+ ] K. K. Jensen, Bc 40 (2001) 938 

PK-C activity J. D. Violin. J Cell Biol 161 (2003) 899 

Tyr-kinase Sato, Nat Biotech (2002) 278 

Redox potential C. T. Dooley, JBC 279 (2004) 22284 

Androgens Awais, Angew Chem 45 (2006) 2707 

and many, many other analytes!! 

77


GFP analogues have revolutionized life sciences. 

But they are not perfect: 

Non-ideal fluorophores 

Wrongly targeted proteins 

Stoichiometry of co-expressed interacting fusion proteins 

Moreover, many fluorescent proteins and their use have been patented 

=> commercial applications need permission & 

license fees have to be paid. 

Fortunately monthly new fluorescent proteins are being found in 

different species !! 

78

Biosynthetic labeling: Nonnatural amino acids 

Normal protein synthesis 

DNA 

mRNA 

Stop codon 

Stop codon 

Each amino acid is encoded in the DNA by 3 

bases forming a codon. 

The end of a protein is indicated by the stopcodons. 

P. G. Schultz, D. Dougherty 

79


Site-specific introduction of artificial amino acids 

with a suppressor tRNA complementary to the stop codon “AUC” 

UGA 

Stop codon 

P. G. Schultz, D. Dougherty 

80


Sofar, only small, relatively 

weak fluorophores could be 

introduced directly: 

• Dansyl 

• NBD 

• Coumarine 

• Bodipy FL 

Wang, Annu Rev Biophys Biomol Struct 35 (2006) 225 

81


Site-specific incorporation of NBD-amino acids in a G protein-coupled receptor. 

• In vivo NBD was introduced at different sites 

• A rhodamine labeled ligand was bound to the receptor 

• Distances between labels was measured by FRET 

=> A model of the ligand-receptor complex. 

G. Turcatti, JBC 271 (1996) 19991 

82


Problems 

• nnaa-tRNA 

- difficult to synthesis 

- hydrolysis in cell 

- low yield 

- high background 

• Only in vitro or in oocyte 

- tedious 

- mammalian cell system wished 

• Only small, no so ideal fluorophores 

- limited scope of application 

however: 

• Biotin-containing nnaa’s => labeled streptavidin 

• Chemo-selective nnaa’s 

83


Introduction of a chemo-selective aldehyde containing nnaa: 

- reacts specifically with hydrazine derivatives of fluorophores. 

In vivo demonstration: m-acetyl-L-phenylalanine 

was introduced in LamB using suppressor tRNA 

methods in E. coli, and post-biosynthetically 

labeled with 3 hydrazine derivatives. 

Z. Zhang, Bc 42 (2003) 6735 

84


Introduction of a chemo-selective alkynyl amino acids, which react specifically 

with azido-derivatives. (or the inverse!): 

+ 

Met Phe Azido-coumarine 

In vivo demonstration: 

labeling of barnstar in expressed E. coli, 

and post-biosynthetically 

• - o/n labeling 

• - random 

Beatty, J Am Chem Soc 127 (2005) 14150 

85

Post-biosynthesis labeling of a genetic tag 

The protein of interest can be genetically modified to contain a 

sequence that can be labelled once the protein has been made. 

Such a sequence can be: 

Protein 

Enzyme 

Substrate 

Binding site 

Peptide 

Binding partner 

Substrate 

86

Post-biosynthesis labeling: Enzyme tag - hAGT 

• O 6 -Alkylguanine-DNA Alkyltransferase (hAGT) is a DNA-repair “enzyme”. 

• The nucleotide-modifying LABEL is covalently bound to AGT. 

-/+ Not all substrates are membrane permeable. 

- Cells need to be devoid of endogenous interfering AGTs => Mutant versions of AGT 

Keppler, Nat BioTech 21 (2003) 86 Commercially available as SNAP-tag from 

87

Post-biosynthesis labeling: Enzyme tag - hAGT 

• Development of AGT-mutants: 

- no more interference of endogenous AGT 

- “CLIP” recognizing cytosine-based substrates 

Keppler (2003)Nat BioTech; (2004)PNAS; Juillerat (2005) ChemBioChem; Gautier (2008)Chem & Biol 

88

Post-biosynthesis: Substrate protein - Carrier proteins 

Intermediates in cellular biosynthesis are in several cases attached to so-called 

“carrier proteins” (CP) to enhanced their solubility or preserve reactive form. 

÷ Carrier proteins are small 70-100 residues, compact and fold autonomously. 

÷ Enzymes couple the CoA-activated intermediates () to the carrier proteins 

+ 

CP 

PPTase 

CP 

4’-phosphopantetheine (PP) group of coenzyme A (CoA) is coupled covalently to 

an OH-group of a serine on CP by PP-transferase (PPTase). 

Modification of the SH-group of CoA has no or little effect on the reaction. 

± Only cell surface molecules 

Examples are: 

Peptide Carrier Proteins (PCP) J. Yin, Chem & Biol 12 (2005) 999 

Acyl Carrier Proteins (ACP) George, JACS 126 (2004) 8896 

89


Important for labeling: 

• Genetically fuse CP to protein of interest 

• Express in cells 

• Incubate with fluorescently labeled CoA and PPTase 

• Wash out free substrate before imaging 

-+ Surface protein labeling only 

as labeled CoA and PPTase are membrane impermeable. 

+- CoA-fluorophores nicely water-soluble. 

Commercially available from 

90

Post-biosynthesis: Substrate protein - ACP 

Two-color labeling for FRET: Comparison with fluorescent proteins 

NK1_eCFP NK1_EYFP Merge 

FPs: 

Co-transfect 

mixed DNA 

ACP: 

Label with 

mixed CoA’s 

ACP-NK1_Cy3 ACP-NK1_Cy5 Merge 

=> ACP is by far better for investigations on cell surface proteins. 

Meyer, PNAS 103 (2006) 2138 & FEBS (2006) 

91


Possibility for specific labeling of several proteins using both ACP and PCP fusions, 

employing specific PPT enzymes: 

AcpS from E. coli will only modify ACP 

Sfp from B. subtilis will modify both ACP & PCP 

Example: cell wall proteins in yeast labeled with Cy3 on ACP and Cy5 on PCP. 

Vivero-Pol, JACS 127 (2005) 12770 

92

Post-Biosynthetic labeling: Peptide substrate - Biotin-ligase 

• Fusion of biotin acceptor sequence GLNDIFEAQKIEWHE to protein of 

interest 

• Enzymatic coupling of a biotin analogue which reacts specifically with 

O 

hydrazine derivatives of fluorophores. 

O 

N 

N 

H 

S 

S 

Biotin ligase 

Ketone 1 

ATP 

H 2 N 

pH 6-7 

N 

H 

O 

+ Highly specific for Biotin tag 

+ Only surface exposed proteins 

- Hydrazine coupling slow 

- Bond can hydrolyse 

Chen, Nature Meth (2005) 

93

Post-Biosynthetic labeling: Peptide substrate - Biotin-ligase 

Alternatively, coupling with normal biotin and labelling with QD-streptavidin 

- Huge size of label 

Howarth, PNAS (2005) 

94

Post-Biosynthetic labeling: Peptide tags - Epitopes 

Epitopes are sequences of 5 to about 20 amino acids against 

which antibodies can be raised or engineered. These 

sequences can be introduce into protein of wish. 

Examples are: 

HA Tyr - Pro - Tyr - Asp - Val - Pro - Asp -Tyr - Ala 

VSV Tyr - Thr - Asp - Ile - Glu - Met - Asn - Arg - Leu - Gly - Lys 

FLAG Asp -Tyr -Lys -Asp - Asp - Asp - Asp - Lys 

His6 His - His - His - His - His - His 

Fluorescently labelled specific & high affinity antibodies are 

commercially available. 

