Analyzing conformational changes in single FRET-labeled A1 parts of archaeal A1AO-ATP synthase

TL;DR

This study investigates conformational changes in the archaeal A1AO-ATP synthase's subunit F using single-molecule FRET, providing insights into its regulation mechanisms to prevent ATP hydrolysis.

Contribution

It introduces a method to distinguish genuine conformational changes from artifacts by analyzing fluorescence lifetimes in archaeal ATP synthase.

Findings

Identified conformational states of subunit F via smFRET.

Differentiated true conformational signals from artifacts using fluorescence lifetime analysis.

Enhanced understanding of ATP synthase regulation in archaea.

Abstract

ATP synthases utilize a proton motive force to synthesize ATP. In reverse, these membrane-embedded enzymes can also hydrolyze ATP to pump protons over the membrane. To prevent wasteful ATP hydrolysis, distinct control mechanisms exist for ATP synthases in bacteria, archaea, chloroplasts and mitochondria. Single-molecule F\"orster resonance energy transfer (smFRET) demonstrated that the C-terminus of the rotary subunit epsilon in the Escherichia coli enzyme changes its conformation to block ATP hydrolysis. Previously we investigated the related conformational changes of subunit F of the A1AO-ATP synthase from the archaeon Methanosarcina mazei G\"o1. Here, we analyze the lifetimes of fluorescence donor and acceptor dyes to distinguish between smFRET signals for conformational changes and potential artefacts.