LOCALIZATION OF METHIONINE RESIDUES IN BACTERIORHODOPSIN BY CARBONYL C-13-NMR WITH SEQUENCE-SPECIFIC ASSIGNMENTS

High-resolution C-13-NMR experiments have been performed on bacteriorh odopsin biosynthetically labeled with carbonyl-C-13 amino acids and so lubilized in the detergent dodecylmaltoside. C-13-NMR spectra showing good resolution were obtained in the case of labeled amino acids moder ately represented in the BR sequence. For BR labeled with [C-13]carbon yl methionine, several sequence-specific assignment could be performed by co-labeling with N-15 amino acids or proteolysis. These assignment s were used to obtain structural data on BR. Water-exposure of methion ine side chains in the protein was assessed by studying, using NMR, th eir oxidation by hydrogen peroxide. Local secondary structure at the l evel of methionine residues was monitored through the effect of H-1-H- 2 exchange on NMR spectra. It was concluded that Met32, Met68 and Met1 63 are peripheral while all 6 other methionine residues are deeply emb edded within hydrophobic alpha-helices. These results confirm the curr ent model of the BR folding and secondary structure.