+ Small tags 

+ Specific & high affinity 

+ Any color 

+- Multiple labels per antibody 

- Antibodies are huge 

95

Post-Biosynthetic labeling: Peptide tags - FlAsH binding 

• The biarsenical FlAsH is virtually non-fluorescent 

• Binding to a CCxxCC motif results in a 100-fold fluorescence increase 

• Reversed by addition of ethylenedithiol (EDT) 

+ Works also in vivo 

- Binding is slow and has a low affinity 

- Need for high concentration of EDT and non-fluorescent dyes. 

B. A. Griffin, Science 281 (1998) 269 

96


FlAsH labelling, an example: 

Dual color pulse labeling of CCxxCC-connexin using 

- 1 st pulse of FlAsH 

- 2 nd pulse of ReAsH later in time 

J. Zhang, Nat Rev MolCelBiol 3 (2002) 906 & 

G. Gaietta, Science 296 (2002) 503 

97


Improvement of peptide tag 

1998: Initial ..CC-xx-CC.. peptide α-helix 

=> background = specific needed a lot of thiol reagents 

2002: Then ..CC-PG-CC.. loop 

=> less non specific 

2005: Now: ..FLN CC-PG-CC-MEP.. 

=> 1.7-fold increase quantum yield 

=> 20-fold increased resistance dithiol reagents 

- can be washed with 0.75 mM dimercaptopropanol to 

suppress non-specific binding 

- Always tested on highly expressed proteins 

e.g. actin or concentrated proteins like connexins 

- Still very slow 1-2 hours 

Martin, Nat Biotech 23 (2005)1308 

98


Improvement of the system: 

2007: Development of AsCy3 

+ more stable and brighter than previous probes 

+ binds specifically to ..CC-AEAA-CC.. 

and not to ..CC-xx-CC.. 

+ binds faster than FlAsH and ReAsH 

+ orthogonal double labeling possible by using both AsCy3 and FlAsH 

and their respective peptide tags CC-xx-CC and CC-AEAA-CC 

Cao, JACS 129 (2007) 8672 

99

Post-Biosynthetic labeling: Peptide tags - NTA-Probes 

Principle: Selective binding of NTA-Ni 2+ to oligohistidine sequences 

+ Wide choice of chromophores: Quenching, co-localisation, FRET, 

membrane permeability.. 

+ Small label 

-+ Moderate affinity 

Guignet (2004) Nature Biotech 

100


NTA-I binding to a His 6 -tagged GFP is demonstrated in vitro by FRET. 

+ Fast binding and disociation constant K d = 3 µM 

+- Fully reversible by EDTA 

Guignet (2004) Nature Biotech 

101


Reversible labelling of proteins on oligo-histidine tags by NTA-conjugates 

Lata 

2005 JACS 

K d : t 1/2 : 

mono-NTA 5’000 nM 5 sec fits to endurance of Cy5 

tris-NTA 0.1 nM 30 min need a quantum dot 

or a good organic dye 

102

Imaging single 5HT3R receptors using NTA-probes 

• NTA-Atto 647 

Different modes of diffusion were observed 

Guignet, ChemPhysChem 2007 

103

Labeling of receptors with fluorescent ligands 

Example: Conjugation of acetylcholine with dansyl to probe the 

muscle-type acetylcholine receptor 

104

Reversible Sequential single cell ligand binding assays 

A single cell expressing the acetylcholine receptor is pulse labeled & 

washed repeatedly many times 

Schreiter, ChemBioChem 2005 

105

Reversible Sequential labeling with receptor ligands 

A single cell is pulse labeled repeatedly under different conditions, 

e.g. changing concentrations of competing non-fluorescent ligand 

=> Pharmacological profiling on a single cell 

Schreiter, ChemBioChem 2005 

106

Post-Biosynthetic: Chemical labelling 

Reactive groups in proteins: 

• -SH of Cys 

• ε-NH 2 

of Lys or N-terminus 

• -OH of Ser 

• -COOH of C-terminus, 

Asp or Glu 

Abundance of amino acids: 

• Cys low on outside 

• Lys high 

• Ser + Thr high 

• Asp + Glu high 

Special cases: 

• N-terminal Ser 

• N-terminal Cys 

Accessibility 

• Surface exposed 

• Burried 

+ large choice of dyes 

- limited site-specificity, especially in vivo 

⇒ Often performed in vitro using purified proteins!! 

