Structural mapping of chloroplast coupling factor.
Fluorescence resonance energy transfer measurements have been used to investigate the spatial relationships between the nucleotide binding sites and the gamma-subunit of the H+-ATPase from chloroplasts and the orientation of these sites with respect to the membrane surface. Fluorescent maleimides reacted covalently at specific sulfhydryl sites on the gamma-subunit served as energy donors. One sulfhydryl site can be labeled only under energized conditions on the thylakoid membrane surface (light site). The two gamma-sulfhydryls exposed after catalytic activation served as a second donor site (disulfide site). In one set of experiments, the nucleotide analogue 2'(3')-(trinitrophenyl)adenosine triphosphate, selectively bound at each of the three nucleotide binding sites of the solubilized coupling factor, was used as an energy acceptor; in another, octadecylrhodamine with its acyl chain inserted in the vesicle bilayer and the rhodamine fluorophore exposed along the membrane surface was the energy acceptor. The distance between the sulfhydryl and disulfide sites was also obtained by sequentially labeling the sites with coumarin (donor) and fluorescein (acceptor) maleimide derivatives, respectively. The results indicate that all three nucleotide sites are approximately equal to 50 A from the light-labeled gamma-sulfhydryl. Two of the nucleotide sites are very far from the gamma-disulfide (greater than 74 A), while the third site, which binds nucleotides reversibly under all conditions, is 62 A from this sulfhydryl. The light-labeled sulfhydryl and disulfide sites are about 42-47 A apart. Finally, the distance of closest approach between the membrane surface of the reconstituted system and the gamma-disulfide is 31 A.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002736:Chloroplasts / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008301:Maleimides / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D004220:Disulfides; D005456:Fluorescent Dyes; D008301:Maleimides; D009711:Nucleotides; D013438:Sulfhydryl Compounds; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases