INTERACTIONS OF CHAPERONE ALPHA-CRYSTALLIN WITH THE MOLTEN GLOBULE STATE OF XYLOSE REDUCTASE - IMPLICATIONS FOR RECONSTITUTION OF THE ACTIVE ENZYME

alpha-Crystallin is a multimeric protein that has been shown to functi on as a molecular chaperone. Present investigations were undertaken to understand its mechanism of chaperoning. For this functional in vitro analysis of alpha-crystallin we used xylose reductase (XR) from Neuro spora crassa as the model system. Denaturation studies using the struc ture-perturbing agent guanidinium chloride indicated that IIR folds th rough a partially folded state that resembles the molten globule. Fluo rescence and delay experiments revealed that alpha-crystallin interact s with the molten globule state of XR (XR-m) and prevents its aggregat ion. Cold lability of alpha-crystallin XR-m interaction was revealed b y temperature shift experiments implicating the involvement of hydroph obic interactions in the formation of the complex. Reconstitution of a ctive XR was observed on cooling the alpha-crystallin XR-m complex to 4 degrees C or on addition of ATP at 37 degrees C. ATP hydrolysis is n ot a prerequisite for XR release since the nonhydrolyzable analogue 5' -adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active XR. Experimental evidence has been provided for temperature- an d ATP-mediated structural changes in the alpha-crystallin XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone. The relevance of our finding to the role of alpha-c rystallin in vivo is discussed.