Biomolecular condensates represent unique microenvironments that organize intracellular biology and promote biochemical reactions. However, the biomolecular interactions driving condensate phase separation are often weak, transient, and heterogeneous. Investigating the structural biology and chemical properties of condensate interiors has therefore proven experimentally challenging, often requiring the use of perturbative probes. To overcome this challenge, we combine label-free optical scattering and vibrational spectroscopy approaches spanning ultraviolet, visible, mid-infrared, and terahertz wavelengths with deep-learning-based ensemble prediction of intrinsically disordered protein conformations. This suite of label-free approaches provides quantitative insights into protein-protein/protein-solvent interactions and the chemical properties of condensate interiors. Investigating the N-terminal domain of the RNA DEAD-box helicase 4 (DDX4), our experimental and computational results support a model of phase separation involving protein chain collapse, reduced dielectric, and water release. These molecular events are expected to enhance the strength of multivalent protein-protein interactions within condensates, creating a positive feedback loop important for condensate growth and phase separation.
This work investigates how protein folding landscapes are altered inside condensates, using the protein α-helix as a model folded domain and develops a chemically specific, residue-resolution model for quantification of α-helical folding and applies it to characterize diverse helices within condensates of varying physicochemical properties.
Nathaniel Hess, Jerelle A. Joseph· Journal of the American Chem...· 0 citations
The formation of biomolecular condensates has been linked to a broad range of essential cellular processes, including immune signaling cascades, mRNA transport, and autophagy. Conversely, phase-separated condensates have also been associated with aberrant protein misfolding and subsequent aggregation in neurodegenerative disease-related processes. Protein phase separation and phase transitions involve the formation of heterogeneous and dynamic assemblies that can evolve into gel-like or semi-crystalline states, which are challenging to characterize using high-resolution structural biology techniques. Solid-state nuclear magnetic resonance (NMR) spectroscopy now offers a broad arsenal of methods to probe the structural and dynamic features of viscous condensates, elastic solids, coacervates and rigid protein assemblies. This review provides an overview of solid-state NMR approaches that are readily applicable and discusses potential methodological developments to investigate protein condensates and coacervates as well as to study protein phase separation and phase transitions.
Seamoon Deb, Erick J Dufourc, Birgit Habenstein et al.· Biophysical Chemistry· 0 citations
Biomolecular condensates formed by liquid–liquid phase separation create dynamic, water-rich microenvironments that are essential for cellular organization, yet it is challenging to understand their internal aqueous phase. Here, we introduce Brønsted photoacids and photobases as a new class of environmental fluorescent probes to interrogate the water phase inside biomolecular condensates. Using in vitro condensates formed by intrinsically disordered poly lysine or poly arginine peptides with nucleotides, we combine steady-state and time-resolved fluorescence to resolve the probe’s excited-state proton transfer and subsequent proton recombination dynamics. We show that both photoacids and photobases remain largely solvated within poly lysine-based condensates, with only modest slowing of proton transfer relative to bulk water, consistent with a moderately more viscous aqueous environment. We mainly focus on photoacids, which exhibit strongly enhanced geminate proton recombination, attributed to nanometre-scale confinement of water within the condensates, inducing reflective boundaries for the dissociated proton. By modeling the recombination kinetics, we use the photoacid as a molecular ruler to estimate the dimensions of confined aqueous nanocavities, revealing characteristic maximum radii of ∼6 nm. Altering condensate composition systematically modulates these properties, with ATP- and poly arginine–based condensates displaying denser, less hydrated interiors. These findings establish excited-state proton transfer probes as powerful tools for quantifying nanoscale water confinement in biomolecular condensates, with implications for condensate function and molecular sequestration.
Liquid-liquid phase separation of proteins has been observed to occur on biological membranes, where it is thought to play a role in diverse cellular behaviors. Recent work has demonstrated colocalization between protein condensates on opposing leaflets of the bilayer, suggesting that protein phase separation may be coupled across the bilayer. However, the mechanism behind this coupling phenomenon remains poorly understood. Here we seek to understand the protein-protein and protein-membrane interactions that give rise to transbilayer coupling of protein condensates. We perform coarse-grained molecular dynamics simulations of a bilayer with a disordered protein condensate tethered to each leaflet surface. In this system, we observe stable, coupled diffusion of the condensates across the membrane. We find that increasing the protein-protein interaction strength leads to decoupling, driven by competing membrane curvatures induced by each condensate. However, by applying membrane tension we suppress curvature and restore coupling even at higher protein interaction strengths. Under coupling conditions, we find that lipid entropy is reduced upon direct contact with proteins, but this effect is not transferred to the opposing leaflet. Interestingly, further analysis reveals increased transverse lipid packing (interdigitation) beneath the condensates relative to protein-free regions. Based on these observations, we propose that enhanced lipid interdigitation mediates interleaflet communication and serves as the primary mechanism driving transbilayer coupling of condensates in this system. This work provides insight into a potential physical mechanism for transmembrane communication in cellular contexts and suggests directions for future investigation. Significance Statement Liquid-like condensates are active participants at cellular membranes, where they act as organizers and catalysts for various cellular processes. Recent work has demonstrated that protein condensates can couple across the bilayer; however, the molecular mechanism of this transbilayer coupling remained unknown. Here, we investigate the molecular basis of transmembrane condensate coupling through detailed analysis and propose a mechanism for the phenomenon. This work advances our understanding of how information is transmitted across the bilayer, with implications in cellular requiring coordination across the membrane, such as signaling, and more broadly in the field of membrane biophysics.
