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.
Hydrophobic hydration underpins processes central to chemistry, biology, and technology, from protein folding and molecular self-assembly to catalysis and nanomaterial dispersion. However, direct structural information on water surrounding large hydrophobic molecules remains scarce due to their very low solubility. Here, we overcome this limitation by using low-temperature vapor codeposition to form amorphous mixtures of water and anthracene, a hydrophobic “mini-graphene”. We then resolve the structure of anthracene’s first hydration shell by neutron diffraction. The linear polycyclic aromatic hydrocarbon induces distinct structural motifs in its hydration shell. Water molecules above and below the outer aromatic rings exhibit one broken water–water hydrogen bond enabling weak O–H···π interactions with the solute, while a pronounced “dewetting” effect emerges at the central ring. These findings provide experimentally constrained structural insights into the hydration around a large hydrophobe under conditions where conventional liquid-state experiments are not feasible.
Gustavo A. Madrigal, Christos Vasilopanagos, T. Headen et al.· Journal of Physical Chemistr...· 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
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.
Pushpendra Singh, A. Bandyopadhyay· Biosyst.· 0 citations
Water confined in poly(vinyl alcohol) (PVA) films exhibits enhanced basicity toward embedded chromophores, yet whether this anomalous reactivity reflects a broader acid-base duality remains unexplored. Here, using 5-methoxyflavone (5MF) as a photobase probe whose C═O stretch is spectrally isolated in FTIR, we uncover that short hydrogen bonds (SHBs) render the same confined water capable of protonating 5MF without external acid. In low-water-content PVA films, the C═O stretch of 5MF red-shifts substantially, confirming SHB formation. Time-resolved fluorescence reveals two protonated emission bands with identical, short lifetimes, distinct from the long-lived free cation in acidic films─pointing to a tightly bound 5MFH+···OH- complex rather than free 5MFH+. DFT calculations suggest that the distinct emissions can be rationalized as two limiting wave function distributions of the same proton undergoing quantum delocalization within the SHB. This unified SHB picture resolves the conflicting acid-base behaviors of confined water and highlights the potential of PVA films as versatile platforms for exploring acid-base phenomena enabled by nanoconfinement.
Qin Yu, Xiya Peng, Ziyi Wang et al.· Journal of Physical Chemistr...· 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
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
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