Eukaryotic yeast V-ATPase rotary mechanism insights revealed by high-resolution single-molecule studies.

Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the V complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V without C, and V ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly.

FF ATP synthase molecular motor mechanisms.

The F-ATP synthase, consisting of F and F motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F (αβ) ring stator contains three catalytic sites. Single-molecule F rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position.

pH-dependent 11° FF ATP synthase sub-steps reveal insight into the F torque generating mechanism.

Most cellular ATP is made by rotary FF ATP synthases using proton translocation-generated clockwise torque on the F c-ring rotor, while F-ATP hydrolysis can force counterclockwise rotation and proton pumping. The F torque-generating mechanism remains elusive even though the F interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent FF structures. Here, single-molecule FF rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of F to undergo single c-subunit rotational stepping.

ΩqPCR measures telomere length from single-cells in base pair units.

ΩqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision of ΩqPCR were assessed using 800 bp and 1600 bp synthetic telomeres inserted into plasmids, which were measured to be 819 ± 19.6 and 1590 ± 42.

SARS-CoV-2 and mitochondrial health: implications of lifestyle and ageing.

Infection with SARs-COV-2 displays increasing fatality with age and underlying co-morbidity, in particular, with markers of the metabolic syndrome and diabetes, which seems to be associated with a "cytokine storm" and an altered immune response. This suggests that a key contributory factor could be immunosenescence that is both age-related and lifestyle-induced. As the immune system itself is heavily reliant on mitochondrial function, then maintaining a healthy mitochondrial system may play a key role in resisting the virus, both directly, and indirectly by ensuring a good vaccine response.

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases.

F-ATP synthases use proton flow through the F domain to synthesize ATP in the F₁ domain. In , the enzyme consists of rotor subunits γε and stator subunits (αβ)₃δ₂. Subunits or (αβ)₃ alone are rotationally symmetric.

Elastic coupling power stroke mechanism of the F-ATPase molecular motor.

The angular velocity profile of the 120° F-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a Δμ = -31.

Protonation-dependent stepped rotation of the F-type ATP synthase c-ring observed by single-molecule measurements.

The two opposed rotary molecular motors of the FF-ATP synthase work together to provide the majority of ATP in biological organisms. Rotation occurs in 120° power strokes separated by dwells when F synthesizes or hydrolyzes ATP. F and F complexes connect via a central rotor stalk and a peripheral stator stalk.

The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation.

The FF -ATP (F-ATP) synthase is essential for growth of , the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors.

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor ABDF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor αβγ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M.

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase.

Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a.

Anatomy of F1-ATPase powered rotation.

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood.

Microsecond resolution of single-molecule rotation catalyzed by molecular motors.

Single-molecule measurements of rotation catalyzed by the F(1)-ATPase or the F(o)F(1) ATP synthase have provided new insights into the molecular mechanisms of the F(1) and F(o) molecular motors. We recently developed a method to record ATPase-driven rotation of F(1) or F(o)F(1) in a manner that solves several technical limitations of earlier approaches that were significantly hampered by time and angular resolution, and restricted the duration of data collection. With our approach it is possible to collect data for hours and obtain statistically significant quantities of data on each molecule examined with a time resolution of up to 5 μs at unprecedented signal-to-noise.

Padlock probe-mediated qRT-PCR for DNA computing answer determination.

Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5' and 3' ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site.

Direct observation of stepped proteolipid ring rotation in E. coli F₀F₁-ATP synthase.

Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F₀F₁-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F₀ has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F₀ subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli F₀F₁ embedded in lipid bilayer nanodiscs.

Solving the fully-connected 15-city TSP using probabilistic DNA computing.

Implementation of DNA computers has lagged behind the theoretical advances due to several technical limitations. These limitations include the amount of DNA required, the efficiency and accuracy of methods to generate and purify answers, and the lack of a reliable method to read the answer. Here we show how to perform calculations using a reasonable amount of DNA with greater efficiency and accuracy and a new readout method that was used to successfully solve a problem with 15 vertices and 210 edges, the largest problem ever solved with DNA.

Single molecule measurements of F1-ATPase reveal an interdependence between the power stroke and the dwell duration.

Increases in the power stroke and dwell durations of single molecules of Escherichia coli F(1)-ATPase were measured in response to viscous loads applied to the motor and inhibition of ATP hydrolysis. The load was varied using different sizes of gold nanorods attached to the rotating gamma subunit and/or by increasing the viscosity of the medium using PEG-400, a noncompetitive inhibitor of ATPase activity. Conditions that increase the duration of the power stroke were found to cause 20-fold increases in the length of the dwell.

WITHDRAWN: Microsecond resolution of enzymatic conformational changes using dark-field microscopy.

We report a novel method to detect angular conformational changes of a molecular motor in a manner sensitive enough to achieve acquisition rates with a time resolution of 2.5mus (equivalent to 400,000fps). We show that this method has sufficient sensitivity to resolve the velocity of the F(1)-ATPase gamma-subunit as it travels from one conformational state to another (transition time).

Determination of torque generation from the power stroke of Escherichia coli F1-ATPase.

The torque generated by the power stroke of Escherichia coli F(1)-ATPase was determined as a function of the load from measurements of the velocity of the gamma-subunit obtained using a 0.25 micros time resolution and direct measurements of the drag from 45 to 91 nm gold nanorods. This result was compared to values of torque calculated using four different drag models.

Single-molecule detection of DNA via sequence-specific links between F1-ATPase motors and gold nanorod sensors.

We report the construction of a novel biosensing nanodevice to detect single, sequence-specific target DNA molecules. Nanodevice assembly occurs through the association of an immobilized F1-ATPase molecular motor and a functionalized gold nanorod via a single 3',5'-dibiotinylated DNA molecule. Target-dependent 3',5'-dibiotinylated DNA bridges form by combining ligation and exonucleation reactions (LXR), with a specificity capable of selecting against a single nucleotide polymorphism (SNP).

Abundance of Escherichia coli F1-ATPase molecules observed to rotate via single-molecule microscopy with gold nanorod probes.

The abundance of E. coli F1-ATPase molecules observed to rotate using gold nanorods attached to the gamma-subunit was quantitated. Individual F1 molecules were determined to be rotating based upon time dependent fluctuations of red and green light scattered from the nanorods when viewed through a polarizing filter.

Recent developments of bio-molecular motors as on-chip devices using single molecule techniques.

The integration of microfluidic devices with single molecule motor detection techniques allows chip based devices to reach sensitivity levels previously unattainable.

Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit.

F(1)-ATPase mutations in Escherichia coli that changed the strength of hydrogen bonds between the alpha and beta subunits in a location that links the catalytic site to the interface between the beta catch loop and the gamma subunit were examined. Loss of the ability to form the hydrogen bonds involving alphaS337, betaD301, and alphaD335 lowered the k(cat) of ATPase and decreased its susceptibility to Mg(2+)-ADP-AlF(n) inhibition, while mutations that maintain or strengthen these bonds increased the susceptibility to Mg(2+)-ADP-AlF(n) inhibition and lowered the k(cat) of ATPase. These data suggest that hydrogen bonds connecting alphaS337 to betaD301 and betaR323 and connecting alphaD335 to alphaS337 are important to transition state stabilization and catalytic function that may result from the proper alignment of catalytic site residues betaR182 and alphaR376 through the VISIT sequence (alpha344-348).

Microsecond time scale rotation measurements of single F1-ATPase molecules.

A novel method for detecting F(1)-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 micros (equivalent to 400,000 fps) is reported. This is sufficient for resolving the rate at which the gamma-subunit travels from one dwell state to another (transition time).