Transmembrane Protein Structure: Spin Labeling of Bacteriorhodopsin Mutants

American Association for the Advancement of Science (AAAS) - Tập 248 Số 4959 - Trang 1088-1092 - 1990
Christian Altenbach1, Thomas M. Marti2, H. G. Khorana2, Wayne L. Hubbell1
1Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90024-7008
2Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.

Tóm tắt

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 α helix. The orientation of this helical segment with respect to the remainder of the protein was determined.

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American Association for the Advancement of Science (AAAS)

Tập 248 Số 4959

1088-1092

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