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Science 1 June 1990:
Vol. 248. no. 4959, pp. 1088 - 1092
DOI: 10.1126/science.2160734
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Articles

Science, Vol 248, Issue 4959, 1088-1092
Copyright © 1990 by American Association for the Advancement of Science

articles

Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants

C Altenbach, T Marti, HG Khorana, and WL Hubbell

Jules Stein Eye Institute, University of California, Los Angeles 90024-7008.

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.


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