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Proceedings Paper

Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX
Author(s): N. Zarrabi; S. Ernst; M. G. Düser; A. Golovina-Leiker; W. Becker; R. Erdmann; S. D. Dunn; M. Börsch
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Paper Abstract

FoF1-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 Fo 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 Fo 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 Fo and F1.

Paper Details

Date Published: 24 February 2009
PDF: 14 pages
Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 718505 (24 February 2009); doi: 10.1117/12.809610
Show Author Affiliations
N. Zarrabi, Univ. Stuttgart (Germany)
S. Ernst, Univ. Stuttgart (Germany)
M. G. Düser, Univ. Stuttgart (Germany)
A. Golovina-Leiker, Univ. Stuttgart (Germany)
W. Becker, Becker & Hickl GmbH (Germany)
R. Erdmann, PicoQuant GmbH (Germany)
S. D. Dunn, Univ. of Western Ontario (Canada)
M. Börsch, Univ. Stuttgart (Germany)

Published in SPIE Proceedings Vol. 7185:
Single Molecule Spectroscopy and Imaging II
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)

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