Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase.
The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F). CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322. Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45%. The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57. These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted epsilon subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1.
Johnson
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Department of Biology, Johns Hopkins University, Baltimore, MD 21218-2685, USA.
Evron
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McCarty
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D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000378:metabolism; D004252:DNA Mutational Analysis; D004735:Energy Transfer / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008301:Maleimides / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea; D013439:Sulfhydryl Reagents / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D003545:Cysteine; D005456:Fluorescent Dyes; D008301:Maleimides; D010649:Phenylalanine; D011506:Proteins; D006180:Proton-Translocating ATPases; D013439:Sulfhydryl Reagents; D014364:Tryptophan; D000097795:ATPase Inhibitory Protein; C003944:N-(3-pyrene)maleimide