Methyl Groups as Probes of Structure and Dynamics in NMR Studies of High‐Molecular‐Weight Proteins

Methyl groups are of particular interest in NMR studies of proteins since they occur frequently in the hydrophobic cores of these molecules and thus are often sensitive reporters of structure and dynamics. Methyl probes can play a very important role in applications that involve high-molecular-weight proteins because of favorable properties that facilitate the recording of NMR spectra with high sensitivity and resolution. First, the threefold degeneracy of methyl protons in CH3 isotopomers (CH3, CH2D, and CHD2 methyls will be considered in this review) effectively increases the concentration of each group significantly beyond that for, say, backbone amides. Second, because methyl groups are localized at the peripheries of side chains, many tend to be dynamic; this leads to slower relaxation that can be exploited in studies of large systems. Third, in the past few years it has become possible to produce proteins in which methyl groups are selectively protonated in a highly deuterated background; this leads to further enhanced relaxation properties that greatly increase the size of systems that can be studied. Fourth, distances between proximal methyl groups, established on the basis of NOEs, often connect regions of the molecule that are far removed in primary structure. In addition, these moieties serve as probes in investigations of protein–ligand interactions, 10] fast and slow timescale side-chain dynamics, dynamics of protein folding, and in the detection of proteins and complexes in in-cell NMR experiments. In this Minireview, we focus on using methyl groups to study both structure and dynamics in high-molecular-weight proteins. A key aspect has been the interplay between new isotope-labeling methodology and NMR techniques that are specifically designed for a given labeling pattern. Thus, a description of the new labeling approaches is first presented, followed by a brief summary of the NMR experiments that have been developed for site-specific methyl assignments. The relaxation properties of methyl groups are discussed, and basic principles of methyl-TROSY spectroscopy are presented. Finally, a number of practical applications involving global protein-fold determination and studies of side-chain dynamics are described. The approaches and concepts described here are illustrated with applications to the enzyme malate synthase G (MSG) from E. coli—a monomeric 723-residue protein (82 kDa) that has been extensively characterized by NMR in our laboratory over the past several years and whose global fold has been recently derived de novo from NMR data exclusively. MSG is a four-domain enzyme that catalyzes the Claisen condensation of glyoxylate and acetyl-CoA to produce malate and is a part of a biosynthetic bypass (“glyoxylate shunt”) that is activated in many pathogenic microorganisms under anaerobic conditions. Since the glyoxylate shunt is absent in man, the enzymes of this bypass have recently been recognized as potential targets for drug design to improve existing antibiotic agents.