Fast ATP-dependent Subunit Rotation in Reconstituted F o F 1 -ATP Synthase Trapped in Solution
Abstract
F o F 1 -ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their 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. Single-molecule Förster resonance energy transfer (smFRET) has been used to detect subunit rotation within F o F 1 -ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual F o F 1 -liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted F o F 1 -ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F 1 complexes uncoupled from the membrane-embedded F o complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.
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Endorsement statement (21 September 2021)
The preprint by Heitkamp and Börsch describes visualization of the fast ATP-dependent subunit rotation in reconstituted FoF1-ATP synthase using single-molecule FRET techniques. Using a highly innovative method for trapping single molecules, the authors were able to see the static and dynamic disorder of enzymes in solution, not possible in previous studies. The work makes important contributions to both understanding the structural dynamics of FoF1-ATP synthase and the development of methodologies to study single-molecule dynamics of other proteins in solution.
(This endorsement refers to version 5 of this preprint, which was peer reviewed by Biophysics Colab.)
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Consolidated peer review report (18 September 2021)
GENERAL ASSESSMENT
The FoF1-ATP Synthase is the fundamental enzyme that uses the electrochemical potential of protons (pmf) from the electron transport chain to catalyze production of ATP, the currency for energy in our cells. It is also a spectacular nanomachine that works by the rotation of an internal subunit, like an axle, which couples the proton transport by the Fo domain into ATP synthesis by the F1 domain. The coupling is highly efficient, so the enzyme can also act in reverse, as a proton pump that is fueled by ATP hydrolysis. This paper provides a glimpse into the dynamics of the enzyme using single-molecule FRET (smFRET). These authors attached a donor fluorophore (Cy3B) on the rotating ε-subunit and acceptor fluorophore (Alexa Fluor 647) on the static C-terminus of the …
Consolidated peer review report (18 September 2021)
GENERAL ASSESSMENT
The FoF1-ATP Synthase is the fundamental enzyme that uses the electrochemical potential of protons (pmf) from the electron transport chain to catalyze production of ATP, the currency for energy in our cells. It is also a spectacular nanomachine that works by the rotation of an internal subunit, like an axle, which couples the proton transport by the Fo domain into ATP synthesis by the F1 domain. The coupling is highly efficient, so the enzyme can also act in reverse, as a proton pump that is fueled by ATP hydrolysis. This paper provides a glimpse into the dynamics of the enzyme using single-molecule FRET (smFRET). These authors attached a donor fluorophore (Cy3B) on the rotating ε-subunit and acceptor fluorophore (Alexa Fluor 647) on the static C-terminus of the α-subunit. Upon application of ATP, the ε-subunit is expected to rotate relative to the α-subunit, and transport protons into the vesicle. During the three steps of one rotation, the donor fluorophore is expected to move between one position far from the acceptor, and two positions closer to the acceptor, producing transitions from low to high FRET efficiency with each rotation. To hold the enzyme still during the measurements, the authors use an innovative Anti-Brownian electrokinetic trap (ABEL trap), allowing the them to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution for extended periods of time (seconds). What they observe is both interesting and unexpected. While the behavior of single molecules is always stochastic, these authors observed about 10-fold differences in the rotation rates from molecule to molecule, a heterogeneity referred to as static disorder, that is not accounted for by stochastic behavior. Using proton ionophores they show that some of this heterogeneity likely results from the buildup of a pmf across the vesicle membrane due to proton pumping in the presence of ATP. However, much of the heterogeneity is not accounted for and awaits further investigation. This preprint provides the groundwork and methodology for those investigations.
Overall, the preprint is a real tour-de-force, requiring intricate membrane protein biochemistry, site-specific fluorescent labeling, single-vesicle trapping, and single-molecule FRET measurements and analysis. The use of the ABEL trap is a particularly powerful innovation that allows them to record a single molecule for a much longer time than previous studies (1 s vs. 10 ms) and, therefore, capture many more cycles of rotation for each molecule. This allowed them to see the static and dynamic disorder of enzymes in solution, not possible in previous studies.
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REVIEWING TEAM
Curated by:
William N. Zagotta, Professor, University of Washington, USA
(This consolidated report is a result of peer review conducted by Biophysics Colab on version 5 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)
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