Twisting and subunit rotation in single F_OF₁-ATP synthase

Abstract

F O F 1 -ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single F O F 1 -ATP synthases.

Load-dependent destabilization of the γ-rotor shaft in F O F 1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

“…The intact molecular motor has a mass of 525 kDa. F o F 1 investigations are typically conducted under hydrolysis conditions (1,6,7), where γec 10 rotation is driven by the β subunits that cycle through a series of conformations (8). ATP hydrolysis triggers power strokes that involve consecutive interactions of the β-levers with γ (3, 6, 9).…”

“…However, there is scant information on how the conformation of F o F 1 is affected by internal and external forces while working under physiological conditions, where ATP/ADP interconversion is coupled with transmembrane proton transport in the presence of proton-motive force (PMF). With few exceptions (1,3,17), previous experiments on the rotary mechanism have focused on isolated F 1 .…”

Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap

“…This strain was transformed with the plasmid pRA100 carrying the structural genes of the atp operon under control of the atppromoter [54] . The cells were grown in a modified complex medium (0.5 g/l yeast extract, 1 g/l tryptone, 17 mM NaCl, 10 mM glucose, 107 mM KH 2 4 , 0.5 mM arginine, 0.5 mM isoleucine, 0.7 mM valine, 4 µM thiamine and 0.4 µM 2,3-dihydroxybenzoic acid). Cells were grown in a 10 L fermenter (FerMac 320, Electrolab, UK) at 37°C at 800 rpm, with a fresh air supply at 4.5 L/min, to the late logarithmic phase.…”

“…Subunits γ and ε in F 1 form a second motor that is elastically coupled to the c-ring [3,4] . Subunit γ forms the central rotary stalk that contacts the c-ring with its globular portion and intrudes the hexameric barrel of the three αβ-subunits of the stator.…”

“…The rotational states correlate with the crystal structure [10] . Our group studies single F o F 1 -ATP synthase molecules reconstituted in proteoliposomes to show the conformational changes and to resolve the stepsize of the rotor subunits in the holoenzyme via Förster resonance energy transfer (FRET) since 15 years [4, . In a first set of experiments F o F 1 -ATP synthase molecules were labeled with two fluorescent dyes.…”