Site-Specific Protein Internal Motions Revealed by 2H Solid-State NMR Spectroscopy

Abstract

Protein dynamics contribute significantly to biological events such as ligand binding, allosteric regulation, enzyme catalysis and signal transduction. In comparison with the backbone, protein side chains often present more diverse dynamics, which remain poorly understood for insoluble and membrane proteins at atomic resolution due to limitations of experimental techniques. We have designed a new 2H-13C-13C solid-state nuclear magnetic resonance (SSNMR) technique that enables extraction of 2H quadrupolar tensor parameters and relaxation times with high sensitivity and accuracy. It thereby provides access to site-resolved protein side chain motional amplitudes and timescales. Herein we present the backbone and side chain dynamics of microcrystalline β1 immunoglobulin binding domain of protein G (GB1) that are elucidated on a per-site basis with the newly developed 2H SSNMR technique. Complex side chain motional networks were observed and demonstrated, remarkably, to correlate with protein binding activities. For example, large-amplitude side chain motions were observed for regions of GB1 contacting and interacting with immunoglobulin G (IgG). In contrast, rigid side chains were primarily found for residues in the structural core of GB1 that are absent from protein binding and interactions. The revealed protein internal motions provide new insights into the biological roles for side chain dynamics, which directly correlate to conformational entropy and have crucial contributions in energy changes upon the occurrences of biological events.