The free-energy surfaces that underlie the conformational distributions of intrinsically disordered proteins (IDPs) are shallow and lack the deep minima characteristic of stable, folded structures. However, even in the absence of secondary or tertiary structure, sequence patterning can lead to conformational preferences and changes in chain dimensions as a function of solution conditions. While patterning effects have received extensive attention from simulation and theory, there is little corresponding data from experiment. Here we investigate the impact of charge patterning on chain dimensions and dynamics in a set of specifically designed polyampholytic IDP variants across the natural range of charge segregation with single-molecule FRET, nanosecond fluorescence correlation, circular dichroism, and NMR spectroscopy. We find that the conformational ensembles and their cooperative response to salt concentration show prominent and systematic dependencies on charge patterning, and to some extent on residue type. In contrast, the chain dynamics remain in the tens-of-nanosecond range, consistent with the absence of pronounced free-energy barriers. In close combination with molecular simulations, we show how the concept of susceptibility can be used to quantify cooperativity in the absence of barriers and relate it to the shallow free-energy surfaces of IDPs.

Abstract Phosphorylation of intrinsically disordered proteins (IDPs) is essential for regulating biomolecular interactions in many cellular processes. However, a quantitative understanding of how phosphorylation tunes the affinity between highly charged IDPs and nucleic acids is lacking. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 acts as an electrostatic rheostat that governs RNA binding. By combining enzymatic phosphorylation, phosphomimetic variants, and chemically synthesised phosphopeptides with single-molecule Förster resonance energy transfer measurements, we reveal the RS domain to be a potent driver of protein–RNA association. Increasing phosphorylation progressively reduces this interaction, and extensive phosphorylation eliminates detectable RNA binding. Remarkably, the binding free energy depends linearly on RS-domain net charge, regardless of whether the charge arises from phosphorylation or acidic residues introduced as phosphomimetics. Together, our findings uncover a quantitative framework for how phosphorylation tunes the interactions of charged IDPs and rationalize why two acidic residues are required to mimic a single phosphorylation event.

Highly charged chains of poly(ADP-ribose) (PAR) are synthesized in the cell as part of their central role in DNA damage response. However, the effects of PAR on nucleosome structure and dynamics remain incompletely understood. Here we combine droplet-based microfluidic mixing with single-molecule Förster resonance energy transfer spectroscopy to resolve the kinetics of PAR-induced nucleosome decompaction in non-equilibrium measurements with millisecond time resolution. This approach avoids surface-adhesion and enables the tether-free observation of nucleosome remodeling. We find that PAR triggers nucleosome decompaction via a length-dependent kinetic threshold: Chains with less than ten ADP-ribose units act slowly and weakly, whereas longer PAR polymers induce efficient and rapid nucleosome opening. The extent and reversibility of decompaction further depend on PAR concentration and ionic strength, reflecting a mechanism dominated by electrostatic interactions. Enzymatic PAR digestion demonstrates that PAR can promote both reversible linker DNA opening and irreversible nucleosome disassembly. Coarse-grained molecular simulations suggest that these effects arise from a competition between PAR and DNA for histone tail binding. Altogether, our results establish PAR length as a key factor controlling chromatin accessibility during DNA repair and highlight droplet-based microfluidics as a powerful platform for studying such biomolecular interactions.

Single-molecule spectroscopy combined with Förster resonance energy transfer is widely used to quantify distance dynamics and distributions in biomolecules. Most commonly, measurements are interpreted using simple analytical relations between experimental observables and the underlying distance distributions. However, these relations make simplifying assumptions, such as a separation of timescales between interdye distance dynamics, fluorescence lifetimes, and dye reorientation, the validity of which is notoriously difficult to assess from experimental data alone. Here, we use experimentally validated long-timescale, all-atom explicit-solvent molecular dynamics simulations of a disordered peptide with explicit fluorophores for testing these assumptions, in particular the separation of the relevant timescales and the description of chain dynamics in terms of diffusion in a potential of mean force. Our results allow us to quantitatively assess the resulting errors; they indicate that, even outside the simple limiting regimes, the errors from common approximations in data analysis are generally smaller than the systematic uncertainty limiting the accuracy of Förster resonance energy transfer efficiencies. We also illustrate how the direct comparison between measured and simulated experimental data can be employed to optimize force field parameters and develop increasingly realistic simulation models.

