Membrane-protein structural mapping of chloroplast coupling factor in asolectin vesicles.
The spatial relationship of specific sites on chloroplast coupling factor, reconstituted in asolectin vesicles, to the bilayer surface has been studied with fluorescence methods. Fluorescence resonance energy transfer measurements have been used to map the distances of closest approach of the N,N'-dicyclohexylcarbodiimide-binding site and the disulfide on the gamma-polypeptide to the bilayer center. The dicyclohexylcarbodiimide site was labeled with N-cyclohexyl-N'-pyrenylcarbodiimide and the gamma-disulfide site with a coumarinyl derivative. The bilayer center was labeled with 25-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-N-methylamino]-27-norc holesterol. The distances obtained, 15 and 43 A, respectively, were combined with previous measurements of the distance of closest approach between these sites and the membrane surface to estimate the perpendicular distances of the sites from the membrane surface. The depth of the dicyclohexylcarbodiimide site was also determined by studying the quenching of fluorescence by 5-, 7-, 12-, and 16-doxylstearic acids. The model developed suggests that the dicyclohexylcarbodiimide site is 6-10 A below the membrane surface and the gamma-disulfide is 16 A above the membrane surface. The distances measured are subject to a considerable uncertainty, but the proposed model provides a useful starting point for further structural studies.
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Department of Chemistry, Cornell University, Ithaca, New York 14853.
Hammes
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D001665:Binding Sites; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D004024:Dicyclohexylcarbodiimide; C024990:asolectin; D006180:Proton-Translocating ATPases