Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.
Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85----Glu and Arg-82----Ala mutants exist in a pH-dependent equilibrium between purple (lambda max approximately 550/540 nm) and blue (lambda max approximately 600/590 nm) forms of the pigment. Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85----Asn mutant displays a blue chromophore (lambda max approximately 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form. In contrast, the Asp-212----Asn mutant is purple (lambda max approximately 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (i) the pKa of the purple-to-blue transition is directly influenced by the pKa of the carboxylate at residue 85 and (ii) the relative strengths of interaction between the protonated Schiff base, Asp-85, Asp-212, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.
Subramaniam
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S
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Marti
T
T
Khorana
H G
HG
D000409:Alanine; D001120:Arginine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D006217:Halobacterium / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011522:Protons; D013053:Spectrophotometry
D005971:Glutamates; D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D001120:Arginine; D000409:Alanine