2.3. Alanine scanning (mutagenesis)

Alanine scanning is a site-directed mutagenesis method used to identify whether a particular residue contributes to the stability or function of a specific protein. Alanine is used owing to its chemically inert, non-bulky, methyl functional group that nevertheless imitates the secondary structure preferences that certain other amino acids exhibit. This strategy also can be used to discern if the side chain of a particular residue plays an important role in bioactivity or not ^{[25], [26]}. Mutagenesis ^[27] was performed using MOE (Molecular Operating Environment) ^[21]that computes the particular amino acid residue impact upon replacing by alanine. The complete procedure of alanine scanning mutagenesis has been given in the previous study ^[28]. Two parameters dAffinity and dStability were considered while calculating the impact of alanine substitutions. High positive dAffinity and dStability means highly significant substitution. Furthermore, we also used mCSM-NA an online server, to determine the impact of alanine substitution on the structure and affinity of nucleocapsid phosphoprotein-RNA complex. mCSM–NA ^[29] uses the graph-based signature concept, which combines a pharmacophore modeling and information of nucleic acid properties to predict and characterize the effect of a single point missense mutation on protein-nucleic acid binding. To further validate our results, we also used DrugScorePPI ^[30] an online webserver based on the knowledge-based scoring function to predict changes in the binding free energy upon alanine mutations. Combining these three methods predicted the most significant substitutions for RNA interaction with the binding protein.