Anomalous Effect of Denaturant on Protein Unfolding Dynamics Revealed by Single Molecule Manipulation Experiments

High temperatures and chemical denaturants in bulk experiments, as well as mechanical forces in single-molecule studies, typically promote protein unfolding. In this study, we report an unexpected decrease in the unfolding rate of cold shock protein (Csp) at low concentrations of guanidine hydrochloride (GuHCl) in single-molecule magnetic tweezers experiments. This behavior contrasts with that of control protein GB1, which unfolds faster under the same denaturing conditions. Steered molecular dynamics (SMD) simulations indicate that stretching force applied to the N- and C-termini of Csp triggers an allosteric conformational change, converting loop regions into β-strands and reducing the solvent-accessible surface area (SASA). The combination of experimental and simulation data suggests that the unfolding transition state of Csp has a smaller SASA than that of the native state, providing a structural explanation for the observed kinetic anomaly. These results demonstrate that allosteric conformational or dynamical changes, triggered by mechanical or chemical perturbations, can render proteins resistant to denaturation by lowering their unfolding rates, thereby conferring resistance to environmental stress.