Substitution of membrane-embedded aspartic acids in bacteriorhodopsin causes specific changes in different steps of the photochemical cycle.
Millisecond photocycle kinetics were measured at room temperature for 13 site-specific bacteriorhodopsin mutants in which single aspartic acid residues were replaced by asparagine, glutamic acid, or alanine. Replacement of aspartic acid residues expected to be within the membrane-embedded region of the protein (Asp-85, -96, -115, or -212) produced large alterations in the photocycle. Substitution of Asp-85 or Asp-212 by Asn altered or blocked formation of the M410 photointermediate. Substitution of these two residues by Glu decreased the amount of M410 formed. Substitutions of Asp-96 slowed the decay rate of the M410 photointermediate, and substitutions of Asp-115 slowed the decay rate of the O640 photointermediate. Corresponding substitutions of aspartic acid residues expected to be in cytoplasmic loop regions of the protein (Asp-36, -38, -102, or -104) resulted in little or no alteration of the photocycle. Our results indicate that the defects in proton pumping which we have previously observed upon substitution of Asp-85, Asp-96, Asp-115, and Asp-212 [Mogi, T., Stern, L. J., Marti, T., Chao, B. H., & Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4148-4152] are closely coupled to alterations in the photocycle. The photocycle alterations observed in these mutants are discussed in relation to the functional roles of specific aspartic acid residues at different stages of the bacteriorhodopsin photocycle and the proton pumping mechanism.
Stern
L J
LJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Ahl
P L
PL
Marti
T
T
Mogi
T
T
Duñach
M
M
Berkowitz
S
S
Rothschild
K J
KJ
Khorana
H G
HG
D000409:Alanine; D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry
D005971:Glutamates; D008051:Lipid Bilayers; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine; D000409:Alanine