Unfolding and refolding of bovine alpha-crystallin in urea and its chaperone activity.

We undertook an unfolding and refolding study of alpha(L)-crystallin in presence of urea to explore the breakdown and formation of various levels of structure and to find out whether the breakdown of various levels of structure occurs simultaneously or in a hierarchal manner. We used various techniques such as circular dichroism, fluorescence spectroscopy, light scattering, polarization to determine the changes in secondary, tertiary, and quaternary structure. Unfolding and refolding occurred through a number of intermediates. The results showed that all levels of structure in alpha(L)-crystallin collapsed or reformed simultaneously. The intermediates that occurred in the 2-4 M urea concentration range during unfolding and refolding differed from each other in terms of the polarity of the tryptophan environment. The ANS binding experiments revealed that refolded alpha(L)-crystallin had higher number of hydrophobic pockets compared to native one. On the other hand, polarity of these pockets remained same as that of the native protein. Both light scattering and polarization measurements showed smaller oligomeric size of refolded alpha(L)-crystallin. Thus, although the secondary structural changes were almost reversible, the tertiary and quaternary structural changes were not. The refolded alpha(L)-crystallin had more exposed hydrophobic sites with increased binding affinity. The refolded form also showed higher chaperone activity than native one. Since the refolded form was smaller in oligomeric size, some buried hydrophobic sites were available. The higher chaperone activity of lower sized oligomer of alpha(L)-crystallin again revealed that chaperone activity was dependent on hydrophobicity and not on oligomeric size.