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

摘要

The importance and utility of Ala~β methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins is explored. Using ~2H and ~(13)C relaxation measurements in {U-~2H; Ala~β-[~(13)CHD_2]}-labeled Malate Synthase G (MSG)—an 82-kDa monomeric enzyme that contains 73 Ala~β methyl groups—we show that the vast majority of selectively labeled Ala~β 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~β methyls to the protein backbone and their high degree of ordering. In the case of MSG, these applications include the measurement of ~1H-~(13)C residual dipolar couplings in Ala~β methyls, characterization of slow (μs-to-ms) dynamics at the substrates' binding sites, and methyl-TROSY-based NOE spectroscopy performed on {U-~2H; Ala-[~(13)CH_3]; lle~(δ1)-[~(13)CH_3]; Leu,Val-[~(13)CH_3/~(12)CD_2]}-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.