High-resolution 13C NMR study of the topography and dynamics of methionine residues in detergent-solubilized bacteriorhodopsin.
The proton transport membrane protein bacteriorhodopsin has been biosynthetically labeled with [methyl-13C]methionine and studied by high-resolution 13C NMR after solubilization in the detergent Triton X-100. The nine methionine residues of bacteriorhodopsin give rise to four well-resolved 13C resonances, two of which are shifted upfield or downfield due to nearby aromatic residues. Methionine residues located on the hydrophilic surfaces, on the hydrophobic surface, and in the interior of the protein could be discriminated by studying the effects of papain proteolysis, glycerol-induced viscosity increase, and paramagnetic broadening by spin-labels on NMR spectra. Such data were used to evaluate current models of the bacteriorhodopsin transmembrane folding and tertiary structure. T2 and NOE measurements were performed to study the local dynamics of methionine residues in bacteriorhodopsin. For the detergent-solubilized protein, hydrophilic and hydrophobic external residues undergo a relatively large extent of side chain wobbling motion while most internal residues are less mobile. In the native purple membrane and in reconstituted bacteriorhodopsin liposomes, almost all methionine residues have their wobbling motion severely restricted, indicating a large effect of the membrane environment on the protein internal dynamics.
Seigneuret
M
M
URA-CNRS 1290, CEN Saclay, Gif-sur-Yvette, France.
Neumann
J M
JM
Levy
D
D
Rigaud
J L
JL
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D003902:Detergents; D006217:Halobacterium / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008715:Methionine; D008961:Models, Structural; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D011487:Protein Conformation; D012995:Solubility; D013113:Spin Labels; D013816:Thermodynamics
D002247:Carbon Isotopes; D003902:Detergents; D008081:Liposomes; D013113:Spin Labels; D001436:Bacteriorhodopsins; D008715:Methionine; D010206:Papain