Confinement effects on protein stability in a freezing water environment

Abstract

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.

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