bi060177r_si_001.pdf (7.51 kB)
Hydration and Packing along the Folding Pathway of SH3 Domains by Pressure-Dependent NMR†
journal contribution
posted on 2006-04-18, 00:00 authored by Irina Bezsonova, Dmitry M. Korzhnev, R. Scott Prosser, Julie D. Forman-Kay, Lewis E. KayThe volumetric properties associated with protein folding transitions reflect changes in protein
packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation
techniques are employed to probe the folding−unfolding kinetics of two SH3 domains as a function of
pressure so that the changes in partial molar volumes along the folding pathway can be measured. The
two domains fold with rates that differ by ∼3 orders of magnitude, so their folding dynamics must be
probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via
a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is
employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond
time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is
extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial
molar volumes can be obtained using low pressures (1−120 bar), thus minimizing perturbations to any
of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent
with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that
are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic
processes over a wide range of time scales.