title: The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap.
abstract: F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1(or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA, USA ; Department of Physics, Stanford University, Stanford, CA, USA.
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Bockenhauer
authors.affiliation: Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Duncan
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA, USA.
authors.forename: W E
authors.initials: WE
authors.lastname: Moerner
authors.affiliation: Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Börsch
keywords: ABEL trap; F1-ATPase; conformational change; single-molecule FRET; ε subunit