title: Three-color Förster resonance energy transfer within single F₀F₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.
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. F(O)F(1)-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 F(1) 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(O)F(1)-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.
authors.affiliation: University of Stuttgart, 3rd Institute of Physics, Pfaffenwaldring 57, 70550 Stuttgart, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ernst
authors.forename: Monika G
authors.initials: MG
authors.lastname: Düser
authors.forename: Nawid
authors.initials: N
authors.lastname: Zarrabi
authors.forename: Michael
authors.initials: M
authors.lastname: Börsch
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry; D003198:Computer Simulation; D004548:Elasticity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008958:Models, Molecular; D009010:Monte Carlo Method
chemical_list: D002232:Carbocyanines; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C085321:cyanine dye 5; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; C557221:Escherichia coli Proton-Translocating ATPase