Histidine Tags in Human Recombinant Alpha B‐Crystallin (<scp>HSPB5</scp>) Proteins Are Detrimental for Zinc Binding Studies

The stability of α-crystallin, the major protein of the mammalian eye lens and a molecular chaperone, is one of the most crucial factors for its survival and function. The chaperone-like activity and stability of α-crystallin dramatically increased in the presence of Zn². Each subunit of α-crystallin could bind multiple zinc atoms through inter-subunit bridging and cause enhanced stability. Three histidines H104, H111, and H119 of recombinant human αB-crystallin (HSPB5) are found to be the Zn²⁺ binding residues. In this article, we did site-directed mutagenesis of six histidine residues and made five-point mutants and a double mutant of αB-crystallin. We studied the effect of zinc on the chaperone function, surface hydrophobicity, and stability of the histidine mutants. We removed the histidine tag from H18A and H101V mutants and studied the stability and chaperone function in the presence and absence of zinc. H83 and H111 mutations showed similar enhancement in chaperone function like WT in the presence of Zn²⁺. Point mutants having his tags showed similar stability enhancement, but point mutant H18A without his tag showed less enhancement in stability in the presence of zinc. This indicates the significance of the presence of his tags in the study of zinc binding interaction with recombinant human αB-crystallin.