Our in silico analysis confirms that though Arg39 side chain is not involved in holding the protein atoms together, it shows few interactions with Gly38 and Cys40, which are lost when Arg39 is substituted with Ala. It is shown earlier, that Gly38 in HPR has an important function, possibly, it may enhance the role of Arg39 as one of the many noncatalytic phosphate binding residues involved in the interaction of HPR protein with the double-helical substrate. In HPR, Gly38 is present on the surface in a2-b1 loop which forms a part of the V-shaped cleft in which the active site is located. The absence of a larger side chain gives the polypeptide backbone at glycine residue much greater conformation flexibility than at other residues. It appears that this conformational flexibility imparted to HPR around the active site, determines its ability to bind and melt duplex RNA, which is the first step in the hydrolysis of duplex RNA, and the presence of an aspartic acid instead of glycine at position 38, as in RNase A, diminishes the flexibility of the polypeptide backbone. In HPR, though Gly38 is in close vicinity of Tyr92, there is no contact between the two residues. However, mutation of Gly38 to Asp38 introduces one hydrogen bond and one van der Waal interaction with Tyr92. The almost complete loss of dsRNA cleavage activity of R39A/ G38D and Q28A/G38D/R39A can be explained by the absence of Arg39 which makes these XL880 proteins poor in melting the helix, and the presence of aspartic acid at position 38 which results in a compromised flexibility of the protein. The far-UV CD spectra and heat induced denaturation curves showed that R39A, Q28A and G38D had decreased stability. The ABT-263 effect of mutation on the stability of protein was more pronounced when R39A mutation was combined with Q28A, Q28L or G38D alone or Q28A and G38D together. The Q28L variant which was very similar to the native protein in terms of stability, showed similar DNA melting activity and in turn similar catalytic activity towards dsRNA as that of the native enzyme. Thus, stability also appears to be an important contributor in the DNA melting activity of HPR. In BS-RNase, Leu28 is known to increase the propensity of domain swapping, and thus facilitating the formation of noncovalent dimer. However, analysis by native PAGE showed that Q28L mutation in HPR did not promote the process of dimerization. Our study shows that a leucine at position 28 can substitute glutamine as Q28L variant showed similar stability, helix unwinding activity and dsRNA cleavage activity as that ofHPR. The observation is further validated by in silico analysis that shows both Gln28 and Leu28 to have interaction with Thr24 which is lost in Q28A variant.