Features of F(1)-ATPase catalytic and noncatalytic sites revealed by fluorescence lifetimes and acrylamide quenching of specifically inserted tryptophan residues.
Catalytic and noncatalytic nucleotide sites of the F(1) sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F(1) enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg(2+). Residue beta-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F(1). Results here showed that in betaY331W mutant F(1), each of the three beta-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.
Weber
J
J
Department of Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, Rochester, New York 14642, USA. joachim_weber@urmc.rochester.edu
Senior
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AE
D020106:Acrylamide / Q000494:pharmacology; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D009711:Nucleotides; D020106:Acrylamide; D014364:Tryptophan; D006180:Proton-Translocating ATPases; D008274:Magnesium