STRUCTURE-FUNCTION STUDIES ON BACTERIORHODOPSIN .8. SUBSTITUTIONS OF THE MEMBRANE-EMBEDDED PROLINE-50, PROLINE-91, AND PROLINE-186 - THE EFFECTS ARE DETERMINED BY THE SUBSTITUTING AMINO-ACIDS

  • 25 August 1989
    • journal article
    • research article
    • Vol. 264  (24) , 14192-14196
Abstract
To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded .alpha.-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50 .fwdarw. Ala, Pro-91 .fwdarw. Ala and .fwdarw. Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50 .fwdarw. Gly was 10-fold faster than in the wild-type. While, Pro-186 .fwdarw. Ala regenerated the wild-type chromophore, the mutants Pro-186 .fwdarw. Val and Pro-186 .fwdarw. Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186 .fwdarw. Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentrations restored the wild-type purple chromophore in the Pro-186 .fwdarw. Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186 .fwdarw. Ala and Pro-186 .fwdarw. Gly showed about 65%, whereas Pro-186 .fwdarw. Val showed 10-20% of the normal proton pumping.

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