Subunit rotation in a single F o F 1 -ATP synthase in a living bacterium monitored by FRET

We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of FoF1-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F1-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than kBT per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in Fo and F1, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.

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Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2010.03.127 ◽

2010 ◽

Vol 395 (2) ◽

pp. 173-177 ◽

Cited By ~ 6

Author(s):

Mayumi Nakanishi-Matsui ◽

Sachiko Kashiwagi ◽

Masaki Kojima ◽

Takamasa Nonaka ◽

Masamitsu Futai

Keyword(s):

Atp Synthase ◽

Β Subunit ◽

Subunit Rotation ◽

Hinge Domain

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Fast ATP-dependent subunit rotation in reconstituted FoF1-ATP synthase trapped in solution

10.1101/2021.03.21.436299 ◽

2021 ◽

Author(s):

Thomas Heitkamp ◽

Michael Börsch

Keyword(s):

Atp Synthase ◽

Single Molecule ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Atp Synthesis ◽

Electrochemical Potential ◽

Ensemble Averaging ◽

Subunit Rotation ◽

Atp Synthases

ABSTRACTFoF1-ATP synthases are the ubiquitous membrane enzymes which catalyze ATP synthesis or ATP hydrolysis in reverse, respectively. Enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. By utilizing an Anti- Brownian electrokinetic trap (ABEL trap), single-molecule Förster resonance energy transfer (smFRET)-based subunit rotation monitoring was prolonged from milliseconds to seconds. The extended observation times for single proteoliposomes in solution allowed to observe fluctuating rotation rates of individual enzymes and to map the broad distributions of ATP-dependent catalytic rates in FoF1-ATP synthase. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores and uncouplers the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second, i.e. much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 fragments without coupling to the membrane-embedded Fo domain of the enzyme.

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Spotlighting motors and controls of single FoF1-ATP synthase

Biochemical Society Transactions ◽

10.1042/bst20130101 ◽

2013 ◽

Vol 41 (5) ◽

pp. 1219-1226 ◽

Cited By ~ 27

Author(s):

Michael Börsch ◽

Thomas M. Duncan

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Single Molecule ◽

Conformational Changes ◽

Structural Information ◽

E Coli ◽

Single Molecule Fret ◽

Membrane Enzyme ◽

Subunit Rotation ◽

Fof1 Atp Synthase

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.

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