Urea induced characterization & structural dynamics of the Nucleocapsid Protein using biophysical and in silico approaches

• Nucleocapsid protein (N) of SARS CoV-2 is loses its tertiary and secondary structure under urea induced denaturation • Differential scanning calorimetry measured the global changes during the denaturation process, revealing slight increase in Tm with increasing concentration of urea • The increase in Tm along with decrease in Δ Cp value inferred the formation of intermediate like state The Nucleocapsid (N) protein of SARS CoV-2 is multifunctional protein whose role is not limited to replication but also in attenuation of the host immune response. Since N protein is abundantly present and post infection, it becomes essential to understand the structural dynamics of the N protein in absence of high-resolution crystal structure. In this study, the structural and conformational dynamics of the recombinant N protein was studied through circular dichroism (CD), fluorescence spectroscopy and differential scanning calorimetry (DSC) under urea induced denaturing conditions. The biophysical studies revealed that N protein loses its tertiary structure cooperatively starting from 2.00 M of urea induced denaturation with a redshift in λ max . The CD spectroscopy revealed that there is loss in random coil content hence secondary structure content and shifting of θ max towards higher wavelength. The DSC experiments revealed increase in T m along with decrease in Δ C p value inferring formation of intermediate like state. All the in vitro studies were further complimented using 300 ns MD simulation using Gromacs 2022.2. These data provide information of structural characteristics and dynamics of the N protein in absence of high-resolution crystal structure and redefine the current understanding of structure of the N protein. Furthermore, pave way for the development of antiviral drugs targeting the N protein to complement the existing vaccine in case of emergence of neutralization escape mutant.