title: Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence.
abstract: Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gammaT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm2 s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.
authors.affiliation: Institut für Physikalische Chemie, Universität Freiburg, Germany. boersch@ruf.uni-freiburg.de
authors.forename: M
authors.initials: M
authors.lastname: Börsch
authors.forename: P
authors.initials: P
authors.lastname: Turina
authors.forename: C
authors.initials: C
authors.lastname: Eggeling
authors.forename: J R
authors.initials: JR
authors.lastname: Fries
authors.forename: C A
authors.initials: CA
authors.lastname: Seidel
authors.forename: A
authors.initials: A
authors.lastname: Labahn
authors.forename: P
authors.initials: P
authors.lastname: Gräber
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D004926:Escherichia coli; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
chemical_list: D009711:Nucleotides; D006180:Proton-Translocating ATPases