α‐Crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions

α‐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 α‐crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of α‐crystallin is less investigated. Here we studied the refolding of Gdn HCl‐denatured Xyl II in the presence and absence of α‐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 α‐crystallin formed a complex with a putative molten globule‐like intermediate in the refolding pathway of Xyl II. The α‐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 α‐crystallin•Xyl II complex brought about conformational changes in α‐crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of α‐crystallin chaperone functions by metal ions.