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Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophoreThe consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.
Document ID
20050000797
Acquisition Source
Legacy CDMS
Document Type
Reprint (Version printed in journal)
Authors
Lanyi, J. K.
(University of California Irvine)
Tittor, J.
Varo, G.
Krippahl, G.
Oesterhelt, D.
Date Acquired
August 22, 2013
Publication Date
January 30, 1992
Publication Information
Publication: Biochimica et biophysica acta
Volume: 1099
Issue: 1
ISSN: 0006-3002
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: GM 29498
Distribution Limits
Public
Copyright
Other
Keywords
Non-NASA Center
NASA Discipline Exobiology

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