Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore

Janos K. Lanyi, Jörg Tittor, György Váró, Günther Krippahl, Dieter Oesterhelt

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

The 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 pKi values (about 5.4 adn 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. expressed Both blue and red forms of the protein in are in the two-dimensional crystalline state. A single pKi, such as in the E. coli protein, was observed only after solubilization with detergent. The photocyle 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 B-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodepsin 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 pKi lot 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 teh Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.

Original languageEnglish
Pages (from-to)102-110
Number of pages9
JournalBBA - Bioenergetics
Volume1099
Issue number1
DOIs
Publication statusPublished - Jan 30 1992

Keywords

  • (Halobacterium sp. GRB strain 384)
  • Bacteriorhodopsin
  • Bacteriorhodopsin structures
  • Chromophore

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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