Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Eva Hammann; Andrea Zappe; Stefanie Keis; Stefan Ernst; Doreen Matthies; Thomas Meier; Gregory M. Cook; Michael Börsch

doi:10.1117/12.907242

13 February 2012 Step size of the rotary proton motor in single F_oF₁-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Eva Hammann, Andrea Zappe, Stefanie Keis, Stefan Ernst, Doreen Matthies, Thomas Meier, Gregory M. Cook, Michael Börsch

Author Affiliations +

Proceedings Volume 8228, Single Molecule Spectroscopy and Superresolution Imaging V; 82280A (2012) https://doi.org/10.1117/12.907242
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme F_oF₁-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a F_o motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary F_o motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped F_o motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.

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Eva Hammann, Andrea Zappe, Stefanie Keis, Stefan Ernst, Doreen Matthies, Thomas Meier, Gregory M. Cook, and Michael Börsch "Step size of the rotary proton motor in single F_oF₁-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET", Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280A (13 February 2012); https://doi.org/10.1117/12.907242

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