Subunit exchange demonstrates a differential chaperone activity of calf α-crystallin toward βlow- and individual γ-crystallins

Abstract

The chaperone activity of native α-crystallins toward βLOW- and various γ-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 γS. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of α-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 α-crystallin temperature-induced modifications were found necessary to allow for the association with βLOW- and γ-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified α-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 βLOW- (60 °C) or various γ- crystallins (66 °C) resulted at 37 °C in decreased subunit exchange in proportion to bound ligands. Therefore, βLOW- and γ-crystallins were compared for their capacity to associate with α-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual γ-crystallin family members. The strongest effect was observed for γS, followed by hγSrec, γE, γA-F, γD, γB. Moreover, fluorescence properties of α-crystallins in the presence of bound βLOW-and γ-crystallins indicated that the formation βLOW/α- or γ/α-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of α-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated α-crystallin structure.