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Journal of Biomedical Optics • Open Access

Three-color Förster resonance energy transfer within single FOF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time
Author(s): Stefan Ernst; Monika G. Dueser; Nawid Zarrabi; Michael Börsch

Paper Abstract

Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. Förster-type resonance energy transfer, commonly referred to as FRET, is required in order to observe the dynamics of relative movements within the protein. Förster-type resonance energy transfer between two specifically attached fluorophores provides a ruler with subnanometer resolution between 3 and 8 nm, submillisecond time resolution for time trajectories of conformational changes, and single-molecule sensitivity to overcome the need for synchronization of various conformations. FOF1-ATP synthase is a rotary molecular machine which catalyzes the formation of adenosine triphosphate (ATP). The Escherichia coli enzyme comprises a proton driven 10 stepped rotary FO motor connected to a 3-stepped F1 motor, where ATP is synthesized. This mismatch of step sizes will result in elastic deformations within the rotor parts. We present a new single-molecule FRET approach to observe both rotary motors simultaneously in a single FOF1-ATP synthase at work. We labeled this enzyme with three fluorophores, specifically at the stator part and at the two rotors. Duty cycle-optimized with alternating laser excitation, referred to as DCO-ALEX, allowed to control enzyme activity and to unravel associated transient twisting within the rotors of a single enzyme during ATP hydrolysis and ATP synthesis. Monte Carlo simulations revealed that the rotor twisting is larger than 36 deg.

Paper Details

Date Published: 3 February 2012
PDF: 10 pages
J. Biomed. Opt. 17(1) 011004 doi: 10.1117/1.JBO.17.1.011004
Published in: Journal of Biomedical Optics Volume 17, Issue 1
Show Author Affiliations
Stefan Ernst, Univ. Stuttgart (Germany)
Monika G. Dueser, Univ. Stuttgart (Germany)
Nawid Zarrabi, Univ. Stuttgart (Germany)
Michael Börsch, Friedrich-Schiller-Univ. Jena (Germany)

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