Single-molecule spectroscopy of protein folding dynamics—expanding scope and timescales

► Single-molecule spectroscopy can now be used to probe timescales from nanoseconds to weeks. ► Nanosecond-FCS allows the investigation of the submicrosecond dynamics of unfolded and intrinsically disordered proteins. ► Microfluidic mixing methods provide nonequilibrium single-molecule information from submillisecond to second timescales. ► The rigorous analysis of photon statistics has allowed the average transition path time of protein folding to be measured. ► Single-molecule spectroscopy starts to bridge the gap between folding in vitro and in vivo. Single-molecule spectroscopy has developed into an important method for probing protein structure and dynamics, especially in structurally heterogeneous systems. A broad range of questions in the diversifying field of protein folding have been addressed with single-molecule Förster resonance energy transfer (FRET) and photo-induced electron transfer (PET). Building on more than a decade of rapid method development, these techniques can now be used to investigate a wide span of timescales, an aspect that we focus on in this review. Important current topics range from the structure and dynamics of unfolded and intrinsically disordered proteins, including the coupling of folding and binding, to transition path times, the folding and misfolding of larger proteins, and their interactions with molecular chaperones.