[2,3-13C]-labeling of aromatic residues-getting a head start in the magic-angle-spinning NMR assignment of membrane proteins

The assignment of magic-angle spining NMR speara of large membrane proteins such as the 281-residue ouler membrane protein G (OmpG) is difficult because of substantial signal overlap and line-broadening effects. To obtain sequence specific assignments a new labeling sitalegy was applied to OmpG in which alanine and glycine were uniformly (N-15, C-13] labeled and phenylalanine and tyrosine were [N-15, C alpha,C beta-C-13]-labeled. This labeling pattern resulted in many long-range inter-residue cross- peaks being observed in spectra recorded with long mixing times. In addition, a reduction in the number of C' labels resulted in NCO-type spectra with little overlap and provided the basis for unambiguous sequential assignments. In conjuntion with spectra from [1,3-C-13]. and [2-C-13]-glycerol samples, a total of 45 reliable sequence specific assignments could be made for OmpG on the basis of this sample.