A new labeling method for methyl transverse relaxation-optimized spectroscopy NMR spectra of alanine residues

The development of specific methyl labeling schemes and transverse relaxation-optimized spectroscopy (TROSY) has extended the molecular size range for the application of NMR spectroscopy to proteins. Generally, methyl groups of isoleucine, leucine, valine residues are specifically protonated in a highly deuterated background and H-1-C-13 correlation experiments provide a means to study structure and dynamics in multimeric complexes with molecular weights far in excess of 100 kDa. We have extended this approach to alanine residues which offers several potential advantages, including its high abundance and wide distribution in protein sequences together with a high tolerance to mutation. We have developed an efficient method for the synthesis and incorporation of L-alanine-3-C-13,2-H-2 into protein sequences. We also demonstrate the usefulness of specific protonation of alanine residues in combination with methyl TROSY experiments on the 306 kDa fragment of the eukaryotic AAA-ATPase, p97, in complex with one of its many adaptor proteins.