Evidence regarding the hypothesis that the histidine-histidine contact pairs may affect protein stability

It has been lately proposed that the interaction between like-charged residues stabilizes the native state of proteins. To explore this, we created a histidine-histidine pair in the Ca-III binding site of the Bacillus amyloliquefaciens α-amylase (BAA) and then examined the impact of this pairing on the BAA. For this purpose, we used site-directed mutagenesis (SDM) to substitute Pro407 with His, Ala, Gln, Arg, and Glu in the BAA. Subsequently, thermostability, kinetic parameters and structural properties of these variants were measured. Moreover, His-His pairing effect on the BAA thermostability was examined by simultaneous mutation of two residues (P407H/H406A and P407H/H406N). The data exhibited a significant improve in thermostability and structural features of enzyme by His replacement instead of Pro407. Other substitutions in this site did not have a significant effect on the enzyme properties, except for P407R, which yielded a partial improvement. The results also showed that the thermostabilities of double mutants significantly decreased compared with that of the P407H mutant. Moreover, the thermostability of P407H remarkably increased compared with that of other variants even in the absence of Ca2+. Our data clearly demonstrated that His406-His407 pairing was the major cause for improved thermal stability.