Research Report 2000 - MDC

Interactions of
Biopolymers in
Solution
Joachim Behlke
Our group is engaged in the analysis
of the structure of proteins and nucleic
acids in solution and their interactions
using analytical ultracentrifugation
methods. Special programs have been
developed that allow us to determine
the gross conformation of polymers,
self- and hetero-association as well as
parameters of thermodynamic
nonideality. The substances
investigated are of medical and
biotechnological relevance and the
data obtained may help us understand
possible regulatory mechanisms of
transcription or protein folding and
metabolic pathways within the cell.
44
Gross conformation of peptides
To obtain estimates of the possible
shape of angiotensin peptides which
bind to the AT1 receptor (seventransmembrane-helixG-proteincoupled
complex), we have analysed
the gross conformation of these
peptides using measurements of
hydrodynamic mobility and
theoretical calculations. The most
probable, extended structure of
angiotensin 2, about 3 nm in length
with a kink, seems to penetrate
approximately 2 nm into the AT1
receptor where it binds to specific
amino acids and induces the complex
reaction.
Regulation of oligomeric protein
structures and their
consequences
Collaborations with E.-C. Müller,
A. Otto, MDC, and T. Kriegel, TU
Dresden (hexokinase), P. Tavares,
Inst. Pasteur, Paris (portal protein
SPP1) and S. Brantl and
K. Steinmetzer, Univ. Jena (CopR)
Homodimeric hexokinase 2 from
Saccharomyces cerevisiae has one
phosphorylation site at Ser 14. This
modification is triggered in vivo by
glucose exhaustion. We have shown
that, following site-directed
mutagenesis (Ser 14 exchange by Glu)
or phosphorylation, the dimeric
enzyme dissociates completely into
monomers. We assume that the in vivo
phosphorylation at Ser 14, as
transiently occurs in low glucose
states, may be a mechanism to
improve glucose utilization at low
levels and / or that nuclear
localization of the monomer may be
involved in signal transduction
whereby glucose causes catabolite
repression.
Bacteriophage SPP1 portal protein is a
large cyclic homo-oligomer composed
of 13 subunits. It is stable in the
presence of 10-50 mM MgCl 2.
Decreasing electrolyte concentration
leads to a reversible dissociation into
monomers which are partially
unfolded. The reassociation of
monomers into the 13-mers requires a
chaperone-independent folding of
monomers in the presence of Mg ++ .
CopR binds as a dimer with high
affinity to two consecutive major
grooves (site I and site II) of the DNA
(K D = 0.4 nM). The complex
formation is a coupled process and its
analysis requires knowledge of the
preceding CopR dimerization which
has a dissociation constant of 1.4 µM.
Since the cellular concentration of
CopR is about 20-fold higher than the
dimerization constant we can assume
that CopR binds in vivo as a
preformed dimer.
Recognition of peptide
sequences at the interface of
homodimeric proteins
Collaboration with W. Höhne,
Humboldt-Univ., Berlin
To map the putative dimerization site
in the capsid protein p24 (HIV-1) a set
of overlapping peptides spanning the
p24 sequence was synthesized and
tested for the ability to modify the
monomer-dimer equilibrium. Most of
the candidates were inactive.
However, one peptide was found to
compete with the monomers in the
dimerization reaction. This sequence,
therefore, may be part of the contact
region between two monomers.
Nucleic-acid protein interaction
Collaboration with A. Rich, MIT,
Cambridge, MA, and H. Oschkinat,
Inst. of Molecular Pharmacology,
Berlin
The Z domain of the human RNA
editing enzyme double-stranded RNA
deaminase I (ADAR1) binds to lefthanded
Z-DNA with high affinity
(K D = 30 nM). Using sedimentation
equilibrium techniques and CD
spectroscopy, we found that two Z
domains bind to one d(CG) 3T 4(CG) 3
hairpin which contains a stem of six
base pairs in the Z-DNA conformation.
We suggest that short segments (6 bp)
of the Z-DNA within a gene are able
to recruit two ADAR1 enzymes to that
particular site.