title: Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants.
abstract: Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.
authors.affiliation: Jules Stein Eye Institute, University of California, Los Angeles 90024-7008.
authors.forename: C
authors.initials: C
authors.lastname: Altenbach
authors.forename: T
authors.initials: T
authors.lastname: Marti
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
authors.forename: W L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010070:Oxalates; D019815:Oxalic Acid; D010100:Oxygen; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D008565:Membrane Proteins; D010070:Oxalates; D013113:Spin Labels; D001436:Bacteriorhodopsins; D019815:Oxalic Acid; D003545:Cysteine; D010100:Oxygen