Tassilo Hornung

Direct observation of stepped proteolipid ring rotation in E. coli FoF1-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 FoF1‐ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in Fo has been particularly hampered by signal‐to‐noise and time resolution. Here, we show the presence of a transient dwell between Fo subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli FoF1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c‐ring to a limit of ∼36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the Fo motor where c‐ring rotational diffusion is limited to 36°.

Anatomy of F1-ATPase powered rotation

Significance We present a description of the angular velocity of the power stroke as a function of rotational position for the F1-ATPase molecular motor. The angular velocity of this motor, which rotates in 120° power strokes separated by catalytic dwells, is part of the FoF1 ATP synthase in oxidative phosphorylation, and has been thought not to vary. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur, providing direct evidence that angular velocity depends on substrate binding affinity. The data support a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank). 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. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).

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. These results suggest that the order of hydrolysis, product release, and substrate binding may change as the result of external load on the motor or inhibition of hydrolysis.

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.

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. Although the gamma-subunit was able to rotate with a 20x increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63+/-8 pN nm, independent of the load on the enzyme.

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

Background: FoF1 synthesizes ATP by ion gradient-powered Fo c-ring CW rotation viewed from the periplasm. Results: An electrostatic Fo-cR50/aE196 leash and proton gate promotes CW rotation against ATPase-driven CCW rotation by as much as one c subunit. Conclusion: A subunit a “grab and push” mechanism rotates the Fo c-ring CW. Significance: How Fo drives CW rotation for ATP synthesis against F1 ATPase-dependent CCW torque is a major unresolved question. 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. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.

Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach.

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~1011 ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 106 enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.

Energy Transduction by the Two Molecular Motors of the F1Fo ATP Synthase

The F1Fo ATP synthase has nearly universal importance as the major source of ATP among all life forms. These molecular motors couple the energy provided by a transmembrane proton gradient to the production of ATP from ADP and phosphate. The intrinsic membrane complex of ab2c10–15 subunits, known as Fo, functions as a proton channel via a Brownian ratchet mechanism and the F1 peripheral membrane complex of α3β3γδe subunits contains one site for ATP synthesis/hydrolysis per αβ heterodimer. When F1 is purified from Fo and the membrane, it retains the ability to hydrolyze ATP. The ring of three αβ heterodimers form the stator around the γ-subunit rotor that rotates in response to ATP hydrolysis activity producing a torque of 61 pN nm. Rotation occurs via the alternating site mechanism in which ATP binds to one site, while product release occurs at another site. It uses the non-equilibrium transmembrane electrochemical proton gradient derived from the oxidation of metabolites or light during photosynthesis to drive the reaction ADP + Pi ↔ ATP + H2O away from equilibrium, and thereby maintains high cellular concentrations of ATP. Under some conditions, the enzyme can catalyze ATPase-driven proton pumping in the reverse direction across the membrane. However, the enzymes from mitochondria and chloroplasts employ mechanisms to minimize this reverse reaction.

Elastic coupling power stroke mechanism of the F1-ATPase molecular motor

Significance Molecular motor F1-ATPases use the free energy from ATP binding and hydrolysis to ADP and Pi to rotate subunit γ, and can synthesize ATP in the FOF1 ATP synthase. We determined the energetics during each 3° of the power stroke from temperature-dependent changes of angular velocity. Unexpectedly, the power stroke activation energy of phase 1 (0°–60°) was negative and varied parabolically, indicating that it was powered by elastic energy of a torsional spring consistent with unwinding the γ-subunit coiled-coil. Phase 2 rotation had an enthalpic component, indicating additional input of energy is required to complete the 120° power stroke, consistent with energy derived from ATP binding. These results deepen our understanding of these important molecular motors summarized in the proposed elastic coupling mechanism. The angular velocity profile of the 120° F1-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a ΔμATP = −31.25 kBT at a time resolution of 10 μs. Angular velocities during the first 60° of the power stroke (phase 1) varied inversely with temperature, resulting in negative activation energies with a parabolic dependence. This is direct evidence that phase 1 rotation derives from elastic energy (spring constant, κ = 50 kBT·rad−2). Phase 2 of the power stroke had an enthalpic component indicating that additional energy input occurred to enable the γ-subunit to overcome energy stored by the spring after rotating beyond its 34° equilibrium position. The correlation between the probability distribution of ATP binding to the empty catalytic site and the negative Ea values of the power stroke during phase 1 suggests that this additional energy is derived from the binding of ATP to the empty catalytic site. A second torsion spring (κ = 150 kBT·rad−2; equilibrium position, 90°) was also evident that mitigated the enthalpic cost of phase 2 rotation. The maximum ΔGǂ was 22.6 kBT, and maximum efficiency was 72%. An elastic coupling mechanism is proposed that uses the coiled-coil domain of the γ-subunit rotor as a torsion spring during phase 1, and then as a crankshaft driven by ATP-binding–dependent conformational changes during phase 2 to drive the power stroke.