Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F₀F₁-ATP synthase

Abstract

Subunit ε is an intrinsic regulator of the bacterial FoF1-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of ε can extend into the central cavity formed by the α and β subunits, as revealed by the recent X-ray structure of the F1 portion of the Escherichia coli enzyme. This insertion blocks the rotation of the central γ subunit and, thereby, prevents wasteful ATP hydrolysis. Here we aim to develop an experimental system that can reveal conditions under which ε inhibits the holoenzyme FoF1-ATP synthase in vitro. Labeling the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET) allowed monitoring of the conformation of ε in the reconstituted enzyme in real time. New mutants were made for future three-color smFRET experiments to unravel the details of regulatory conformational changes in ε.

The NMR solution structure of Mycobacterium tuberculosis F‐ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine

“…Fluorescence Energy Transfer (FRET) studies have indicated that the EceCTD shows both compact and extended (e e ) conformations in F 1 or in reconstituted F 0 F 1 in liposomes [28]. An e c was shown for the isolated bacterial subunit e structures [11][12][13] as well as in the F 1 -ATPase structure of Caldalkalibacillus thermarum [14], and an e e state was determined in the E. coli and G. stearothermophilus F 1 -ATPase structure [7,16] as well as the E. coli F 1 F 0 ATP synthase [17].…”

Conformational dynamics of the rotary subunit F in the A3B3DF complex of Methanosarcina mazei Gö1 A‐ATP synthase monitored by single‐molecule FRET

“…The variable design of the confocal microscope setup has been described previously . Here, a ps‐pulsed laser (PicoTA 490; Picoquant, Berlin, Germany) as used to excite the FRET donor fluorophore Atto488 with 150 μW at 488 nm using a repetition rate of 40 MHz.…”

Fluoride resistance inStreptococcus mutans: a mini review

“…It is worth mentioning that the development of modern biotechnology makes this type of study possible. The combination of single-molecule fluorescence resonance energy transfer (smFRET) and confocal microscopy allows the observation of regulatory conformational changes of specific proteins [37]. Such an approach has been taken to study the inhibition of Escherichia coli membrane F-ATPase by aurovertin [38] and is thus promising to confirm the fluoride inhibition of F-ATPase in S. mutans cells.…”