Three-color Förster resonance energy transfer within single F[sub O]F[sub 1]-ATP synthases: monitoring elastic deformations of the rotary double motor in real time

Abstract

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.

Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

“…Fluorescence photons were detected by three single-photon counting avalanche photodiodes (SPCM-AQRH-14, Excelitas or Perkin-Elmer) for simultaneous spectrally-resolved FLIM and time-resolved anisotropy measurements 23 . Three synchronized TCSPC cards (SPC 153, Becker&Hickl) recorded the photons [24][25][26] . FLIM and time-resolved anisotropy images were analyzed using the SPCImage software (Becker&Hickl), whereas intensity-based anisotropy imaging was analyzed using counter cards (National Instruments) and custom Matlab scripts (Mathworks) [27][28][29][30][31][32][33][34][35] .…”

Spotlighting motors and controls of single FoF1-ATP synthase

“…SmFRET was also applied to detect a reversible, elastic twisting mode within the rotor subunits ε and c of F o F 1 -ATP synthase during ATP hydrolysis and synthesis [52, 53] . Transient elastic energy storage had been postulated to address the symmetry mismatch of the F 1 and F o motor step sizes and to ensure maximum efficiency of motor operation (experimental details are summarized in [2, 54] ).…”

“…Transient elastic energy storage had been postulated to address the symmetry mismatch of the F 1 and F o motor step sizes and to ensure maximum efficiency of motor operation (experimental details are summarized in [2, 54] ). Using three different specifically attached fluorophores on a single F o F 1 -ATP synthase (EGFP- a fusion on the stator, Alexa532-ε and Cy5- c on rotor), we could show that the distances between markers on residues ε56 and c 2 fluctuated during rotor movement, indicating a twisting up to three single steps of c or 108°, respectively [52] .…”

Confining Brownian motion of single nanoparticles in an ABELtrap

“…The direction of rotation reverses when the enzyme switches from ATP synthesis to ATP hydrolysis. Elastic properties of the central rotor subunits compensate the mismatch of step sizes [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] . Yet, the drawbacks of these initial measurements are short observation times and large intensity fluctuations inherent to single molecules freely diffusing through a confocal detection volume.…”