Thermal stability and hydration dynamics of Chymotrypsin Inhibitor 2 in aqueous amino acid solutions from replica exchange molecular dynamics.

Additives like arginine, while known to enhance protein stability, were observed to improve the solubility of proteins in an equimolar mixture of arginine and glutamic acid. This study examines the conformational stability of a model protein, chymotrypsin inhibitor 2, in arginine, glutamic acid, and their mixture over a temperature range of 300-440 K using Replica Exchange Molecular Dynamics. The study demonstrates that arginine stabilized CI2 through hydrogen bonds and electrostatic interactions with polar and charged residues, thereby restricting conformational fluctuations and reducing solvent penetration. Glutamic acid enhanced its stability by promoting preferential hydration and maintaining a hydration shell that minimized hydrophobic exposure during thermal fluctuations. The arginine-glutamic acid mixture exhibited intermediate stabilizing behavior, where hydrogen bonding interactions between the oppositely charged amino acids reduced arginine self-association, resulting in a balanced hydrophilic-hydrophobic environment around the protein. Free energy landscape analysis showed no significant unfolding transitions across temperatures, supporting a kinetically stable ensemble. Radial distribution and cluster analyses indicated that arginine formed compact clusters near the protein surface, whereas glutamic acid remained dispersed, allowing higher water mobility. Water diffusion and dipole-dipole correlation studies revealed faster hydration dynamics in glutamic acid and slower, more confined motion in arginine, with intermediate behavior in the mixed solution.