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The interactions of intrinsically disordered proteins (IDPs) with their molecular targets are essential for the regulation of many cellular processes. IDPs can perform their functions while disordered, and they may fold to structured conformations on binding. Here we show that the cis / trans isomerization of peptidyl−prolyl bonds can have a pronounced effect on the interactions of IDPs. By single-molecule spectroscopy, we identify a conserved proline residue in NCBD (the nuclear-coactivator binding domain of CBP) whose cis / trans isomerization in the unbound state modulates the association and dissociation rates with its binding partner, ACTR. As a result, NCBD switches on a time scale of tens of seconds between two populations that differ in their affinities to ACTR by about an order of magnitude. Molecular dynamics simulations indicate as a cause reduced packing of the complex for the cis isomer. Peptidyl-prolyl cis / trans isomerization may be an important previously unidentified mechanism for regulating IDP interactions. 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.

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.

The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.

Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu.

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities. 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.

For disordered proteins, the dimensions of the chain are an important property that is sensitive to environmental conditions. We have used single-molecule Förster resonance energy transfer to probe the temperature-induced chain collapse of five unfolded or intrinsically disordered proteins. Because this behavior is sensitive to the details of intrachain and chain–solvent interactions, the collapse allows us to probe the physical interactions governing the dimensions of disordered proteins. We find that each of the proteins undergoes a collapse with increasing temperature, with the most hydrophobic one, λ-repressor, undergoing a reexpansion at the highest temperatures. Although such a collapse might be expected due to the temperature dependence of the classical “hydrophobic effect,” remarkably we find that the largest collapse occurs for the most hydrophilic, charged sequences. Using a combination of theory and simulation, we show that this result can be rationalized in terms of the temperature-dependent solvation free energies of the constituent amino acids, with the solvation properties of the most hydrophilic residues playing a large part in determining the collapse.

Internal friction is an important contribution to protein dynamics at all stages along the folding reaction. Even in unfolded and intrinsically disordered proteins, internal friction has a large influence, as demonstrated with several experimental techniques and in simulations. However, these methods probe different facets of internal friction and have been applied to disparate molecular systems, raising questions regarding the compatibility of the results. To obtain an integrated view, we apply here the combination of two complementary experimental techniques, simulations, and theory to the same system: unfolded protein L. We use single-molecule Förster resonance energy transfer (FRET) to measure the global reconfiguration dynamics of the chain, and photoinduced electron transfer (PET), a contact-based method, to quantify the rate of loop formation between two residues. This combination enables us to probe unfolded-state dynamics on different length scales, corresponding to different parts of the intramolecular distance distribution. Both FRET and PET measurements show that internal friction dominates unfolded-state dynamics at low denaturant concentration, and the results are in remarkable agreement with recent large-scale molecular dynamics simulations using a new water model. The simulations indicate that intrachain interactions and dihedral angle rotation correlate with the presence of internal friction, and theoretical models of polymer dynamics provide a framework for interrelating the contribution of internal friction observed in the two types of experiments and in the simulations. The combined results thus provide a coherent and quantitative picture of internal friction in unfolded proteins that could not be attained from the individual techniques.

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. Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro . Their application in vivo , however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

RNA chaperones are proteins that aid in the folding of nucleic acids, but remarkably, many of these proteins are intrinsically disordered. How can these proteins function without a well-defined three-dimensional structure? Here, we address this question by studying the hepatitis C virus core protein, a chaperone that promotes viral genome dimerization. Using single-molecule fluorescence spectroscopy, we find that this positively charged disordered protein facilitates the formation of compact nucleic acid conformations by acting as a flexible macromolecular counterion that locally screens repulsive electrostatic interactions with an efficiency equivalent to molar salt concentrations. The resulting compaction can bias unfolded nucleic acids towards folding, resulting in faster folding kinetics. This potentially widespread mechanism is supported by molecular simulations that rationalize the experimental findings by describing the chaperone as an unstructured polyelectrolyte. 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.

The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex. Here we 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. The results reveal the formation of a metastable encounter complex that is electrostatically favored and transits to the final bound state within tens of microseconds. Such measurements thus open a new window into the microscopic events governing biomolecular interactions. 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.