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Crowding leads to enhanced interactions between ring polymers and promotes adsorption of ring polymers to surfaces.

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ABSTRACT Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. Additionally, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation. SIGNIFICANCE Phase separation has emerged as a process of significant relevance to sorting macromolecules into distinct compartments, thereby enabling spatial and temporal control over cellular matter. Considerable effort is being invested into uncovering the driving forces that enable the separation of macromolecular solutions into coexisting phases. At its heart, this process is governed by the balance of macromolecule-solvent, inter-macromolecule, and solvent-solvent interactions. We show that the driving forces for phase separation, including the coefficients that measure interaction strengths between macromolecules, can be extracted by titrating the concentrations of crowders that enable macromolecules to phase separate at lower concentrations. Our work paves the way to leverage specific categories of measurements for quantitative characterizations of driving forces for phase separation.

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Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation.

Multiple factors drive biomolecular condensate formation. In plants, condensation of the transcription factors AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 attenuates response to the plant hormone auxin. Here, we report that actin-mediated movement of cytoplasmic ARF condensates enhances condensation. Coarse-grained molecular simulations of active polymers reveal that applied forces drive the associations of macromolecules to enhance phase separation while giving rise to dense phases that preferentially accumulate motile molecules. Our study highlights how molecular motility can drive phase separation, with implications for motile condensates while offering insights into cellular mechanisms that can regulate condensate dynamics.

Macromolecular crowding can significantly impact the behavior of biopolymers, with crowding-induced depletion interactions influencing both the conformations and surface adsorption of individual polymers. Although previous studies have explored the influence of homogeneous polymer stiffness in crowded conditions, biomolecules such as DNA can exhibit sequence-dependent stiffness, and DNA origami nanoparticles can be designed with alternating stiff and flexible domains. In this work, we use Langevin dynamics simulations to characterize how nonuniform bending stiffness modulates the conformations and adsorption of polymers in crowded environments. By systematically varying the relative length and arrangement of flexible and semiflexible domains along a linear chain, we show that increasing osmotic pressure leads to a pattern-dependent collapse of the polymer, as revealed by a decrease in the radius of gyration. In general, large flexible regions promote polymer collapse, although flexible domains separating extended semi-flexible regions can facilitate their contact, leading to stable folded conformations. When a surface is present, large semiflexible domains promote adsorption, and the pattern of stiffness can be used to control the adsorption threshold. Our findings provide insight into the impact of spatially varying stiffness on the behavior of polymers in crowded environments, highlighting mechanisms relevant to biopolymers and deformable nanoparticles in both cellular and cell-free contexts.

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Intrinsically disordered prion-like low complexity domains (PLCDs) drive phase transitions that underlie the biogenesis of many biomolecular condensates. Here, we report results from large-scale Monte Carlo simulations on lattices aided by computations of Binder cumulants and rigorous finite-size scaling. These approaches enable accurate mapping of the critical regime and computations of the full binodal of an archetypal PLCD. This weakly associating polymer undergoes phase separation coupled to percolation. Between the lowest temperature and the critical point, the concentrations along the left arm of the binodal vary by four orders of magnitude. The overlap line intersects the left arm of the binodal well below the critical point. This, taken together with the intersection of the percolation line and the left arm of the binodal, leads to demarcation of the binodal into three regimes. Regime I is farthest from the critical point. Here, the coexisting dilute phase is akin to a gas of dispersed polymers. The dilute arm of the binodal lies above the overlap line in Regimes II and III. Here, the semidilute nature of dilute phases enables clustering of polymers that is enhanced by intermolecular associations. The coexisting dense phases form confined percolated networks in Regimes I and II. In Regime III, which is closest to the critical point, the dense phase becomes unconfined and fragmented, and the system is defined by two interconnected, system-spanning networks. In addition to mapping the critical point accurately, we evaluated methods for identifying the theta temperature. We find that scaling approaches based on assumptions from two-parameter theories for homopolymers yield erroneous estimates of the theta temperature of an archetypal PLCD. Accurate estimation of the theta temperature requires direct calculation of the temperature dependence of the two-body interaction coefficient. We discuss implications for inferring solvent quality from scaling analysis of segmental distances of disordered proteins.

Microgels, a microscale variant of hydrogels (1–100 µm), exhibit high surface area and responsiveness to external stimuli while retaining the soft, viscoelastic nature of their macroscale counterparts. While microgels can be derived from both synthetic and natural polymers, protein‐based microgels offer significant advantages due to their diverse function and activities. However, traditional fabrication methods, such as microfluidics and emulsion‐based techniques, often involve trade‐offs between scalability, structural integrity, and functionality. To overcome these limitations, liquid‐liquid phase separation is leveraged to fabricate microgels using globular supercharged fluorescent protein and a terminal epoxy derivative of PEG polymer – poly(ethylene glycol)diglycidyl ether (PEGDE). The presence of terminal epoxy groups on PEGDE facilitates internal crosslinking with lysine residues of supercharged proteins, resulting in stable microgels. The microgels are characterized with fluorescence microscopy, SEM, and FTIR. Fluorescence recovery after photobleaching experiments suggest the encapsulation of the polymers within the dense phase and are dependent on the polymer chain length. The results are further supported by coarse‐grained MD simulations providing mechanistic insights. Finally, the utility of the microgels in dye and nanoparticle adsorption, along with biomineralization of fluorinated calcium phosphate, is shown. These highlight the ability of microgels to potentially open avenues for biomimetic material synthesis.