4D 1H-13C NMR spectroscopy for assignments of alanine methyls in large and complex protein structures.

Alanine (13)CH(3) methyl groups can serve as a useful addition to the Ile, Leu, Val (ILV) selective isotope labeling methodology adopted for NMR studies of high-molecular-weight protein systems. A four-dimensional (4D) methyl-detected "out-and-back" NMR experiment has been developed that allowed us to obtain practically complete (1)H-(13)C assignments of more than 70 alanine methyl sites in a 723-residue enzyme Malate Synthase G. It can be anticipated that the developed NMR methodology will promote the use of alanine methyls as important probes of molecular structure and dynamics in large proteins.