Alpha-crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions.

Alpha-crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of alpha-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of alpha-crystallin is less investigated. Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. The alpha-crystallin.Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%-35% recovery of activity. The nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca(2+) was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the alpha-crystallin.Xyl II complex brought about conformational changes in alpha-crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of alpha-crystallin chaperone functions by metal ions.