Experimental and In Silico Analysis of the Structural Dynamics of Dengue NS2B-NS3 Protease

The Dengue virus, a significant global health issue, primarily impacts tropical and subtropical regions, leading to dengue fever. The NS2B-NS3 protease complex within the virus plays a crucial role in viral replication, positioning it as a promising target for antiviral drug development. A recent study explored the structural dynamics of this protease across different pH levels, combining both experimental and computational methodologies.

Recombinant NS2B-NS3 protease was produced in E. coli and purified using affinity chromatography, followed by purity assessment through SDS-PAGE. Circular dichroism (CD) spectroscopy indicated that the protein undergoes pH-dependent alterations in secondary structure, exhibiting stability at neutral to mildly basic pH conditions, while demonstrating destabilization under both acidic and highly basic pH levels.

Dynamic light scattering (DLS) further confirmed protein aggregation and structural heterogeneity at extreme pH levels.

In conjunction, in silico techniques, such as homology modelling and molecular dynamics (MD) simulations, were employed to offer detailed perspectives on the conformational shifts within the protease. The modelling structure was verified and enhanced using computational resources, unveiling structural stability at physiological pH, contrasted with pronounced disruptions at extreme pH levels.

Secondary structure analyses, conducted via DSSP (Dictionary of Secondary Structure of Proteins) and principal component analyses, highlighted substantial transitions, particularly at acidic pH where helices and beta-sheets reorganized into random coils.

Docking and MD simulations were conducted with small molecules for therapeutic evaluation, focusing on interactions between the Dengue NS2B-NS3 protease and potential inhibitors. The findings underscore the pH-dependent conformational flexibility of the protease, enhancing our comprehension of its functional mechanisms. This research lays the groundwork for the rational design of pH-specific inhibitors, presenting a robust foundation for the development of targeted antiviral therapies against dengue fever.