A ²H NMR Relaxation Experiment for the Measurement of the Time Scale of Methyl Side-Chain Dynamics in Large Proteins

An NMR experiment is presented for the measurement of the time scale of methyl side-chain dynamics in proteins that are labeled with methyl groups of the ¹³CHD₂ variety. The measurement is accomplished by selecting a magnetization mode that to excellent approximation relaxes in a single-exponential manner with a T₁-like rate. The combination of R₁(¹³CHD₂) and R₂(¹³CHD₂) ²H relaxation rates facilitates the extraction of motional parameters from ¹³CHD₂-labeled proteins exclusively. The utility of the methodology is demonstrated with applications to proteins with tumbling times ranging from 2 ns (protein L, 7.5 kDa, 45 °C) to 54 ns (malate synthase G, 82 kDa, 37 °C); dynamics parameters are shown to be in excellent agreement with those obtained in ²H NMR studies of other methyl isotopomers. A consistency relationship is found to exist between R₁(¹³CHD₂) and the relaxation rates of pure longitudinal and quadrupolar order modes in ¹³CH₂D-labeled methyl groups, and experimental rates measured for a number of proteins are shown to be in excellent agreement with expectations based on theory. The present methodology extends the applicability of ²H relaxation methods for the quantification of side-chain dynamics in high molecular weight proteins.