Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multiphase systems defined by coexisting dense and dilute phases. Here, we show that solution ions partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules. Our findings were enabled by direct measurements of the activities of cations and anions within coexisting phases of protein and RNA condensates. Asymmetries in ion partitioning between coexisting phases vary with protein sequence, macromolecular composition, salt concentration, and ion type. The Donnan equilibrium set up by the asymmetrical partitioning of solution ions generates interphase electric potentials known as Donnan and Nernst potentials. Our measurements show that the interphase potentials of condensates are of the same order of magnitude as membrane potentials of membrane-bound organelles. Interphase potentials quantify the degree to which microenvironments of coexisting phases are different from one another. Importantly, and based on condensate-specific interphase electric potentials, we reason that condensates are akin to capacitors that store charge. Interphase potentials should lead to electric double layers at condensate interfaces, thereby explaining recent observations of condensate interfaces being electrochemically active.

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates are two- or multi-phase systems defined by coexisting dense and dilute phases. Here, we report the discovery of asymmetrical partitioning of ions across coexisting phases. These asymmetries lead to the generation of interphase electric potentials known as Donnan and Nernst potentials. Strikingly, these potentials are of the same order of magnitude as membrane potentials of membrane-bound systems. Our findings were enabled by direct measurements of the activities of cations and anions within coexisting phases that define condensates formed by protein and RNA molecules. Asymmetries in ion partitioning between coexisting phases vary with condensate type, salt concentration, and ion type. Our discovery of condensate-specific interphase electric potentials, which are membrane-like potentials of membraneless bodies, provides a physical basis for condensates being responsive elements to osmotic stress and modulators of electrochemical equilibria in cells.

Flow cytometry-based fluorescence measurements are a promising approach for high-throughput quantitative analysis of the kinetic properties and thermodynamic driving forces of biomolecular phase transitions in cellular environments. Whether monitored by FRET or pulse shape analysis, many biomolecular systems that undergo phase transitions manifest as two-state discontinuous transitions. We have previously demonstrated that such transitions likely represent nucleated phase transitions. The simplest mechanism for such transitions is that of homogeneous nucleation, described in terms of classical nucleation theory. Experimental constraints make it difficult to gather continuous kinetic traces or collect data such that the concentration of the protein of interest is either held constant or continuously measured, and these are necessary prerequisites for the application of classical nucleation theory. Here, we present advances in experimental design, statistical analysis and the development of a theoretical framework based on extending classical nucleation theory to overcome many of the aforementioned challenges and enable the quantification of saturation concentrations and the underlying thermodynamic and kinetic properties that drive observed phase transitions in live cells, namely the height of the energetic barrier for nucleation, and the rate of crossing that barrier. These advances will enable future systematic and quantitative comparisons of a variety of proteins, including prion-like domains and other aggregating proteins under a wide range of cellular conditions. This offers a path forward for quantitative understanding of the determinants of protein self-assembly, nucleated phase transitions, phase separation dynamics, and the impact of cellular factors as modulators of intracellular phase transitions.

Whi3 is an RNA binding protein from Ashbya gossypii that phase separates to form RNA-protein condensates. Observations in cells show that certain RNA molecules co-localize with Whi3 and other RNA molecules into a single condensate, while some others form apparently demixed condensates. Is a there a thermodynamic basis for different ternary combinations of Whi3 and RNA molecules to form demixed condensates? Simulations show that RNA molecules must have strong repulsive interactions for one another, or strong homotypic interactions if demixing is to be thermodynamically feasible. We tested this using in vitro reconstitutions to quantify the phase behaviors of binary and ternary mixtures. A novel analysis of the measured phase boundaries show that heterotypic protein-RNA interactions are the main drivers of phase separation. This was further tested by deletion of cognate binding sites on RNA molecules, showing that there are several ways for Whi3 to associate with RNA molecules. Investigations of phase behaviors internary mixtures show that demixed condensates are dynamically arrested phases. Therefore, in vitro studies suggest that demixing is likely to be under dynamical rather than thermodynamic control. To test for this in live cells, we generated a mutant where differentially expressed RNAs are constitutively expressed under a single promoter. We find that RNA molecules that demix in a wildtype background formed colocalized condensates when they are expressed concomitantly. Our studies show that the non-equilibrium aspects of phase separation, specifically the ability to form dynamically arrested phases, can give rise to demixed condensates that achieve long-livedspatial segregation of RNA molecules within distinct condensates.

