Jul 2026· International Journal of Molecular Sciences· Vol 27· 0 citations· 268 references
Medicine
TL;DR
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.
Abstract
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.
An overview of solid-state NMR approaches that are readily applicable and potential methodological developments to investigate protein condensates and coacervates as well as to study protein phase separation and phase transitions are discussed.
Seamoon Deb, Erick J Dufourc, B. Habenstein et al.· Biophysical Chemistry· 0 citations
Biomolecular condensates formed by intrinsically disordered proteins require molecular models that accurately describe proteins in both dilute solution and condensed phases. Explicit-solvent coarse-grained models offer an attractive balance between chemical resolution and computational efficiency. Yet, it remains unclear whether improving dilute-state properties is sufficient to obtain an accurate description of condensates. Here, we address this question by introducing minimal modifications to the Martini 3 force field that combine recent advances in bonded interactions with refined protein–water interactions and strengthened glycine self-interactions, while preserving the underlying chemical transferability of the model. The resulting model substantially improves the description of single-chain conformations across a diverse benchmark of disordered proteins. We then investigate phase separation of the well-characterized low-complexity domain of heterogeneous nuclear ribonucleoprotein A1 and its sequence variants. The model reproduces several key physicochemical properties of biomolecular condensates, including chain expansion in the dense phase, sequence-dependent intermolecular contacts, protein diffusion and its relation to single-chain dimensions, and hydration, while revealing quantitative limitations in condensate density, phase equilibria, and ion partitioning. Our results show that improving dilute-state behaviour translates into a better description of condensed-phase properties, including condensate density, but is not sufficient to quantitatively reproduce the equilibrium between the dilute and dense phases.
Fran Bačić Toplek, Luís Borges-Araújo, Kresten Lindorff-Larsen et al.· bioRxiv· 0 citations
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.
Ethan A. Perets, Jacob A. Spies, Justin H. Cheong et al.· Biophysical Journal· 0 citations
Buffers are commonly selected for their compatibility with biochemical measurements or their specific capabilities, yet buffer interactions with proteins and the surrounding water can actively reshape protein activity, structure, and dynamics. Here, we compare the influences of three widely used electrospray ionization (ESI) buffers on the dynamics and stability of wild-type transthyretin (wtTTR) and TTR mutants (V30M, L55P, T119M, V122I) using native mass spectrometry (nMS). Ammonium acetate (AmAc), ethylenediammonium diacetate (EDDA), and triethylammonium acetate (TEAA) are commonly employed in nMS because they stabilize the sample in solution, facilitate gentle ionization, and minimize adduct formation on proteins and protein complexes. In comparison to AmAc, EDDA and TEAA reduce the average charge state (Zavg) of ions, consistent with conformational changes that decrease the solvent-accessible surface area (SASA) of proteins. Intact protein hydrogen–deuterium exchange experiments indicate significantly lower deuterium uptake for all TTR proteoforms in EDDA compared with AmAc or TEAA. Ion mobility MS revealed that each TTR proteoform has a larger average CCS and broader CCS distributions in AmAc, indicating greater conformational heterogeneity and dynamics than in EDDA or TEAA. Measurements of TTR tetramer disassembly and reassembly further demonstrated that the buffer identity strongly influences tetramer stability in solution. The buffer-dependent effects arise from differences in buffer-protein and buffer-solvent interactions that alter the hydration of the protein. These results highlight that buffer composition can significantly influence experimentally observed protein dynamics and stability, with important implications for interpreting measurements across biochemical and biophysical techniques.
Emily Burningham, Carter Lantz, R. Rider et al.· Analytical Chemistry· 0 citations
Amino acid behavior in water-mediated processes is not well captured by hydrophobicity or side-chain class alone, because residues with similar hydropathy can differ in charge localization, geometry, surface exposure, and hydration response. The objective of this study is to determine whether integrating complementary molecular representations can reveal a chemically interpretable molecular coordinate that organizes amino acid behavior across distinct water-mediated processes. We fused electronic-, structural-, and solvation-level representations into a similarity network and embedded it spectrally to obtain a one-dimensional coordinate, Z F, which captures coupled variation in electrostatics-, geometry-, and hydration-related features. Across gas hydrate formation, ice recrystallization inhibition, and CaCO3 crystallization, Z F provides a consistent residue-level coordinate and captures interaction trends that differ in direction from those described by the Kyte–Doolittle hydropathy scale in the latter two systems.
Yusung Ok, Y. Park· Journal of Chemical Informat...· 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.
Ayat Bdarneh, N. Amdursky· JACS Au· 0 citations
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