Sulfation-dependent mechanism of dextran sulfate enhancing ovotransferrin thermal stability: Structural and molecular dynamics insights

Sulfated polysaccharides have emerged as chaperone-like agents for enhancing the thermal stability of ovotransferrin (OVT), the most heat-sensitive protein in egg white. However, the molecular mechanism by which dextran sulfate (DXS) enhances the thermal stability of OVT remains unclear. Here, we synthesized DXS with three degrees of sulfation (0.3, 1.0, and 2.1) to investigate how the sulfation level influences its interaction with OVT and thereby affects the structure-stability relationship. The specific interaction between DXS and OVT was investigated through molecular dynamics simulations. Turbidity, particle size distribution, and morphology results revealed that DXS acts as a molecular chaperone, effectively inhibiting thermal-induced amorphous aggregation of OVT and promoting the formation of soluble oligomeric structures in a sulfation-dependent manner. Thermal shift assay further showed that DXS significantly delayed the melting temperature of OVT, with a 22.8 % improvement at DXS 1.0 and beyond the instrument's detection limits at DXS 2.1. Besides, spectroscopic characterization revealed that DXS (particularly DXS 2.1) well preserved OVT's secondary and tertiary structures during thermal treatment, with a higher sulfate substitution degree leading to greater stabilization. Furthermore, molecular dynamics simulation results identified a preferential binding site at the pocket formed by ASN-672, TRP-464, and SER-674 residues, where DXS binding promoted the transformation of the flexible loop region into stable helical structures. Electrostatic interactions and hydrogen bonds dominated the binding between DXS and OVT. These findings provide new insights into the molecular basis of polysaccharide-mediated protein stabilization and support the application of sulfated polysaccharides as effective thermal protectants in protein-based food systems.