Chromophore of Bacteriorhodopsin is Closer to the Cytoplasmic Surface of Purple Membrane: Fluorescence Energy Transfer on Oriented Membrane Sheets

Jun Otomo, Akihiro Tomioka, Kazuhiko Kinosita, Hidetake Miyata, Yuko Takenaka, Tsutomu Kouyama, Akira Ikegami

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10 Citations (Scopus)

Abstract

Transmembrane location of the retinal chromophore, either native or reduced in situ to a fluorescent derivative, of the purple membrane of Halobacterium halobium was investigated with fluorescence energy transfer techniques. Single sheets of purple membrane, either native or reduced with borohydride, were adsorbed on polylysine-coated glass; the orientation, whether the exposed surfaces were cytoplasmic or extracellular, was controlled by adjusting the pH of the membrane suspension before the adsorption. On the exposed surface of the reduced membrane, a layer of cytochrome c, hemoglobin, or ferritin was deposited. The rate of excitation energy transfer from the fluorescent chromophore in the membrane to the colored protein was greater when the protein was on the cytoplasmic surface of the membrane than when it was on the extracellular surface. Analysis in which uniform distribution of the protein on the surface was assumed showed that the reduced chromophore is situated at a depth of <1.5 nm from the cytoplasmic surface. The location of the native retinal chromophore was examined by depositing a small amount of tris(2,2′-bipyridyl)ruthenium(II) complex on the native membrane adsorbed on the glass. Energy transfer from the luminescent complex to the retinal chromosphore was more efficient on the cytoplasmic surface than on the extracellular surface, suggesting that the native chromophore is also on the cytoplasmic side. From these and previous results we conclude that the chromophore, whether native or reduced, of bacteriorhodopsin is located at a depth of 1.0 ± 0.3 nm from the cytoplasmic surface of purple membrane.

Original languageEnglish
Pages (from-to)57-64
Number of pages8
JournalBiophysical Journal
Volume54
Issue number1
DOIs
Publication statusPublished - 1988 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics

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