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a-Crystallin, an abundant mammalian lens protein made up of two subunits (aA- and aB-crystallin), is involved in the maintenance of the optimal refractive index in the lens. The protein is implicated in the pathophysiology of a large number of retinal diseases including cataract, age-related macular degeneration, diabetic retinopathy, and uveitis. a-Crystallin belongs to the small heat shock protein (sHSP) family, forms large oligomeric structures, and functions as a molecular chaperone appearing very early during embryonic development. To gain mechanistic insight into the structural and functional role of a-crystallin and its alterations in various retinal diseases, it is important to study the interaction chemistry with its known partners. The hydrophobic sites in a-crystallin have been studied extensively using environmentally sensitive fluorescent probes such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) that interacts with both subunits of a-cystallin in 1:1 stoichiometry at 37 [degrees]C and diminishes the chaperone-like activity of the protein. Furthermore, it has been shown that ATP plays a crucial role in the association of [alpha]-crystallin with substrate proteins. We use surface plasmon resonance (SPR) to monitor the interactions of immobilized oligomeric recombinant [alpha]A subunit of human a-crystallin protein with bis-ANS and ATP. We assess the thermodynamic parameters and kinetics of such interactions at various temperatures. Our results indicate that bis-ANS binds to [alpha]A-crystallin with higher affinity when compared with ATP, although both [alpha]A-crystallin and [alpha]B-crystallin display fast interaction kinetics. Key words: [alpha]A-crystallin, ATP, bis-ANS, surface plasmon resonance (SPR). La cristalline [alpha], une proteine presente en abondance dans le cristallin des mammiferes, est constituee de deux sous-unites ([alpha]A et [alpha]B) et joue un role dans le maintien d'un indice de refraction optimal a l'interieur du cristallin. La proteine est impliquee dans la pathophysiologie d'un grand nombre de maladies de la retine, notamment la cataracte, la degenerescence maculaire liee a l'age, la retinopathie diabetique et l'uveite. La cristalline [alpha] appartient a la famille des petites proteines de choc thermique (sHSP). Elle forme de grandes structures oligomeriques et fonctionne comme un chaperon moleculaire, apparaissant tres tot dans le developpement embryonnaire. Afin de mieux comprendre les aspects mecanistiques du role fonctionnel et structural de la cristalline [alpha] et de ses anomalies dans diverses maladies retiniennes, il importe d'etudier la chimie de ses interactions avec les partenaires qu'on lui connait. Nous avons etudie en detail les sites hydrophobes de la cristalline [alpha] a l'aide de sondes fluorescentes sensibles a l'environnement telles que le sel dipotassique de l'acide 4,4'-dianilino-1,1'-binaphtyl-5,5'-disulfonique (bis ANS). Celui-ci interagit avec les deux sous-unites de la cristalline a dans un rapport stoechiometrique de 1:1 a 37 [degrees]C et reduit l'activite de type chaperon de la proteine. De plus, nous avons montre que l'ATP joue un role crucial dans l'association de la cristalline-a avec ses substrats proteiques. Nous avons utilise la resonance plasmonique de surface (SPR) pour observer les interactions avec le bis ANS et l'ATP d'un oligomere recombinant immobilise de la sous-unite [alpha]A de la cristalline [alpha] humaine. Nous avons evalue les parametres thermodynamiques et la cinetique de ces interactions a differentes temperatures. Nos resultats indiquent que le bis ANS se lie a la cristalline [alpha]A avec une plus grande affinite comparativement a l'ATP, bien que les deux presentent une cinetique d'interaction rapide. [Traduit par la Redaction] Mots-cles: cristalline [alpha]A, ATP, bis ANS, resonance plasmonique de surface (SPR)., Introduction [alpha]-Crystallin is a predominant protein present in the eye lens and comprises up to 40% of the total lens proteins. (1,2) It is made up of two subunits, [alpha]A [...]

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein. [ABSTRACT FROM AUTHOR]

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.)