An investigation into the stability of alpha-crystallin by NMR spectroscopy; evidence for a two-domain structure.

The stability of bovine lens alpha-crystallin with respect to temperature, pH and urea has been investigated by 1H and 31P-NMR spectroscopy. The 1H and 31P-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. 1H 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 alpha-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 1H and 31P-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.