Locations of Arg-82, Asp-85, and Asp-96 in helix C of bacteriorhodopsin relative to the aqueous boundaries.
The amino acids Asp-96, Asp-85, and Arg-82, which are important for proton transport by bacteriorhodopsin, are located in helix C. Site-directed spin labeling has been used to map their positions relative to the aqueous boundaries of the membrane. Selected amino acids in helix C, in the B-C loop on the extracellular side, and in the C-D loop on the intracellular side of the membrane were replaced by cysteine residues and derivatized with a sulfhydryl-specific spin label. The topographical locations of the nitroxide groups were determined by electron paramagnetic resonance spectroscopy in terms of both motional restriction and collision frequencies with dissolved molecular oxygen and membrane-impermeable chromium oxalate. The results show that in dark-adapted bacteriorhodopsin, Tyr-79 is at the aqueous-protein interface on the extracellular side of helix C whereas Val-101 is close to the aqueous boundary on the intracellular side of the protein. Further, Asp-96 is estimated to be within 7 A of the aqueous medium on the intracellular side of the membrane, whereas Arg-82 and Asp-85 are within 5 A and 9 A, respectively, of the aqueous boundary on the extracellular side of the membrane.
Greenhalgh
D A
DA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Altenbach
C
C
Hubbell
W L
WL
Khorana
H G
HG
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000648:ultrastructure*; D001693:Biological Transport, Active; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D006217:Halobacterium; D008055:Lipids / Q000737:chemistry; D008698:Mesylates / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013113:Spin Labels; D013329:Structure-Activity Relationship
D008055:Lipids; D008698:Mesylates; D013113:Spin Labels; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001120:Arginine; D003545:Cysteine