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RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP‐85, ASP‐96, AND ARG‐82
Author(s) -
LIN STEVEN W.,
FODOR STEPHEN P. A.,
MIERCKE LARRY J. W.,
SHAND RICHARD F.,
BETLACH MARY C.,
STROUD ROBERT M.,
MATHIES RICHARD A.
Publication year - 1991
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1991.tb03638.x
Subject(s) - bacteriorhodopsin , raman spectroscopy , mutant , chemistry , resonance (particle physics) , spectral line , crystallography , stereochemistry , biochemistry , physics , optics , gene , atomic physics , membrane , astronomy
— Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site‐directed mutagenesis (Asp‐96→Asn, D96N; Asp‐85→Asn, D85N; and Arg‐82→Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light‐adapted (BR LA ) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BR LA states of D85N and especially R82Q contain more of the 13‐ cis , C=N syn (BR 555 ) species under ambient illumination compared to solubilized native BR. Replacement of Asp‐85 with Asn causes a 25 nm red‐shift of the absorption maximum and a frequency decrease in both the ethylenic (‐7 cm −1 ) and the Schiff base C=NH + (‐3 cm −1 ) stretching modes of BR LA . These changes indicate that Asp‐85 is located close to the protonated retinal Schiff base. The BR LA spectrum of R82Q exhibits a slight perturbation of the C=NH + band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp‐85 − , Arg‐82 + and presumably Asp‐212 − . These data are consistent with a model where the strength of the protein‐chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.