Probing side-chain dynamics in high molecular weight proteins by deuterium NMR spin relaxation: An application to an 82-kDa enzyme

New NMR experiments for the measurement of side-chain dynamics in high molecular weight (similar to 100 kDa) proteins are presented. The experiments quantify H-2 spin relaxation rates in (CH2D)-C-13 or (CHD2)-C-13 methyl isotopomers and, for applications to large systems, offer significant gains both in sensitivity (2-3-fold) and resolution over previously published HSQC schemes. The methodology has been applied to investigate Ile dynamics in the 723-residue, single polypeptide chain enzyme, malate synthase G. Methyl-axis order parameters, S-axis, characterizing the amplitudes of motion of the methyl groups, have been derived from both (CH2D)-C-13 and (CHD2)-C-13 probes and are in excellent agreement. The distribution of order parameters is trimodal, reflecting the range of dynamics that are available to Ile residues. A reasonable correlation is noted between S-axis(2), and inverse temperature factors from X-ray studies of the enzyme. The proposed methodology significantly extends the range of protein systems for which side-chain dynamics can be studied.