Ubiquitin Conformational Dynamics and Hydration Shell Dynamics by Solid State NMR
Knowledge of protein dynamics is essential for understanding the enzyme catalysis, protein molecular recognition, and the entropy changes during protein-ligand binding. Nuclear Magnetic Resonance (NMR) is an ideal biophysical experimental tool for the study of protein dynamics because it can be used to study motions with timescales from picoseconds to seconds. NMR studies of solids are different to those in liquids in that the spatial anisotropy of magnetic interactions such as quadrupolar couplings, dipolar couplings, and chemical shift anisotropy (CSA) are retained. The amplitude of motion in the timescale faster than microseconds can be quantified by the time averaged anisotropic interactions, so solid state NMR has many of unique opportunities to study dynamics. Studies of protein dynamics using deuterium spectra are powerful because the deuterium quadrupolar coupling is larger than any other anisotropic interactions and dominates the deuterium spectra. However, large quadrupolar interactions also introduce complexity for site-resolved dynamics experiments such as 2H-13C CP correlation experiments during magic angle spinning. In this work, we developed a method to quantify protein site-specific amplitude of motions (order parameters) using 2H-13C CP correlation experiments and tested this method on the amino acid model compounds and ubiquitin microcrystalline samples. The ubiquitin backbone order parameters we obtained are very similar to the previously reported order parameters except that we cannot obtain the order parameter information for some dynamical loop regions and the N-terminal part of the á helix. We also solved the ubiquitin crystal structure at the crystallization condition similar to the SSNMR sample condition. These structures allow us to correlate SSNMR parameters with the high resolution X-ray structure and will be of benefit to further method development in SSNMR field. We also found an interesting ubiquitin conformational switch. This conformational switch region shows inherent plasticity and probably plays a role when ubiquitin binds with deubiquinating enzymes. In the last part of the work, we study the dynamical properties of ubiquitin's hydration shell in frozen solution using deuterium spectra. We also study the effect of some Hofmister ions on the dynamical properties of ubiquitin's hydration water and on bulk ice.
Chemistry, Biochemistry, Nuclear magnetic resonance, Catalysis, Enzymes, Deuterium, Ubiquitin
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