Direct observation of stepped proteolipid ring rotation in E. coli FoF1-ATP synthase

Abstract

Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F o F 1 -ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F o has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F o subunits a and c by improving the time resolution to 10 ls at unprecedented S/N, and by using Escherichia coli F o F 1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 ls to form and 175 ls 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 B361 while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F o motor where c-ring rotational diffusion is limited to 361.

Anatomy of F 1 -ATPase powered rotation

“…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).…”

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

“…4B). Because the dependence of light intensity as a function of nanorod orientation relative to the axis of the polarizer has been shown to be sinusoidal (28), the observation of three offset sinusoidal curves in the histograms when the axis of rotation was close to the orthonormal position relative to the microscope slide indicates the presence of three dwells per rotation. Thus, these data show that subunit D rotates on a microsecond time scale in 120°steps inside the MmA 3 B 3 DF complex separated by dwells that occur on a millisecond time scale.…”

“…A comparison of the error inherent in the determination of the rotational position of a nanorod from scattered light intensity by the arcsine 1/2 versus the arcsine function used previously (8,28) is shown in Fig. 6.…”

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F 1 F 0 ATP synthase gate H + transport to the cytoplasm

“…The mutant plasmids described previously were transferred into the chromosomal atp operon deletion strain JWP292 for biochemical characterization (27,28). To purify mutant F 1 F 0 complexes, the Cys substitutions were excised from the pCMA113 derivative plasmid and transferred into plasmid pFV2 (30). Similar to pCMA113, pFV2 codes a Cys-less F 1 F 0 complex and a His tag is attached at the N terminus of subunit β to facilitate purification (32).…”