Abstract: In this study, circular dichroism (CD) and molecular dynamics (MD) simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B (SEB) at temperatures of 298–371 and 298–500 K, and the relationship between the experimental and simulation results were explored. Our computational findings on the secondary structure of SEB showed that at room temperature, the CD spectroscopic results were highly consistent with the MD results. Moreover, under heating conditions, the changing trends of helix, sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD. In order to gain a deeper understanding of the thermal stability mechanism of SEB, the MD trajectories were analyzed in terms of root mean square deviation (RMSD), secondary structure assignment (SSA), radius of gyration (Rg), free energy surfaces (FES), solvent-accessible surface area (SASA), hydrogen bonds and salt bridges. The results showed that at low heating temperature, domain I without loops (omitting the mobile loop region) mainly relied on hydrophobic interaction to maintain its thermal stability, whereas the thermal stability of domain II was mainly controlled by salt bridges and hydrogen bonds. Under high heating temperature conditions, the hydrophobic interactions in domain I without loops were destroyed and the secondary structure was almost completely lost, while domain II could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure. These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level, and provide a direction for establishing safer and more effective food sterilization processes.

Key words: staphylococcal enterotoxin B; circular dichroism; molecular dynamics simulations; temperature-induced unfolding