Aug 2026· Journal of Physical Chemistry B· Vol 130 34, pp.
8599-8615
· 0 citations· 77 references
Medicine
TL;DR
It is demonstrated that hydrophobic amino acids enhance protein thermal stability through a coupled modulation of solvation structure, solvent dynamics, and hydrogen-bonding interactions, with distinct mechanisms depending on side-chain size and aggregation propensity.
Abstract
Thermal stability of proteins is critically influenced by solvation structure and dynamics. Here, we present extensive all-atom molecular dynamics simulations of ubiquitin in pure water and in 2 M aqueous solutions of hydrophobic amino acids-alanine, valine, isoleucine, and phenylalanine at 300 and 450 K. Structural analyses (RMSD, radius of gyration, native contacts, and secondary structure) reveal severe unfolding in pure water at 450 K, whereas all amino acid solutions markedly suppress thermal destabilization. Solvation analyses using pair correlation functions, minimum-distance distribution functions, and Kirkwood-Buff integrals show preferential accumulation of amino acids near the protein surface, forming a mixed solvation shell that competes with water. Alanine exhibits the strongest and most uniform interaction with ubiquitin through its small side chain, enabling efficient packing and enhanced contact interactions. Valine and isoleucine provide intermediate stabilization, balancing hydrophobic interactions with steric constraints that limit surface accessibility. In contrast, phenylalanine shows weaker direct interactions due to its bulky aromatic side chain and aggregation tendency, instead stabilizing the protein indirectly by promoting a structured, long-lived hydration network. Dynamical analyses reveal suppressed translational and rotational motion of water, prolonged protein-water hydrogen-bond lifetimes, and partial retention of tetrahedral ordering at elevated temperature, with phenylalanine inducing the slowest solvent dynamics. These findings demonstrate that hydrophobic amino acids enhance protein thermal stability through a coupled modulation of solvation structure, solvent dynamics, and hydrogen-bonding interactions, with distinct mechanisms depending on side-chain size and aggregation propensity.
Naturally occurring deep eutectic solvents (NADES) offer a tunable microenvironment for biomolecular stabilization. Yet, the atomistic mechanisms governing the protein-solvent interactions remain poorly understood. Here, we present microsecond-long atomistic molecular dynamics simulations investigating lysozyme (Lys) stability in Choline Chloride (ChCl)-polyol (erythritol, xylitol, and sorbitol) aqueous deep eutectic solvents as a function of water wt. % (fw=0,25,50,75). Lys maintains its native structure across all conditions, with stable Rg, RMSD, RMSF, and secondary structure populations. The stabilization mechanism involves three coupled effects. First, solvent-solvent radial distribution functions (RDFs) and inter-component hydrogen bond (H-bond) analysis demonstrate that each NADES component preserves its characteristic H-bond network even in the presence of Lys, confirming that the bulk solvent structure is not disrupted. Second, the poorly structured protein-NADES RDFs are consistent with the preferential exclusion of NADES components from the protein surface with increasing hydration. Third, protein-polyol H-bonds remain persistently high and largely independent of water content, revealing a stable surface association layer that is not displaced by water. Among the polyols, xylitol exhibits significantly higher protein-alcohol H-bonds at low and intermediate hydration levels, owing to its intermediate flexible geometry and hydroxyl density. These findings demonstrate that the Lys stabilization in ChCl-polyol NADES arises from a polyol-dependent interplay among the preserved solvent network structure, inferential evidence for preferential exclusion, and persistent interfacial interactions. Overall, the study provides a molecular framework for rational NADES design for biomolecular stabilization applications.
Roja Satvika Puvvada, Ivy Das Sarkar, Suman Das· Journal of Chemical Physics· 0 citations
Protein stabilizers and denaturants can be used to elucidate the fundamental principles of hydration, which is crucial for biological functions and biotechnology. Despite decades of work, existing molecular models of such stabilizers and denaturants have not yet been fully validated because few experimental methods can detect water structures within the hydration shell in situ under ambient conditions. Here, we devise a molecular model guided by the application of chiral-selective vibrational sum frequency generation spectroscopy to a solvated protein. We show that urea disrupts the first hydration shell, weakening the protein's hydrogen bonds. Conversely, trimethylamine N-oxide resides outside the shell and stabilizes the protein by strengthening water hydrogen bonds within the first hydration shell. In mixtures, trimethylamine N-oxide drives urea out of the shell, neutralizing urea's destabilizing effect. We conclude that protein stability directly correlates with first hydration shell integrity. These insights have broad implications for understanding solvent effects on biocatalysis and heterogeneous cellular environments.
Zhijie Wang, Matthew Tremblay, Nicholas Hatzis-Schoch et al.· Journal of the American Chem...· 0 citations
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
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
Condensates formed by oppositely charged intrinsically disordered proteins provide model systems for understanding how transient electrostatic interactions govern structure and dynamics in biomolecular assemblies. Here we investigate a nearly charge-neutral condensate composed of 50 Prothymosin alpha (ProTalpha) and 40 Histone H1 molecules using a single-bead-per-residue coarse-grained model combining the HPS hydropathy model for disordered regions with a Go model for the globular domain of Histone H1 under NPT conditions at pressures from 2 to 12 bar. We find that chain dimensions, including the radius of gyration (Rg), end-to-end distance (Ree), and their ratio R, are insensitive to pressure, indicating that chain conformations remain largely unchanged over the pressure range studied. Histone H1 exhibits systematically larger values of R than ProTalpha because of its globular-core plus disordered-tail architecture. Translational diffusion coefficients decrease monotonically with pressure, from approximately 0.22 to 0.06 nm^2/ns, with substantial chain-to-chain heterogeneity comparable to the mean diffusion coefficient. Chain relaxation follows a stretched exponential with beta less than 1 that decreases with pressure. ProTalpha relaxation times of approximately 12 to 40 ns obey Rouse scaling, whereas Histone H1 deviates because of the internal constraint imposed by its globular domain. ProTalpha-Histone H1 contact lifetimes of approximately 0.43 to 0.56 ns are much shorter than the Rouse relaxation time, placing the system firmly in the fast-exchange regime where transient electrostatic contacts renormalize chain friction rather than acting as permanent cross-links, consistent with the moderate stretching exponent beta of approximately 0.55 to 0.70 observed across all pressures.
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
