Thermal Stabilization of Ubiquitin in Hydrophobic Amino Acid Solutions via Coupled Modulation of Solvation Structure and Dynamics.

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.