Uv-visible spectroscopy of bacteriorhodopsin mutants: substitution of Arg-82, Asp-85, Tyr-185, and Asp-212 results in abnormal light-dark adaptation.
The light-dark adaptation reactions of a set of bacteriorhodopsin (bR) mutants that affect function and color of the chromophore were examined by using visible absorption spectroscopy. The absorbance spectra of the mutants Arg-82 in equilibrium Ala (Gln), Asp-85 in equilibrium Ala (Asn, Glu), Tyr-185 in equilibrium Phe, and Asp-212 in equilibrium Ala (Asn, Glu) were measured at different pH values during and after illumination. None of these mutants exhibited a normal dark-light adaptation, which in wild-type bR causes a red shift of the visible absorption maximum from 558 nm (dark-adapted bR) to 568 nm (light-adapted bR). Instead a reversible light reaction occurs in the Asp-85 and Asp-212 mutants from a blue form with lambda max near 600 nm to a pink form with lambda max near 480 nm. This light-induced shift explains the appearance of a reversed light adaptation previously observed for the Asp-212 mutants. In the case of the Tyr-185 and Arg-82 mutants, light causes a purple-to-blue transformation similar to the effect of lowering the pH. However, the blue forms observed in these mutants are not identical to those formed by acid titration or deionization of wild-type bR. It is suggested that in all of these mutants, the chromophore has lost the ability to undergo the normal 13-cis, 15-syn to all-trans, 15-anti light-driven isomerization, which occurs in native bR. Instead these mutants may have as stable forms all-trans,syn and 13-cis,anti chromophores, which are not allowed in native bR, except transiently.
Duñach
M
M
Physics Department, Boston University, MA 02215.
Marti
T
T
Khorana
H G
HG
Rothschild
K J
KJ
D001120:Arginine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D003624:Darkness; D006217:Halobacterium / Q000378:metabolism; D008027:Light; D016297:Mutagenesis, Site-Directed; D012172:Retinaldehyde / Q000302:isolation & purification; D013053:Spectrophotometry; D014443:Tyrosine
D001224:Aspartic Acid; D014443:Tyrosine; D001436:Bacteriorhodopsins; D001120:Arginine; D012172:Retinaldehyde