Effect of introducing different carboxylate-containing side chains at position 85 on chromophore formation and proton transport in bacteriorhodopsin.
During the initial stages of the bacteriorhodopsin photocycle, a proton is transferred from the Schiff base to the deprotonated carboxylate of Asp85. Earlier studies have shown that replacement of Asp85 by Asn completely abolishes proton transport activity, whereas extension of the side chain by an additional carbon-carbon bond (Asp85-->Glu) results in a functional proton pump. Here we show that extension of the Asp85 side chain by two additional bond lengths also results in a functional proton pump as long as the terminal group is a carboxylate moiety. These side chains were created by modification of the cysteine residue in the Asp85-->Cys mutant with either iodoacetic acid or iodoacetamide. In vitro chromophore formation studies show that the rate of Schiff base protonation in mutants that contain a carboxylate at residue 85 is invariably faster than in mutants that contain neutral substitutions at this position. We conclude that in bacteriorhodopsin, there is considerable tolerance in the volume of the side chain that can be accommodated at position 85 and that the presence of a carboxylate at residue 85 is important both for proton pumping and for stabilizing the protonated Schiff base.
Greenhalgh
D A
DA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Subramaniam
S
S
Alexiev
U
U
Otto
H
H
Heyn
M P
MP
Khorana
H G
HG
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D005971:Glutamates; D018698:Glutamic Acid; D019617:Halobacterium salinarum / Q000378:metabolism*; D007460:Iodoacetamide / Q000378:metabolism; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D005971:Glutamates; D007461:Iodoacetates; D011522:Protons; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D003545:Cysteine; D019807:Iodoacetic Acid; D007460:Iodoacetamide