Single-molecule fluorescence spectroscopy of fast protein dynamics

Single-molecule experiments have become an integral part of modern structural biology. Unlike other methods, single-molecule Förster resonance energy transfer (smFRET) spectroscopy opens direct access to distance-based temporal trajectories of protein motions. Recent innovations in analysing smFRET experiments with correlation and photon-trajectory based methods have pushed the time resolution of dynamics to much faster than milliseconds. Here, we review these methods, together with their most recent applications and their impact on our understanding of the function of proteins. Important current topics range from the dynamics of intrinsically disordered proteins in complex with their binding partners or in biomolecular condensates, to the conformational dynamics of proteins during their function, from enzymes to molecular machines. We focus particularly on the determination of the timescales of motions and how the utmost information can be gleaned from single-molecule data at the single-photon level. • Single-molecule FRET spectroscopy probes faster-than-millisecond protein dynamics. • Single-molecule spectroscopy unravels the coupling between dynamics and function. • Correlation functions expose motions in both disordered and folded proteins. • Rates can be determined from the shape modulation of FRET efficiency histograms. • Maximum likelihood methods analyze signals on a photon-by-photon basis.