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3 February 2012 Three-color Förster resonance energy transfer within single FOF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time
Stefan Ernst, Monika G. Dueser, Nawid Zarrabi, Michael Börsch
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Journal of Biomedical Optics, Vol. 17, Issue 1, 011004 (February 2012). https://doi.org/10.1117/1.JBO.17.1.011004
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.
© 2012 Society of Photo-Optical Instrumentation Engineers (SPIE) 0091-3286/2012/$25.00 © 2012 SPIE
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Stefan Ernst, Monika G. Dueser, Nawid Zarrabi, and Michael Börsch "Three-color Förster resonance energy transfer within single FOF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time," Journal of Biomedical Optics 17(1), 011004 (3 February 2012). https://doi.org/10.1117/1.JBO.17.1.011004
Published: 3 February 2012
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KEYWORDS
Fluorescence resonance energy transfer
Acquisition tracking and pointing
Photons
Luminescence
Resonance energy transfer
Pulsed laser operation
Confocal microscopy
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