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The authors show how to compute the autocorrelation function of FRET efficiencies and obtain dynamic information from as little as a few thousand molecules.

Although not widely appreciated, measured smFRET efficiencies can vary depending on illumination intensity. Self-healing fluorophores offer robust triplet state suppression to enable accurate smFRET measurements under broad illumination regimes.

The interaction between the polyelectrolytic IDP ProTα and a highly charged folded protein domain is investigated by integrating NMR, single-molecule FRET and MD simulations. Net charge and charge distribution are implicated in binding and selectivity.

Histone H1 binds to nucleosomes with ultrahigh affinity, implying residence times incompatible with efficient biological regulation. Now it has been shown that the disordered regions of H1 retain their large-amplitude dynamics on the nucleosome, which enables a charged disordered histone chaperone to invade the H1–nucleosome complex and vastly accelerate H1 dissociation.

RNA chaperones, such as the hepatitic C virus (HCV) core protein, are proteins that aid in the folding of nucleic acids. Here authors use single‐molecule spectroscopy and simulation to show that the HCV core protein acts as a flexible macromolecular counterion which facilitates nucleic acid folding.

By combining single-molecule spectroscopy, nanophotonic enhancement, and molecular simulations, the authors reveal the extremely rapid chain dynamics of single-stranded nucleic acids.

Pore-forming toxins are expressed as monomers and assemble into multimeric pores. Here, Benke et al. follow the kinetics of pore formation for the bacterial toxin ClyA with single-molecule methods and show that pore formation progresses through the assembly of oligomeric intermediates, rather than by the addition of monomers to a nascent pore.

Two highly charged disordered human proteins phase-separate into viscous complex coacervates while retaining their rapid conformational dynamics through pico- to nanosecond exchange of short-lived side-chain interactions.

Single molecule kinetics investigations and molecular simulations are useful tools in elucidating protein assembly mechanisms. Here, the authors use these to show that even naturally occurring tandem repeats undergo transient misfolding and that assembly is much more complex than we previously understood.

The intrinsically disordered linker histone H1.0 and prothymosin α form a complex which exhibits slow exchange between bound and unbound populations at low protein concentrations and fast exchange at high concentrations. Here authors explain this observation by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange.

A high-affinity complex of histone H1 and prothymosin-α reveals an unexpected interaction mechanism, where the large opposite net charge enables the two proteins to remain highly disordered even in the complex.

Internal friction affects the kinetics of protein folding. Borgiaet al. investigate how this friction affects the folding dynamics of the protein spectrin, revealing a potential role in the rate-limiting conformational changes.

How interactions between binding partners form or break is hidden in the transition paths from the encounter to the formation of a stable complex. Here authors use single‐molecule spectroscopy to measure the transition path times for the association of two intrinsically disordered proteins that form a folded dimer upon binding and identify a metastable encounter complex.

How intrinsically disordered proteins (IDPs) undergo a coupled folding and binding reaction with their molecular targets remains to be understood. Here authors use single-molecule FRET to assess the contribution of cis/trans isomerization of peptidyl-prolyl bonds in regulating IDP interactions.

The combination of single-molecule fluorescence detection with Förster resonance energy transfer provides a powerful probe of biomolecular dynamics on timescales ranging from nanoseconds to days. This Review outlines single-molecule Förster resonance energy transfer spectroscopy with a focus on dynamics and highlights future developments and enhanced capabilities.

Individual protein dynamics can be studied in live eukaryotic cells from the millisecond to nanosecond level using an integrated approach to confocal single-molecule FRET spectroscopy.

Droplet-based microfluidics enable rapid mixing with millisecond dead times and allow single-molecule measurements of non-equilibrium binding kinetics on even challenging, strongly adsorptive samples, such as intrinsically disordered proteins.

SARS-CoV-2 nucleocapsid (N) protein is responsible for viral genome packaging. Here the authors employ single-molecule spectroscopy with all-atom simulations to provide the molecular details of N protein and show that it undergoes phase separation with RNA.

A multi-laboratory study finds that single-molecule FRET is a reproducible and reliable approach for determining accurate distances in dye-labeled DNA duplexes.