Intrinsically disordered proteins (IDPs) are often rich in charged residues, and electrostatic interactions have a pronounced effect on their conformational distributions, interactions and functions. However, attaining quantitative understanding of electrostatics is challenging because of the sequence‐specific arrangement of charges in the chain, the long‐range nature of electrostatic interactions, charge screening, and the condensation of counterions—effects that all need to be taken into account self‐consistently. Here, analytically tractable quantitative models are developed to predict ensemble average distances between any pair of residues in IDPs as a function of sequence and salt concentration, explicitly considering charge patterning. These models are tested systematically against extensive single‐molecule Förster resonance energy transfer (FRET) data mapping intrachain distances for a range of charged IDPs with different sequence compositions, as a function of salt concentration, and with different labeling positions and fluorophores. The resulting polymer model with a minimal set of adjustable parameters accounts for counterion condensation, the resulting effective charges, as well as dipolar interactions, and can be used to predict detailed intrachain distance maps between all residues. Analytical models of this kind offer a valuable complement to simulations and can provide fundamental insight into the interactions underlying the conformational distributions of IDPs.

Biomolecular condensates form by phase separation of biological polymers and have important functions in the cell - functions that are inherently linked to their physical properties at different scales. A notable aspect of such membraneless organelles is that their viscoelastic properties can vary by orders of magnitude, but it has remained unclear how these pronounced differences are rooted in the nanoscale dynamics at the molecular level. Here we investigate a series of condensates formed by complex coacervation of highly charged disordered proteins and polypeptides that span about two orders of magnitude in bulk viscosity. We find that their viscosity is highly correlated with protein translational diffusion and nano- to microsecond chain dynamics. Remarkably, analytical relations from polymer physics can predict condensate viscosity from diffusivity and chain dynamics, and vice versa, even for more hydrophobic disordered proteins and for synthetic polyelectrolytes, indicating a mechanistic link across several decades of length- and timescales. Atomistic simulations reveal that the observed differences in friction - a key quantity underlying these relations - reflect differences in inter-residue contact lifetimes as a function of arginine content and salt concentration, leading to the vastly different dynamics among condensates. The rapid exchange of inter-residue contacts we observe may be a general mechanism for preventing dynamic arrest in compartments densely packed with polyelectrolytes, such as the cell nucleus.

Photon-by-photon analysis tools for diffusion-based single-molecule Förster resonance energy transfer (smFRET) experiments often describe protein dynamics with Markov models. However, FRET efficiencies are only projections of the conformational space such that the measured dynamics can appear non-Markovian. Model-free methods to quantify FRET efficiency fluctuations would be desirable in this case. Here, we present such an approach. We determine FRET efficiency correlation functions free of artifacts from the finite length of photon trajectories or the diffusion of molecules through the confocal volume. We show that these functions capture the dynamics of proteins from nano- to milliseconds both in simulation and experiment, which provides a rigorous validation of current model-based analysis approaches.

It has become increasingly evident that the conformational distributions of intrinsically disordered proteins or regions are strongly dependent on their amino acid compositions and sequence. To facilitate a systematic investigation of these sequence-ensemble relationships, we selected a set of 16 naturally occurring intrinsically disordered regions of identical length but with large differences in amino acid composition, hydrophobicity, and charge patterning. We probed their conformational ensembles with single-molecule Forster resonance energy transfer (FRET), complemented by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as small-angle X-ray scattering (SAXS). The set of disordered proteins shows a strong dependence of the chain dimensions on sequence composition, with chain volumes differing by up to a factor of 6. The residue-specific intrachain interaction networks that underlie these pronounced differences were identified using atomistic simulations combined with ensemble reweighting, revealing the important role of charged, aromatic, and polar residues. To advance a transferable description of disordered protein regions, we further employed the experimental data to parametrize a coarse-grained model for disordered proteins that includes an explicit representation of the FRET fluorophores and successfully describes experiments with different dye pairs. Our findings demonstrate the value of integrating experiments and simulations for advancing our quantitative understanding of the sequence features that determine the conformational ensembles of intrinsically disordered proteins.

