Understanding how proteins behave at low temperatures remains a central challenge in biophysics, with direct implications for cold denaturation and cryopreservation. While cold denaturation of proteins in the supercooled liquid regime has been studied extensively, the behavior of a protein embedded in a growing ice lattice remains largely inaccessible to experiments. Here we use molecular dynamics simulations that explicitly capture ice Ih formation to characterize the conformational dynamics of yeast frataxin (Yfh1) as its aqueous environment crystallizes. Using four independent ice-seeded replicas and liquid-water controls at three temperatures, we first validate the liquid-solid transition through convergent changes in solvent density, potential energy, and local bond-order parameters (W4, W6). Principal component analysis (PCA), dihedral PCA (dPCA), and free-energy landscapes then reveal that crystallization of the solvent markedly reshapes the accessible conformational space, shifting it from a continuous, highly connected regime in liquid water toward a discretized landscape dominated by confined states. Complementary analyses of solvent-accessible surface area (SASA), radius of gyration, and hydrogen bonding indicate a solvent-driven reorganization of protein-water interactions: although first-shell water remains liquid-like, its surface density increases under freezing, while conformational sampling contracts. Together, these results indicate that protein behavior at low temperatures is governed not by temperature alone but by the structural organization of the surrounding water. By imposing geometrical constraints on the solvent, ice formation restricts conformational sampling while preserving -- and even densifying -- the interfacial hydration layer, highlighting the role of water structure as a determinant of protein stability under freezing conditions.
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
Uncrystallized water (UCW) refers to the fraction of water in hydrated materials that remains uncrystallized upon cooling. Rather than forming bulk ice, this water retains molecular mobility even below the nominal freezing point. In polymers and proteins, UCW includes strongly bound water associated with specific functional groups and more weakly confined water residing in nanoscale environments. As a result, it plays an important role in mechanical behavior, structural stability, and biological function. Because UCW is heterogeneous, no single technique can fully capture its properties, and multiple complementary methods are required. This review highlights how dielectric spectroscopy (DS), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), neutron scattering, particularly quasielastic neutron scattering (QENS), and terahertz and infrared spectroscopies each probe different aspects of UCW across distinct time and different sample sizes. We outline what each technique measures, the models commonly used to interpret the data, and how combining these approaches helps distinguish between “free,” “bound,” and “non-freezable” water. Studies that apply multiple methods to the same samples, with consistent control of hydration and temperature, are essential for developing a coherent and quantitative picture of UCW. This integrated approach is also key to translating UCW behavior into practical design principles for cryopreservation, pharmaceutical freezing, and biomaterials. Importantly, complementarity arises from differences in timescale and the distinct correlation functions each method probes. Computational spectroscopy–ranging from classical molecular dynamics to emerging machine learning approaches–provides an atomistic link between dielectric, NMR, neutron, and vibrational measurements and shared descriptors of hydration dynamics.
Kitsakorn Locharoenrat· Frontiers in Analytical Scie...· 0 citations
Understanding how biological membranes retain integrity under extreme dehydration is vital for explaining anhydrobiotic survival and defining the physicochemical limits of life. Here, we use molecular dynamics simulations to investigate how progressive dehydration (30-2 water molecules per lipid) affects a liquid-disordered (Ld) DPPC/DOPC/cholesterol membrane. Dehydration reduces the area per lipid and membrane thickness, increases acyl-chain ordering, and majorly suppresses membrane lateral diffusion, with trends comparable to those observed in the liquid-ordered (Lo) phases, indicating a largely phase-independent response. The hydrogen-bond analysis shows that the phosphate-associated oxygen atoms retain the strongest and most persistent interactions, maintaining nearly constant free energy of bond disruption and thereby stabilizing the lipid membrane. Overall, both the Ld and Lo lipid membranes rely on localized head group hydration to withstand water loss, offering molecular insight into lipid membrane resilience in anhydrobiosis, extremophile biology, and water-limited environments.
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
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
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
