Alanine Methyl Groups as NMR Probes of Molecular Structure and Dynamics in High-Molecular-Weight Proteins

The importance and utility of Ala(beta) methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins is explored. Using H-2 and C-13 relaxation measurements in {U-H-2; Ala(beta)-[(CHD2)-C-13]}-labeled Malate Synthase G (MSG)-an 82-kDa monomeric enzyme that contains 73 Ala(beta) methyl groups we show that the vast majority of selectively labeled Ala(beta) methyls are highly ordered. A number of NMR applications used for solution studies of structure and dynamics of large protein molecules can benefit from proximity of Ala(beta) methyls to the protein backbone and their high degree of ordering. In the case of MSG, these applications include the measurement of H-1-C-13 residual dipolar couplings in Ala(beta) methyls, characterization of slow (mu s-to-ms) dynamics at the substrates' binding sites, and methyl-TROSY-based NOE spectroscopy performed on {U-H-2; Ala(beta)-[(CH3)-C-13]; Ile(delta 1)-[(CH3)-C-13]; Leu,Val-[(CH3)-C-13/(CD3)-C-12]}-labeled samples where the number of methyl probes for derivation of distance restraints is maximized compared to the state-of-the-art ILV labeling methodology.