Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore. Multivalent interactions of modular binding domains and their target motifs can drive phase separation. However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect. Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins. We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions. We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing.

Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid "membraneless organelles" that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in [Formula: see text] are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in [Formula: see text] are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.

Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18-R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers)1–3. However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules4 and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta7 model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

Post-translational modifications (PTMs) produce significant changes in the structural properties of intrinsically disordered proteins (IDPs) by affecting their energy landscapes. PTMs can induce a range of effects, from local stabilization or destabilization of transient secondary structure to global disorderto-order transitions, potentially driving complete state changes between intrinsically disordered and folded states or dispersed monomeric and phase-separated states. Here, we discuss diverse biological processes that are dependent on PTM regulation of IDPs. We also present recent tools for generating homogenously modified IDPs for studies of PTMmediated IDP regulatory mechanisms.The amino acid sequences of intrinsically disordered proteins or protein regions (often simply referred to as IDPs) 3 determine their inability to fold into stable tertiary structures under physiological conditions and instead enable them to rapidly interconvert between distinct conformations to mediate critical biological functions (1, 2). The amino acid compositions of IDPs range from very low complexity with little diversity in residue types to much higher sequence complexity that enables disorder-to-order transitions upon binding or post-translational modifications (PTMs) (3-5). This range of sequence composition leads to heterogeneous ensembles with variable hydrodynamic properties and fluctuating secondary and tertiary structure, with some IDPs able to self-associate in phaseseparated protein-dense droplets (6 -11). Structural heterogeneity and dynamic fluctuations endow IDPs with unique advantages over folded proteins for certain roles (12-14). IDPs expose, at least transiently, their entire primary sequence for binding, enabling multiple interactions along their polypeptide chains. This makes them hubs in protein complexes and integrators in signaling networks (15)(16)(17)(18)(19). IDPs also facilitate the multivalent interactions that drive phase separation, underlying cellular membraneless organelles and signaling puncta (20 -22).Due to their accessibility to modifying enzymes, IDPs are the predominant sites of PTMs, which significantly expands their functional versatility (3,23). By changing the physicochemical properties of the primary sequence, PTMs induce a range of structural changes, from local stabilization or destabilization of transient secondary structure to more global conformational changes in disorder-to-order transitions (24, 25). As the hydrodynamic properties of IDPs are strongly affected by electrostatic effects, PTMs that change charge (e.g. phosphorylation and acetylation) can modulate compactness (9 -11). PTMs can also lead to complete state changes, between disordered and folded states (26 -28) or dispersed monomeric and phase-separated states (6,8,22). The scope of PTM-mediated structural and dynamic changes described in recent biophysical and biochemical studies of IDPs is similar to those due to binding to other proteins, nucleic acids, lipids, carbohydrates, ions, cofactors, and other small molecul...

Activity-dependent translation requires the transport of mRNAs within membraneless protein assemblies known as neuronal granules from the cell body toward synaptic regions. Translation of mRNA is inhibited in these granules during transport but quickly activated in response to neuronal stimuli at the synapse. This raises an important question: how does synaptic activity trigger translation of once-silenced mRNAs? Here, we demonstrate a strong connection between phase separation, the process underlying the formation of many different types of cellular granules, and in vitro inhibition of translation. By using the Fragile X Mental Retardation Protein (FMRP), an abundant neuronal granule component and translational repressor, we show that FMRP phase separates in vitro with RNA into liquid droplets mediated by its C-terminal low-complexity disordered region (i.e., FMRPLCR). FMRPLCRposttranslational modifications by phosphorylation and methylation have opposing effects on in vitro translational regulation, which corroborates well with their critical concentrations for phase separation. Our results, combined with bioinformatics evidence, are supportive of phase separation as a general mechanism controlling activity-dependent translation.

The kinetic mechanism of Na ؉ binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na ؉ binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k obs decreases hyperbolically with increasing [Na ؉ ]. The rapid phase is due to Na ؉ binding to the enzyme E to generate the E:Na ؉ form. The slow phase is due to the interconversion between E* and E, where E* is a form that cannot bind NaTemperature studies in the range from 5 to 35°C show significant enthalpy, entropy, and heat capacity changes associated with both Na ؉ binding and the E to E* transition. As a result, under conditions of physiologic temperature and salt concentrations, the E* form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na ؉ (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na ؉ binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na ؉ binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na ؉ binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.

Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). Whereas the binding of 4E-BP peptides containing the eIF4E-binding ⁵⁴YXXXXLΦ⁶⁰ motif has been studied, atomic-level characterization of the interaction of eIF4E with full-length 4E-BPs has been lacking. Here, we use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to characterize the dynamic, structural and binding properties of 4E-BP2. Although disordered, 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical ⁵⁴YXXXXLΦ⁶⁰ and ⁷⁸IPGVT⁸² sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. This dynamic interaction facilitates exposure of regulatory phosphorylation sites within the complex. The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.