The conformational dynamics of single-stranded nucleic acids are fundamental for nucleic acid folding and function. However, their elementary chain dynamics have been difficult to resolve experimentally. Here we employ a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and nanophotonic enhancement to determine the conformational ensembles and rapid chain dynamics of short single-stranded nucleic acids in solution. To interpret the experimental results in terms of end-to-end distance dynamics, we utilize the hierarchical chain growth approach, simple polymer models, and refinement with Bayesian inference to generate structural ensembles that closely align with the experimental data. The resulting chain reconfiguration times are exceedingly rapid, in the 10-ns range. Solvent viscosity-dependent measurements indicate that these dynamics of single-stranded nucleic acids exhibit negligible internal friction and are thus dominated by solvent friction. Our results provide a detailed view of the conformational distributions and rapid dynamics of single-stranded nucleic acids.

The highly charged IDPs ProTα and H1 (−44 and +53, respectively) form a ultrahigh-affinity disordered complex that represents a new paradigm for biomolecular recognition[1-4]. Single molecule Föster resonance energy transfer (smFRET) experiments reveal pico-nanomolar affinity for ProTα-H1 dimer formation at near physiological salt concentrations and an exquisite sensitivity of the affinities to salt concentration[1-4], suggesting that counter-ion release is an important driving force. Temperature-dependent smFRET and isothermal titration calorimetry (ITC) reveal that ProTα-H1 complexation is enthalpically unfavorable.

We present an analytical polymer model of polyelectrolyte complexation (PEC) [1,2], based on the Edwards-Muthukumar Hamiltonian [3,4] and counterion condensation and release [1,2], to quantitatively describe the driving forces of interaction of the ProTα- H1 complexation. This model of PEC takes into account conformational properties of the individual IDPs, counterions associated to the IDPs, and the free salt ions, resulting in several free energy contributions of enthalpic and entropic origin.

Single-molecule spectroscopy is a powerful method for studying the physics of molecular systems, particularly biomolecules, such as proteins and nucleic acids. By avoiding ensemble averaging, single-molecule techniques can resolve structural distributions and fluctuations even for complex and conformationally heterogeneous systems; they also reveal the close link between biological function and the statistical mechanics of the underlying processes. The combination of single-molecule fluorescence detection with Förster resonance energy transfer has become an essential tool for probing biomolecular dynamics on timescales ranging from nanoseconds to days. This Review briefly outlines the state of the art of single-molecule Förster resonance energy transfer spectroscopy and then highlights some of the most important physics-based developments that are expected to further expand the scope of the technique. Key areas of progress include improved time resolution, access to nonequilibrium dynamics and synergies with advances in data analysis and simulations. These developments create new opportunities for attaining a comprehensive understanding of the dynamics and functional mechanisms of biological processes at the nanoscale. 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.

Quantifying biomolecular dynamics has become a major task of single-molecule fluorescence spectroscopy methods. In single-molecule Förster resonance energy transfer (smFRET), kinetic information is extracted from the stream of photons emitted by attached donor and acceptor fluorophores. Here, we describe a time-resolved version of burst variance analysis that can quantify kinetic rates at microsecond to millisecond timescales in smFRET experiments of diffusing molecules. Bursts are partitioned into segments with a fixed number of photons. The FRET variance is computed from these segments and compared with the variance expected from shot noise. By systematically varying the segment size, dynamics at different timescales can be captured. We provide a theoretical framework to extract kinetic rates from the decay of the FRET variance with increasing segment size. Compared to other methods such as filtered fluorescence correlation spectroscopy, recurrence analysis of single particles, and two-dimensional lifetime correlation spectroscopy, fewer photons are needed to obtain reliable timescale estimates, which reduces the required measurement time.

