AN INVESTIGATION INTO THE STABILITY OF ALPHA-CRYSTALLIN BY NMR-SPECTROSCOPY, EVIDENCE FOR A 2-DOMAIN STRUCTURE

The stability of bovine lens a-crystallin with respect to temperature, pH and urea has been investigated by H-1 and P-31-NMR spectroscopy. The H-1 and P-31-NMR spectra of alpha-crystallin show little change with temperature up to 75-degrees-C, indicating that alpha-crystallin has great thermal stability and does not undergo any major change in structure with temperature. H-1 spectral studies of alpha-crystallin and its isolated alpha(A) and alpha(B) subunits reveal a marked difference in the stability of these species. It is found that, at pH 2.5, alpha(A)-crystallin adopts a native conformation whereas alpha(B)-crystallin is denatured. On the other hand, the two subunits when part of the total alpha-crystallin aggregate adopt a native conformation at pH 2.5, but in the presence of 0.1 M glycine the alpha(B) subunits become denatured. Thus, alpha(A)-crystallin and total a-crystallin are more stable species than alpha(B)-crystallin and, in total alpha-crystallin, there is an interaction between the compact domains of the alpha(A) and alpha(B) subunits that leads to enhanced stability. Finally, changes in the H-1 and P-31-NMR spectra of alpha(A)-Crystallin and alpha(B)-cryStallin in the presence of varying concentrations of urea are consistent with a two-domain model for alpha-crystallin subunits with the C-terminal domain being less stable and unfolding first in the presence of urea.