Robert R. Ishmukhametov

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.

Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states.

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E. coli F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk’s ε subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides. DOI: http://dx.doi.org/10.7554/eLife.21598.001

Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu2+ catalyzed cross‐linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross‐linking with 6 Cys pairs introduced into these regions. Additional disulfide cross‐linking was observed between the N‐terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone‐4‐maleimide derivatized Cys in the 2–3 periplasmic loop of subunit a were shown to cross‐link with the periplasmic N‐terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase.

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.

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.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes.

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.