Three-color Förster resonance energy transfer within single FOF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time

Ernst, Stefan; Düser, Monika G.; Zarrabi, Nawid; Börsch, Michael

doi:doi:10.1117/1.JBO.17.1.011004

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Journal of Biomedical Optics / Volume 17 / Issue 1 / Special Section on FRET at 65

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Three-color Förster resonance energy transfer within single F_OF₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time

J. Biomed. Opt. 17, 011004 (Feb 03, 2012); http://dx.doi.org/10.1117/1.JBO.17.1.011004

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Abstract

References (51)

Stefan Ernst, Monika G. Düser, Nawid Zarrabi, and Michael Börsch

University of Stuttgart, 3rd Institute of Physics, Pfaffenwaldring 57, 70550 Stuttgart, Germany

Friedrich Schiller University Jena, Single-Molecule Microscopy Group, Jena University Hospital, Nonnenplan 2-4, 07743 Jena, Germany

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. F_OF₁-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 F_O 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 F_OF₁-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.

History

Received Jun 04, 2011
Revised Jul 15, 2011
Published online Feb 03, 2012

Digital Object Identifier

http://dx.doi.org/10.1117/1.JBO.17.1.011004

Citation

Stefan Ernst, Monika G. Düser, Nawid Zarrabi and Michael Börsch, "Three-color Förster resonance energy transfer within single F_OF₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time", J. Biomed. Opt. 17, 011004 (Feb 03, 2012); http://dx.doi.org/10.1117/1.JBO.17.1.011004