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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 Zn 2 . 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 Zn 2+ 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 Zn 2+ . 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., (© 2025 Wiley Periodicals LLC.)

α-Crystallin, the major protein of mammalian eye lens, is a member of the small heat shock protein family and is a molecular chaperone. We previously reported that its molecular chaperone function as well as stability increased in presence of Zn+². Despite the effect of Zn+² on the structure and function of α-crystallin, evidence for direct interaction between them remained elusive. We now present the MALDI mass spectrometric data that shows direct evidence of Zn+² binding to recombinant αA- and αB-crystallin. The binding stoichiometry was over three Zn+² per subunit of α-crystallin at zinc/protein molar ratio of 20. Observation of multiple Zn+² binding is consistent with the large increase in thermodynamic stability. Sequence-based analysis of αA- and αB-crystallin predicted both proteins to be nonzinc binding proteins. Our dynamic light scattering data shows that Zn+² stabilizes the oligomeric structure of α-crystallin by bridging neighboring subunits in multiple centers. Despite the low affinity binding, the intersubunit bridging by multiple Zn+² makes the oligomer so stable that oligomer breakdown does not occur even at 6M urea. The subunit bridging has been supported by our FRET data that showed absence of subunit exchange in presence of zinc. MALDI data also showed that the interaction of α-crystallin with Zn+² is quite different from other bivalent metal ions. Bound Zn+² could be easily removed by dialysis of the complex. The relevance of such weak interaction on the stability of the oligomeric structure of α-crystallin and its function in the eye lens has been discussed., (© 2010 Wiley Periodicals, Inc.)