HEAT-INDUCED CONFORMATIONAL CHANGE AND INCREASED CHAPERONE ACTIVITY OF LENS ALPHA-CRYSTALLIN

Purpose. Alpha-crystallin is the major structural protein of the eye l ens known to have chaperone-like activity. Our objective is to elucida te the nature of the thermal transition that alpha-crystallin undergoe s at 60 degrees C and the effect of this transition on the chaperone a ctivity. Methods. FPLC size exclusion chromatography, far- and near-ul traviolet circular dichroism, and tryptophan (Trp) and 1-anilino-8-nap hthalenesulfonate (ANS) fluorescence were used to study conformational change. Turbidity of dithiothreitol (DTT)-reduced insulin was used to study chaperone activity. Results. The thermal transition was identif ied as a conformational change in mainly tertiary (partial unfolding) and quaternary high-molecular-weight (HMW) aggregation structures, alo ng with a loss of 10 percentage points of secondary structure (beta-sh eet). Initial partial perturbation in tertiary structure increased cha perone activity, but the increase was less in the HMW aggregate. Simil ar results were observed in in vivo-formed HMW alpha-crystallin. Concl usions. The conformational change and HMW aggregation of alpha-crystal lin observed at 60 degrees C, as well as in vivo-formed HMW aggregates , increased chaperone activity.