Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High-Molecular-Weight Proteins

摘要

Progress in NMR spectroscopy of high-molecular-weight proteins in the last decade has been strongly linked to the development of new isotopic-labeling strategies. A combination of the selective protonation of methyl groups in fully perdeuterated proteins with methyl transverse relaxation optimized spectroscopy (methyl-TROSY) has enabled local structure and dynamics to be studied in protein assemblies of up to 1 MDa in size by solution NMR spectroscopic techniques. Labeling procedures in which specifically [~1H,~(13)C]methyl-labeled biosynthetic precursors are added as the sole proton source in a perdeuterated culture medium can provide a high level of methyl protonation without detectable isotopic scrambling.'41 Maximal deuteration is critical for the optimization of resolution and the intensity of [~1H, ~(13)C]methyl signals in methyl-TROSY experiments.