The present study clearly demonstrated that the pH sensitivity of the HA protein is determined by the charged residues especially those involved in salt bridge formation in and around the hinge region (interface region; therefore the residue easily gets exposed to solvent). The destabilising mutants showed a lower pH dependency for the conformational change in agreement with those of amantadine resistant mutants. On contrary, stability of the protein was increased with introduction of a possible salt bridge in the neighbourhood of hinge region (I89R or I89E-Y308R). Not only in the hinge region, stability to the protein could also be conferred by introduction of an ion-pair (T212E-N216R) in the distal region of the HA trimer.

The present research work provided an explanation for the stability of A/JPN/305/57 strain at pH 5.0. Very likely, the stability of the A/JPN/305/57 strain is enhanced by at least 2 –3 complexes of salt bridges around the hinge region. Indeed, this present study proved that introduction of attractive forces (possibly salt bridges) as for example in the distal region stabilised the HA ectodomain. In addition, the salt bridge network of A/JPN/305/57 strain with additional two ring like salt networks connecting either the HA1 monomers (R206-E210) or HA2 (R83-E85) provides the stability of the HA ectodomain at pH 5.0.