Packing-induced conformational and functional changes in the subunits of alpha-crystallin

The heteroaggregate alpha -crystallin and homoaggregates of its subunits, a lphaA- and alphaB-crystallins, function like molecular chaperones and preve nt the aggregation of several proteins. Although modulation of the chaperon e-like activity of alpha -crystallin by both temperature and chaotropic age nts has been demonstrated in vitro, the mechanism(s) of its regulation in v ivo have not been elucidated. The subunits of alpha -crystallin exchange fr eely, resulting in its dynamic and variable quaternary structure. Mixed agg regates of the zeta -crystallins and other mammalian small heat shock prote ins (sHSPs) have also been observed in vivo. We have investigated the time- dependent structural and functional changes during the course of heteroaggr egate formation by the exchange of subunits between homoaggregates of alpha A- and alphaB-crystallins. Native isoelectric focusing was used to follow t he time course of subunit exchange. Circular dichroism revealed large terti ary structural alterations in the subunits upon subunit exchange and packin g into heteroaggregates, indicating specific homologous and heterologous in teractions between the subunits. Subunit exchange also resulted in quaterna ry structural changes as demonstrated by gel filtration chromatography. Int erestingly, we found time-dependent changes in chaperone-like activity agai nst the dithiothreitol-induced aggregation of insulin, which correlated wit h subunit exchange and the resulting tertiary and quaternary structural cha nges. Heteroaggregates of varying subunit composition, as observed during e ye lens epithelial cell differentiation, generated by subunit exchange disp layed differential chaperone-like activity. It was possible to alter chaper one-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit excha nge and the resulting structural and functional changes observed could cons titute a mechanism of regulation of chaperone-like activity of alpha -cryst allin (and possibly other mammalian sHSPs) in vivo.