APS March Meeting 2011
Volume 56, Number 1
Monday–Friday, March 21–25, 2011;
Dallas, Texas
Abstract: Q4.00001 : Formation of protein-complexes in crowded environments: from in vitro to in vivo
11:15 AM–11:51 AM
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Author:
Rates of protein interactions are one to five orders of magnitude
slower
than the theoretically calculated collision rate of spheres of
the same
size. The rates can be increased by favorable electrostatic
forces between
the two proteins. Recent studies have established that the
association
reaction proceeds through transient complexes, which may be
specific or
diffusive in nature. To bring binding studies closer to the in vivo
environment, we investigated the role of crowding on binding. For
crowding
we added various polymers to the solution, including Dextran and
PEGs of
different molecular weights. While crowding enhances
oligomerization and
polymerization of macromolecules, it has only a small effect on
the binding
rates and affinities of transient protein-protein interactions.
We suggest
that the limited effect of crowders, which is much bellow the
expected from
the increased viscosity of the solutions, is a result of the
occluded volume
effect in high crowder concentrations. Direct measurements of the
stability
of the encounter complex shows that crowders slow both k1 and
k-1, resulting
in an increased half-life of the encounter complex. High crowder
concentrations also slow k2, suggesting an increased size of the
encounter
region. These results fit double-mutant cycle measurements on the
activated
complex, which suggest an increased size of the fruitful
encounter region.
These results are in line with the suggested occluded volume
effect of
crowders. We contrasted these with the effect of crowding on the
weak
binding pair CyPET-YPET. On this pair, aggregation, and not enhanced
dimerization, was detected in PEG solutions. The results suggest
that
typical crowding agents have only a small effect on specific
protein-protein
dimerization reactions while promoting aggregation. To further
validate
these results, we performed real time binding assays in living
cells,
showing that even in the crowded cellular environment binding can
be fast
and specific.
To cite this abstract, use the following reference: http://meetings.aps.org/link/BAPS.2011.MAR.Q4.1