⇒ the only option is Cys labeling !! 

107

Chemical labeling of cysteines 

Maleimide and methylthiosulfonates (MTS) are usefull to react 

with SH-groups in aqueous conditions and on live cells. 

+ large commercial offer 

- bulky thioeter linkage 

+- in general slow to react 

- require large excess of reagent 

- unwanted side reactions 

Methylthiosulfonate 

OH 

S 

O 

- limited commercial offer 

- hydrolyses in water 

+ react extremely rapid 

+ highly selective 

+ quantitative without excess 

+ reversible 

108

Post-Biosynthetic: Site-specific single Cys-labeling 

.Strategy: 

i) Remove native Cys 

ii) Introduce single Cys 

iii) Fluorescence labeling of Cys 

iv) Fluorescence measurements 

NB: - step i) is not always needed 

- steps i) and ii) might kill protein 

- step iii) too!! 

109

Post-Biosynthetic: Cys-labeling of cell surface receptor 

Rhodamine-MTS labeling of a single Cys-mutant in channel of the receptor. 

• red: 

• green: 

rhodamine labeling 

fluorescent receptor ligand 

=> Specific labeling. 

Guignet 

110

Channel gating monitored by fluorescence 

Addition of agonist induces a reversible change in fluorescence intensity. 

1.4x10 6 

Agonist 

Wash 

Fluorescence intensity 

1.3 

1.2 

1.1 

1.0 

0 

100 

200 

300 

Time (s) 

400 

500 

600 

=> Structural rearrangements during channel gating. 

Guignet 

111

Post-Biosynthetic labeling: Comparison 

Method Tag Partner In cell Rev Fast 

HaloTag 33 kDa < 1 kDa Y N m - h 

hAGT 207 aa < 1 kDa Y N m 

DHFR 18 kDa < 1 kDa Y Y m - d 

FKBP 12 kDa < 1 kDa Y N/Y m - h 

ACP (PCP) 77 aa < 1 kDa + E N N m - h 

Epitope 5-30 aa 150 kDa N N/Y m - h 

Biotin-ligase 15 aa < 1 kDa + E N N m - h 

NTA 6-10 aa < 1 kDa Y N/Y s 

FlAsh 6-10 aa < 1 kDa Y N/Y m - d 

React (nn)AA 1 aa < 1 kDa N N m - h 

Ligand - 1 - 1000 kDa Y Y s - m 

112

Reversible vs covalent labeling 

Covalent 

+ once it’s there it is there to stay 

- upon photo bleaching your labeled molecule is invisible 

Reversible 

Depending on affinity and off rate complexes can have a 

• short lifetime of seconds to minutes 

- Cannot wash or label in advance 

+ Upon photobleaching can be replaced 

+ Repetitive labeling under same or different conditions 

• long lifetime of hours to days => almost as covalent labeling 

113

Labeling 

The ideal label and labeling method do not exist. 

=> What do you would like to learn and where 

÷ Localisation and movement ÷ In live cells 

÷ Molecular interactions ÷ Reconstituted systems 

÷ Structural changes 

=> Each protein & each process needs an individual approach. 

=> Combine orthogonal methods for multiple specific labeling. 

=> Beware of nonspecific labeling and artefacts. 

=> Combine measuring techniques. 

=> Do lots of careful experiments. 

114

Protein A - esonn

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