Assigning methyl resonances for protein solution-state NMR studies

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has helped to substantially expand the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to expand, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (> 50 kDa) proteins. In this practical review, we present strategies for efficiently isotopically labeling proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.