Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.
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Journal: 1988/July - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 3288985
Abstract:
We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -112 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their lambda max varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85----Asn mutant showed not detectable proton pumping, the Asp-96----Asn and Asp-212----Glu mutants showed less than 10% and the Asp-115----Glu mutant showed approximately equal to 30% of the normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.
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Proc Natl Acad Sci U S A 85(12): 4148-4152
PMID: 3288985

Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

Abstract

We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -112 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their lambda max varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85----Asn mutant showed not detectable proton pumping, the Asp-96----Asn and Asp-212----Glu mutants showed less than 10% and the Asp-115----Glu mutant showed approximately equal to 30% of the normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
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Abstract
We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -112 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their lambda max varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85----Asn mutant showed not detectable proton pumping, the Asp-96----Asn and Asp-212----Glu mutants showed less than 10% and the Asp-115----Glu mutant showed approximately equal to 30% of the normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.
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