title: Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.
abstract: H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.
authors.affiliation: Department of Physical Chemistry, University of Freiburg, Albertstrasse 23A, 79104 Freiburg, Germany. peter.graeber@physchem.uni-freiburg.de
authors.forename: Roland
authors.initials: R
authors.lastname: Bienert
authors.forename: Boris
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Verena
authors.initials: V
authors.lastname: Rombach-Riegraf
authors.forename: Peter
authors.initials: P
authors.lastname: Gräber
mesh_terms: D055162:Biocatalysis; D002736:Chloroplasts / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D018724:Spinacia oleracea / Q000201:enzymology; D013997:Time Factors
chemical_list: D006180:Proton-Translocating ATPases