The chaperone activity of native ␣-crystallins toward  LOW -and various ␥-crystallins at the onset of their denaturation, 60 and 66°C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human ␥S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of ␣-crystallins at temperatures above 60°C. Further increase is observed at 66°C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These ␣-crystallin temperature-induced modifications were found necessary to allow for the association with  LOW -and ␥-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)-and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified ␣-crystallins retained their ability to exchange subunits and that, at 37°C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66°C. Association with  LOW -(60°C) or various ␥-crystallins (66°C) resulted at 37°C in decreased subunit exchange in proportion to bound ligands. Therefore,  LOW -and ␥-crystallins were compared for their capacity to associate with ␣-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual ␥-crystallin family members. The strongest effect was observed for ␥S, followed by h␥Srec, ␥E, ␥A-F, ␥D, ␥B. Moreover, fluorescence properties of ␣-crystallins in the presence of bound  LOW -and ␥-crystallins indicated that the formation of  LOW /␣-or ␥/␣-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of ␣-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated ␣-crystallin structure.␣-Crystallins and small heat shock proteins have in common a conserved C-terminal domain of about 115 residues, the ␣-crystallin domain, the structure of which is organized around an eight-stranded -sandwich (1, 2). The small heat shock proteins usually associate into high molecular weight monodisperse or polydisperse oligomers, able to protect against stress through the binding of a variety of partially unfolded substrates. ␣-Crystallin was demonstrated by Horwitz in 1992 (3) to also exhibit chaperone properties in vitro. It is able to bind -and ␥-crystallins at the onset of their thermal denaturation, thus preventing further denaturation and aggregation, yet not refolding. Following this pioneering work, the chaperone-like activity of ␣-crystallin has been the subject of numerous studies and functional models have been suggested (4 -30). Essentially, hydrophobic patches on the surface, either present in the native state or revealed after structural modifications, would associate w...
A unique clone from a human pancreatic cDNA library was isolated and sequenced. Examination of the deduced polypeptide sequence of the clone showed a new form of cysteine-rich domain that included a region with the form of a Cys4 zinc-finger-like metal binding site followed by a complex Cys-His region. Searches of the Swiss-Protein data bank found a similar 48-residue domain in fifteen open reading frames deduced from A. thaliana, C. elegans, S. cerevisiae and S. pombe genomic sequences. The high degree of conservation of this domain (13 absolutely conserved and 17 highly conserved positions) suggests that it has an important function in the cell, possibly related to protein-protein or protein-DNA interactions. The gene recognized by the clone is is localized to human chromosome 16, and is conserved in vertebrates. The 2 Kb message is expressed in various human fetal and adult tissues. An antibody made to a peptide sequence of the deduced protein showed reactivity in immunoblots of monkey lung and retinal subcellular fractions and immunohistochemically in late fetal mouse tissues and a limited number of adult mouse tissues, including pancreatic islets, Leydig cells of the testis, and the plexiform layers of the retina.
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