Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

N. Zarrabi; S. Ernst; M. G. Düser; A. Golovina-Leiker; W. Becker; R. Erdmann; S. D. Dunn; M. Börsch

doi:10.1117/12.809610

24 February 2009 Simultaneous monitoring of the two coupled motors of a single F_oF₁-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

N. Zarrabi, S. Ernst, M. G. Düser, A. Golovina-Leiker, W. Becker, R. Erdmann, S. D. Dunn, M. Börsch

Author Affiliations +

Proceedings Volume 7185, Single Molecule Spectroscopy and Imaging II; 718505 (2009) https://doi.org/10.1117/12.809610
Event: SPIE BiOS, 2009, San Jose, California, United States

Abstract

F_oF₁-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound F_o part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ε subunits of the F₁ sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F₁ motor, another one to the F_o motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of F_o and F₁.

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N. Zarrabi, S. Ernst, M. G. Düser, A. Golovina-Leiker, W. Becker, R. Erdmann, S. D. Dunn, and M. Börsch "Simultaneous monitoring of the two coupled motors of a single F_oF₁-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX", Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 718505 (24 February 2009); https://doi.org/10.1117/12.809610

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