Microscopic mechanisms of helical protein unfolding under thermal and confinement effects

Protein folding and unfolding remain central problems in biological sciences, yet a unified understanding of native folding mechanisms is still lacking due to structural diversity and complex folding pathways. In this work, molecular dynamics simulations were employed to investigate the high-temperature unfolding behavior of a helical protein. The results reveal that unfolding is initiated preferentially in regions characterized by weaker residue-residue interactions, thereby predefining the unfolding pathway at the molecular level. Comparative simulations further demonstrate that confinement within a silicon nitride nanopore significantly accelerates protein unfolding through electrostatic interactions with the pore surface, while simultaneously suppressing refolding. Beyond confinement effects, the chemical environment of the solution plays a decisive regulatory role. Monovalent cations with small hydration shells facilitate unfolding, whereas high electrolyte concentrations or multivalent ions enhance conformational stability via electrostatic screening. With respect to chemical denaturants, urea promotes unfolding in a monotonic manner, while guanidine hydrochloride exhibits a dual effect, accelerating unfolding at moderate concentrations but stabilizing protein conformations at high concentrations due to dominant screening effects. Overall, this study provides a microscopic picture of how temperature, spatial confinement, and solution chemistry cooperatively regulate protein conformational dynamics, offering new insights into the physical basis of protein folding and stability.