Inhibitory effect and mechanism of Nb2C nanochaperone on bovine serum albumin unfolding

Bovine serum albumin (BSA) was used as a model protein to investigate the mechanism of guanidine hydrochloride (GdnHCl) induced unfolding and the protective effect of Nb 2 C nanosheets. Spectroscopic and structural analyses showed that GdnHCl induced concentration-dependent unfolding of BSA through a native to intermediate to unfolded transition, accompanied by fluorescence quenching, emission red shift, secondary structure rearrangement, and decreased thermal stability. Mechanistic studies indicated that this process was closely associated with disruption of the water hydrogen-bond network rather than simple direct denaturant protein binding. Raman analysis showed that GdnHCl decreased strong hydrogen bonds while increasing weak hydrogen bonds, and the resulting water-structure reorganization was highly correlated with the unfolding degree of BSA, with a Pearson correlation coefficient of 0.978. Nb 2 C nanosheets effectively suppressed GdnHCl-induced unfolding, increasing the unfolding midpoint concentration from 3.66 to 4.71 mol/L in the presence of 50 μg/mL Nb 2 C. Nb 2 C also alleviated the loss of ordered secondary structure and improved the thermal stability of unfolded BSA, increasing the melting temperature from 43.9 to 61.0 °C. Fluorescence quenching and isothermal titration calorimetry demonstrated that Nb 2 C interacted spontaneously with BSA mainly through hydrogen-bond-dominated interfacial interactions. This work provides a solvent centered perspective on chemical denaturation and identifies Nb 2 C as a promising nanochaperone for protein conformational protection.