Matching Items (3)
Description
The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation.
ContributorsMartin, James (Author) / Frasch, Wayne D (Thesis advisor) / Chandler, Douglas (Committee member) / Gaxiola, Roberto (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2012
Description
The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
ContributorsYanagisawa, Seiga (Author) / Frasch, Wayne D (Thesis advisor) / Misra, Rajeev (Committee member) / Redding, Kevin (Committee member) / Singharoy, Abhishek (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
Across the tree of life, rotary molecular motors like the F1FO ATP synthase utilize a transmembrane nonequilibrium proton gradient to synthesize adenosine triphosphate (ATP), the biological energy currency. The catalytic portion of rotary motors, such as the F1 complex from E. coli and the V1 complex from S. cerevisiae, was purified and studied during ATP hydrolysis. Single-molecule assays utilized gold nanorods to investigate the kinetics of the F1-ATPase catalytic dwell, the biophysics of V1-ATPase, and the kinematics of the F1-ATPase power stroke. Observation of oscillatory rotor motion during the F1 catalytic dwell provided new insight as to how energy from ATP binding is stored during its three stages. That motion indicated a ratchet mechanism, in which F1 changed states according to first-order kinetics with a time constant τ = 0.182, showing that Stage-1 represents a pre-hydrolysis state and Stage-2 represents a post-hydrolysis state. F1 was then observed to return to 0° prior to its next power stroke (Stage-3), which explained why the three catalytic dwells remain 120° apart after many revolutions. Analysis of the 120° power stroke following Stage-3 was conducted in both V1 and F1, allowing comparative biology to elucidate defects in the ATPase mechanism, such as ADP inhibition and faltering rotation. It is noteworthy that the V1 rotary positions of ADP release and ATP binding are the opposite of F1, and that less elastic energy is stored in the V1 rotor due to differences in its catch loop. In both rotary ATPases, energy contributed by binding and hydrolysis can dissipate at multiple points. When the F1 catch loop contact between F1 βD305 and γQ269 was mutated, the elastic energy stored in the rotor dissipated dramatically. Dissipation was clearly shown by sustained Phase-1 decelerations, the distribution of ATP-binding dwells, and high-amplitude oscillations in γQ269L. These findings clarify evolutionary similarities and differences between eukaryotic V1, which is exclusively a hydrolase, and F1, which can both hydrolyze and synthesize ATP.
ContributorsBukhari, Zain Aziz (Author) / Frasch, Wayne D (Thesis advisor) / Gaxiola, Roberto A (Committee member) / Presse, Steve (Committee member) / Wideman, Jeremy G (Committee member) / Arizona State University (Publisher)
Created2024