Functional expression, monodispersity and conformational changes in the SBMV virus viral VPg on binding TFE

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Southern bean mosaic virus (SBMV) is a plant pathogen.
• The viral genome-linked protein (VPg) is crucial for viral replication.
• Understanding VPg structure-function relationships is essential for developing antiviral strategies.

Purpose of the Study:

• To clone and express the SBMV VPg in Escherichia coli.
• To investigate the conformational changes and folding properties of SBMV VPg.
• To determine the effect of trifluoroethanol (TFE) on VPg structure and nucleotide binding.

Main Methods:

• Cloning and expression of SBMV VPg in E. coli.
• Circular dichroism (CD) spectroscopy to assess protein secondary structure.
• Dynamic light scattering (DLS) to evaluate protein monodispersity.
• Nuclear magnetic resonance (NMR) with saturation transfer difference (STD) to study protein-ligand interactions.
• 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence to probe protein unfolding.

Main Results:

• SBMV VPg was successfully cloned and expressed.
• Trifluoroethanol (TFE) induced a concentration-dependent increase in helical content of VPg.
• TFE influenced VPg folding but did not affect its ability to bind nucleotides.
• Local solvent hydrophobicity was identified as a key factor driving conformational changes in VPg.

Conclusions:

• The study provides insights into the structural dynamics of SBMV VPg.
• TFE-induced conformational changes highlight the protein's flexibility.
• VPg folding is distinct from its nucleotide-binding mechanism, suggesting independent regulatory processes.