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E4K4 repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residue-specific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. uncharged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAH-forming sequences. The stabilities of alpha helical conformations increase with the number of E4K4 repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

Charge regulation, defined as the regulation of charge states of ionizable residues through the uptake or release of protons, can influence the conformational ensembles and distributions of charge states that are accessible to intrinsically disordered proteins. These influences are exerted through sequence- and conformational-context dependent pKa values. We recently developed and deployed a q-canonical ensemble that allows for the linked analyses of separate measurements, of charge and conformation as a function of pH. When combined with atomistic, q-canonical Monte Carlo simulations based on the ABSINTH implicit solvation model, we were able to uncover the pH-dependent interplay of charge state and conformational heterogeneity. However, the combinatorial increase in the number of charge states to consider has remained a persistent challenge. Here, we introduce a breakthrough that allows us to predict the extent of charge regulation to be expected for IDPs arbitrary numbers of ionizable residues. The algorithm leverages prior information regarding the effects of local sequence contexts on charge regulation. Longer sequences with arbitrary numbers of ionizable residues are built up using a chain-growth Monte Carlo procedure, each step building on extant information for peptide fragments. We have deployed the new methodology in a high-throughput investigation of sequences drawn from the IDRome, which is the sub-proteome of intrinsically disordered regions (IDRs). We find that the apparent charge, obtained by assuming model compound pKa values, is rarely the same as the real charge that one predicts based on an accounting of charge regulation effects. This work paves the way to understanding the sequence features that result in charge regulation effects, and their importance to biomolecular processes.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" important for regulation. For the mitochondrial fission dynamin, dynamin-related protein 1, a regulatory role for the variable domain (VD) is demonstrated by gain- and loss-of-function mutations, yet the basis for this is unclear. Here, the isolated VD is shown to be intrinsically disordered and undergo a cooperative transition in the stabilizing osmolyte trimethylamine N-oxide. However, the osmolyte-induced state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, which appears to promote phase separation. Since dynamin-related protein 1 is found assembled into discrete punctate structures on the mitochondrial surface, the inference from the present work is that these structures might arise from a condensed state involving the VD that may enable rapid tuning of mechanoenzyme assembly necessary for fission.

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here, we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems.

Single Alpha Helices are repetitive, highly charged polyampholytes that exhibit a stable helical structure, despite having many characteristics of intrinsically disordered proteins. Previous studies have proposed that i, i+4 salt-bridges may be a key to the stabilization of the helical structure. However, measurements have also shown that SAHs can be stable over a large range of pH values, calling into question whether salt-bridges persist as stabilizing factors away from neutral pH values, and if they are the only contributors at neutral pH.

ABSTRACTIntrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations. In the past several decades, IDRs have emerged as a core component of many proteomes, comprising ∼30% of all eukaryotic protein sequences. IDRs are ubiquitous throughout different biological pathways, with a notable enrichment in responses to environmental stimuli such as abiotic stress. However, the diversity of IDR-based systems that biology has evolved to respond to different stimuli is expansive, warranting the exploration of IDRs present in unique molecular contexts. Here, we identify and characterize intrinsic disorder in the soluble, cytoplasmic N-terminal domains of three members of the MscS-Like (MSL) family of mechanosensitive ion channels, MSL8, MSL9 and MSL10. In plants, MSL channels are proposed to mediate the reactions to cell swelling, pathogenic invasion, and touch. A series of bioinformatic tools unanimously predicted that the cytosolic N-termini of MSLs are intrinsically disordered. We confirmed this prediction for the N-terminus of MSL10 (MSL10N) via circular dichroism spectroscopy. MSL10Nadopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE) and underwent structural changes and alterations to homotypic interaction favorability in the presence of molecular crowding agents. Lastly,in vitroimaging of condensates indicated that MSL8N, MSL9Nand MSL10Nhave sharply differing propensities for condensate formation both inherently and in response to salt, temperature, and molecular crowding. Altogether, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond disparately to changes in their physiochemical environment.

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

For proteins with multiple ionizable residues, the canonical assumption is that ionization states of residues are fixed, as dictated by their intrinsic pKa values. Recently, we have applied the formalism of the q-canonical ensemble to analyze and interpret potentiometric measurements of protein net charge as a function of pH. These measurements showed that shifted pKa values and heterogeneous distributions of charge states are required to account for all thermodynamically relevant species in the ensembles that contribute to potentiometric profiles.

The fungal protein Whi3 forms cytoplasmic biomolecular condensates with various types of RNA molecules. Strikingly, Whi3 condensates formed with one type of RNA will either mix or remain unmixed from condensates formed with other RNA molecules. Nanoscale structural differences among different RNA molecules have been implicated as determinants of this differential, RNA-dependent mixing vs. unmixing behavior of Whi3-RNA condensates, although how these features impact emergent, micron-scale phase behavior is unclear. Here, we use a combination of microscopy and spectroscopy measurements to map full phase boundaries of Whi3 molecules with four different RNA molecules. For each Whi3 and RNA pair, the phase boundaries are closed loops. This points to phase behavior being driven by RNA-specific interplay between heterotypic protein-RNA and homotypic protein-protein and RNA-RNA interactions. Importantly, we uncover significant RNA dependence to the shapes of the closed loops, the areas encompassed by closed loops, and the orientations of these closed loops in the plane defined by the concentrations of Whi3 and RNA nucleotides. We annotate each of the closed loops with tie lines derived from a combination of experiments and theoretical approaches. Together, with morphological characterizations, the mapping of RNA-specific two-component phase diagrams allows us to parameterize a stickers-and-spacers model that we deploy in LASSI-based simulations to study the phase behavior of a quinary system comprising each of the four distinct RNAs and the Whi3 protein. The results suggest that the selective mixing vs. unmixing of Whi3-RNA condensates can be explained in terms of RNA-dependent interplay between heterotypic and homotypic interactions. Our findings have broad implications for the spontaneous sorting of RNA into distinct protein-RNA granules, which is relevant for RNA trafficking.