Ketsia Zinga, Jeanne C. Stachowiak, Pengyu Ren· bioRxiv· 0 citations
Aqueous two-phase systems (ATPSs) provide a versatile, fully aqueous platform for probing solute–water interactions and protein structure. This review first surveys the diversity and phase behavior of biphasic aqueous systems formed by polymers and salts. We describe how phase diagrams characterize ATPS formation and composition and how both polymer chemistry and salt identity, rather than molecular size alone, govern phase separation by modulating the solvent properties of water. Building on a modified binodal model, we show that phase separation and solute partitioning can be understood in terms of changes in aqueous solvent dipolarity/polarizability, hydrogen-bond donor/acceptor properties, hydrophobicity, and electrostatics, quantified via solvatochromic probes and homologous solute series. These measurements underpin solvent interaction analysis (SIA), in which the partition coefficients of small molecules and proteins across panels of ATPSs are used to generate “structural signatures” that sensitively report on amino acid substitutions, conformational changes, aggregation, ligand binding, osmolyte effects, and post-translational modifications, independent of protein size. We discuss how SIA can be implemented in vial-, plate-, and microfluidic formats and combined with diverse analytical readouts (HPLC, MS, colorimetric assays, and immunoassays), and we contrast this structure-focused approach with conventional concentration-only proteomic and biomarker strategies. Particular emphasis is placed on structure-based biomarker discovery, where disease-relevant shifts in proteoform distributions—especially glycosylation changes—are often more informative than bulk protein levels and where SIA can complement or simplify complex glycomics and top-down proteomics workflows. As a case study, we describe the recently FDA-approved IsoPSA assay, which applies SIA principles to prostate-specific antigen by measuring cancer-associated structural alterations in circulating PSA via its partition behavior in a proprietary ATPS. IsoPSA generates a single index that discriminates between high-grade prostate cancer and benign and low-grade conditions. Prospective, longitudinal, and MRI-integrated clinical studies demonstrate that IsoPSA improves pre-biopsy risk stratification, reduces unnecessary biopsies, and provides robust negative and positive predictive values within the PSA “gray zone.” Collectively, the data support aqueous solvent interaction analysis as a broadly applicable, mechanistically grounded technology for protein characterization, drug–protein interaction studies, and structure-centric biomarker development, exemplified by the clinical translation of IsoPSA.
B. Zaslavsky, M. Stovsky, V. Uversky· International Journal of Mol...· 0 citations
We propose a theoretical and computational framework in which ordered hydration layers at biomolecular interfaces act as multi-scale coupled, phase-sensitive degrees of freedom that self-organize biological information processing at all scales. When biomolecular surfaces approach nanometer-to-subnanometer separations, overlapping hydration structures are represented by a shared-water coupling Hamiltonian incorporating evanescent and protonic channels. Coarse-graining yields a nonlocal complex-field description for locally connected hydration regions, while slow conformational variation introduces geometric-phase terms define vortex-like phase textures and routing-like correlation patterns. We further use a driven-dissipative Gross-Pitaevskii-type model as a phenomenological, not definitive, description of nonlinear hydration-sensitive collective modes. The model generates testable observables, including step-like phase responses, band-limited correlation windows, multi-timescale pump-probe relaxation, and sensitivity to temperature, osmolytes, and hydration state. We outline a GHz vector-network-analyzer and near-infrared pump-probe validation protocol on hydrated biomolecular films, together with negative controls for phase wrapping, RF leakage, standing waves, ionic conduction, and thermal drift. The framework is built as systems-level modeling that links hydration structure at atomic scale to macroscale, translating molecular recognition at the highest scales with information-like phase organization where continuity of water channels alone spontaneously emerges quantization and fractal-like phase conservation. Water as a singular unifying entity governing a bio-system has been neglected thus far.
Pushpendra Singh, A. Bandyopadhyay· Bio Systems· 0 citations