The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL trap

Samuel D. Bockenhauer; Thomas M. Duncan; W. E. Moerner; Michael Börsch

doi:10.1117/12.2042688

1 April 2014 The regulatory switch of F₁-ATPase studied by single-molecule FRET in the ABEL trap

Samuel D. Bockenhauer, Thomas M. Duncan, W. E. Moerner, Michael Börsch

Proceedings Volume 8950, Single Molecule Spectroscopy and Superresolution Imaging VII; 89500H (2014) https://doi.org/10.1117/12.2042688
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

F₁-ATPase is the soluble portion of the membrane-embedded enzyme F_oF₁-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F₁ part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F₁ (or F_oF₁) 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 F₁ specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F₁ molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F₁ in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F₁ and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

Citation Download Citation

Samuel D. Bockenhauer, Thomas M. Duncan, W. E. Moerner, and Michael Börsch "The regulatory switch of F₁-ATPase studied by single-molecule FRET in the ABEL trap", Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 89500H (1 April 2014); https://doi.org/10.1117/12.2042688

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