Glycine-rich regions feature prominently in intrinsically disordered regions (IDRs) of proteins that drive phase separation and the regulated formation of membraneless biomolecular condensates. Interestingly, the Gly-rich IDRs seldom feature poly-Gly tracts. The protein fused in sarcoma (FUS) is an exception. This protein includes two 10-residue poly-Gly tracts within the prion-like domain (PLD) and at the interface between the PLD and the RNA binding domain. Poly-Gly tracts are known to be highly insoluble, being potent drivers of self-assembly into solid-like fibrils. Given that the internal concentrations of FUS and FUS-like molecules cross the high micromolar and even millimolar range within condensates, we reasoned that the intrinsic insolubility of poly-Gly tracts might be germane to emergent fluid-to-solid transitions within condensates. To assess this possibility, we characterized the concentration-dependent self-assembly for three non-overlapping 25-residue Gly-rich peptides derived from FUS. Two of the three peptides feature 10-residue poly-Gly tracts. These peptides form either long fibrils based on twisted ribbon-like structures or self-supporting gels based on physical cross-links of fibrils. Conversely, the peptide with similar Gly contents but lacking a poly-Gly tract does not form fibrils or gels. Instead, it remains soluble across a wide range of concentrations. Our findings highlight the ability of poly-Gly tracts within IDRs that drive phase separation to undergo self-assembly. We propose that these tracts are likely to contribute to nucleation of fibrillar solids within dense condensates formed by FUS.

Ribonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms compositionally distinct ribonucleoprotein condensates that are implicated in key processes such as cell-cycle control and cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region and a tandem RNA recognition module. Here, we uncover localized order-to-disorder transitions within a 21-residue stretch of the Q-rich region. This region, which can form alpha-helical conformations, is shown to modulate protein density within Whi3-RNA condensates by driving dilute phase oligomerization. Specifically, enhancing helicity within this region enhances oligomerization in the dilute phase. This weakens the associations among disordered Q-rich regions thereby diluting the concentration of Whi3 in condensates. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, dilute phase oligomers, driven by a specific sequence motif, lead to negative regulation of the stoichiometry of protein versus RNA in the dense phase. Our findings stand in contrast to other systems where oligomerization is known to enhance the drive for phase separation. Our results highlight distinctive regulatory effects over phase behavior due to local order-to-disorder transitions within intrinsically disordered regions. This provides a way to leverage molecular scale conformational preferences and coupled intermolecular associations to regulate mesoscale phase behavior and material properties of condensates.A large sub-class of biomolecular condensates are linked to RNA regulation and known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces of biomolecular condensates, relatively little is known about negative regulation of assembly. Here, using a fungal RNP component, Whi3, we show that its intrinsically-disordered, Q-rich region exerts regulatory control over condensate formation through a cryptic helical region that enables the formation of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational equilibria can enable control over micron-scale phase equilibria.

Ionizable residues can release and take up protons and this has an influence on protein structure and function. The extent of protonation is linked to the overall pH of the solution and the local environments of ionizable residues. Binding or unbinding of a single proton generates a distinct charge microstate defined by a specific pattern of charges. Accordingly, the overall partition function is a sum over all charge microstates and Boltzmann weights of all conformations associated with each of the charge microstates. This ensemble-of-ensembles description recast as a q-canonical ensemble allows us to analyze and interpret potentiometric titrations that provide information regarding net charge as a function of pH. In the q-canonical ensemble, charge microstates are grouped into mesostates where each mesostate is a collection of microstates of the same net charge. Here, we show that leveraging the structure of the q-canonical ensemble allows us to decouple contributions of net proton binding and release from proton arrangement and conformational considerations. Through application of the q-canonical formalism to analyze potentiometric measurements of net charge in proteins with repetitive patterns of Lys and Glu residues, we determine the underlying mesostate pKa values and, more importantly, we estimate relative mesostate populations as a function of pH. This is a strength of using the q-canonical approach that cannot be replicated using purely site-specific analyses. Overall, our work shows how measurements of charge equilibria, decoupled from measurements of conformational equilibria, and analyzed using the framework of the q-canonical ensemble, provide protein-specific quantitative descriptions of pH-dependent populations of mesostates. This method is of direct relevance for measuring and understanding how different charge states contribute to conformational, binding, and phase equilibria of proteins.

The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.