Single Molecule Measurements of F₁-ATPase Reveal an Interdependence between the Power Stroke and the Dwell Duration

Abstract

Increases in the power stroke and dwell durations of single molecules of E. 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 γ 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. KeywordsPower Stroke; F 1 -ATPase; Molecular Motors; Single Molecule Measurements Mechanical work at the sub-cellular level is performed by enzymes called molecular motors that convert chemical energy in the form of either ATP or a transmembrane electrochemical gradient into mechanical energy by initiating conformational changes capable of performing work. Molecular motors are classified as either linear or rotary, depending upon the type of work they perform. The effect of external load on some linear molecular motors has been found to alter the relationship between the mechanical output and the kinetics of nucleotide binding, hydrolysis, and product release (1-8).Application of a load to several motors perturbs the mechanical transitions in the catalytic cycle, causing a force-dependent shift in both the dwell time distribution and mechanical transitions (1). Several groups have observed changes in these distributions resulting from intra-molecular strain due to an applied external load that altered the conformations of the active site, and affected nucleotide binding, ATP hydrolysis, and product release (2,5,(8)(9)(10). Single molecule studies using a laser trap revealed a 100-fold increase in the rate of ADP binding when 2 pN of load was applied to the linear molecular motor myosin VI (7). A biochemical mechanism was derived from this application of an external force that caused this motor to stop its transport function, and become a structural linker. Spudich (1) that the use of mechanical strain to alter protein function may serve as a widespread means of enzyme activity regulation.The F 1 F o ATP synthase is composed of the transmembrane F o complex and extrinsic membrane F 1 complex that couples the energy from a transmembrane proton gradient to synthesize ATP from ADP and phosphate. Both complexes are rotary molecular motors that use a common drive shaft composed of the γ and ε subunits. Crystal structures show that the F 1 γ subunit is composed of two domains, a coiled-coil domain that protrudes through the center cavity of the (αβ) 3 ring, and an open α-helix/β-sheet domain known as the foot that binds to the ε subunit and the c-subunit ring of F o (11,12). The F 1 -ATPase-driven and F o -proton gradient-driven rotation of the γε subunits occur in opposite directions (11,12).The rotary cycle of the F 1 -ATPase ...

Anatomy of F 1 -ATPase powered rotation

“…We had previously determined that the viscous load on F 1 attached to nanorods under the same conditions as those presented here did not limit the average angular velocity of ∼360°·ms −1 measured during the first 90°of rotation (22,24,25). Because the phase-2 angular velocity sometimes substantially exceeds that average, more experiments are necessary to establish the point at which viscous load becomes rate-limiting.…”

“…Single-molecule rotation assays were performed by attaching the biotinylated F 1 -ATPase to a coverslip, followed by assembly of the avidin-coated nanorod to the biotin on the γ-subunit as described (25). Light scattered from a rotating nanorod for single-molecule experiments was collected at 200 kHz for analysis using a single-photon counting avalanche photodiode as described by Ishmukhametov et al (30).…”

Theory for rates, equilibrium constants, and Brønsted slopes in F 1 -ATPase single molecule imaging experiments

“…refs. 2 and 3), and much information has been extracted by elaborate techniques at the single molecule level (3)(4)(5)(6). Complementing experimental tools such as X-ray spectroscopy (7) and ensemble biochemical methods (8), single molecule experiments reveal key details of the coupling between enzymatic processes and rotation (9)(10)(11)(12).…”

What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

“…Subsequently gold nanorods (Spetzler et al 2009) were attached to the rotor shaft, now permitting the angular velocity during the transition between steps to be observed. In particular, the gold nanorods do not limit the rotation velocity via their friction with the water molecules.…”

“…A particular example would be to use a collision theory picture (Volkán-Kacsó & Marcus, 2015) of ATP binding to the weak binding site of an empty β subunit in which the pre-exponential term A is a collision frequency Z, and a term related to an ATP binding outside of the β subunit. For the binding and release of ATP there are extensive data obtained by different methods (Adachi et al 2012;Braig et al 2000;Spetzler et al 2009;Watanabe et al 2010Watanabe et al , 2012Yasuda et al 2001). In the ATP binding an ATP first forms a collision complex (Oster & Wang, 2000) with the F 1 -ATPase, followed by an ATP binding as the next step (an 80°step) in the overall hydrolysis.…”