Improving FRET-based monitoring of single chemomechanical rotary motors at work

Protein dynamics are observable in real time using internal distance measurements of reference positions. Based on Förster resonance energy transfer (FRET) as the distance ruler between two fluorescent markers, investigations of single molecules one at a time have modified our views of protein conformational changes. X-ray crystallography provides trapped conformations at atomic resolution and NMR can add information about flexible parts of proteins, but the pathways between these structures including unknown conformations are often not directly accessible. Especially for membrane enzymes comprising multiple subunits, limited structural information requires alternative experimental methods to follow conformational pathways. Herein we summarize the knowledge gathered from single-molecule FRET studies of the membrane-embedded rotary nanomotor F(o)F(1)-ATP synthase. In addition, new ideas and concepts to shift and extend the current limitations of the confocal FRET detection approach using freely diffusing, liposome-reconstituted, individual enzymes are discussed: nanodiamonds as new fluorophores, optimized laser excitation schemes, Hidden Markov Models for software-based FRET trajectory analysis, and application of the anti-Brownian electrokinetic trap (ABELtrap) to keep the single enzyme in focus.