A wide range of biomolecules in solution can phase-separate and form membraneless organelles in the cell. These assemblies often have liquid-like properties, and the corresponding dynamics and exchange of molecules with the environment are important for biological function. The dynamics and materials properties of these systems are commonly assessed based on translational diffusion or rheological properties, typically covering timescales of milliseconds and longer. However, information on the structure and dynamics at the molecular level is lacking.

Many proteins contain large structurally disordered regions or are entirely disordered under physiological conditions. The functions of these intrinsically disordered proteins (IDPs) often involve interactions with other biomolecules. An important emerging effort has thus been to identify the molecular mechanisms of IDP interactions and how they differ from the textbook notions of biomolecular binding for folded proteins. In this review, we summarize how the versatile tool kit of single-molecule fluorescence spectroscopy can aid the investigation of these conformationally heterogeneous and highly dynamic molecular systems. We discuss the experimental observables that can be employed and how they enable IDP complexes to be probed on timescales from nanoseconds to hours. Key insights include the diverse structural and dynamic properties of bound IDPs and the kinetic mechanisms facilitated by disorder, such as fly-casting; disorder-mediated encounter complexes; and competitive substitution via ternary complexes, which enables rapid dissociation even for high-affinity complexes. We also discuss emerging links to aggregation, liquid-liquid phase separation, and cellular processes, as well as current technical advances to further expand the scope of single-molecule spectroscopy.

Dynamic and multivalent interactions are hallmarks of intrinsically disordered proteins (IDPs) acting within complex cellular regulatory pathways. Thanks to their astonishing molecular plasticity, IDPs often interact with numerous binding partners. While the interaction networks are expected to be highly regulated, disentangling the underlying regulatory mechanisms remains challenging. The nuclear coactivator binding domain (NCBD) is a known transcriptional hub in regulatory networks. At equilibrium, two conformations of NCBD exist due to the cis/trans isomerization of a single proline residue: an effect previously shown to expand the tunability of complex formation with the activator for thyroid hormone (ACTR) by orders of magnitude. Interestingly, proline residues are often located, as in the case of NCBD, within phosphorylation motifs.

The highly charged IDPs ProTα and H1 (−44 and +53, respectively) form a high-affinity, yet disordered complex and thus define a limiting case in the spectrum of disorder in IDP complexes [1-3]. Temperature-dependent single-molecule FRET (smFRET) experiments and isothermal titration calorimetry (ITC) reveal ProTα-H1 complexation to be enthalpically unfavorable. Using smFRET, we also identify stoichiometrically defined ternary complexes between ProTα and H1 at equilibrium and quantitate their salt-dependent stabilities, allowing us to accurately analyze ensemble experiments such as ITC performed with micromolar protein concentrations, where ternary complex formation is pronounced. Salt-dependent measurements show that counterion release entropy is a dominant driving force of complexation. An analytical polymer theory for polyelectrolyte complexation that explicitly accounts for counterions explains the thermodynamic results and suggests that the positive binding enthalpy is dominated by the relative exothermicities of counterion condensation and interchain ion-pair formation. Despite its high affinity, the disordered complex shows remarkable plasticity and expands continuously with increasing salt concentration, as seen from inter- and intramolecular FRET using various ProTα and H1 labeling variants. However, the salt-dependent expansion is non-uniform, which can be explained by differences in charge densities in different ProTα and H1 segments. Our investigations provide a comprehensive framework for the unconventional behavior of polyelectrolyte-like biomolecular complexes.

Probing non-equilibrium dynamics with single-molecule spectroscopy is important for dissecting biomolecular mechanisms. However, existing microfluidic rapid-mixing systems for this purpose are incompatible with surface-adhesive biomolecules, exhibit undesirable flow dispersion and are often demanding to fabricate. Here we introduce droplet-based microfluidic mixing for single-molecule spectroscopy to overcome these limitations in a wide range of applications. We demonstrate its robust functionality with binding kinetics of even very surface-adhesive proteins on the millisecond timescale.