CATARACT DEVELOPMENT ON MOLECULAR LEVEL: FT-NMR STUDY

Senile cataract etiology may be regarded as caused by pathological cytosol-protein interactions of sufficient magnitude to manifest light scattering. Early biobhemical changes correlated with morphological changes in cataract appears to involve protein-membrane interactions. These interactions are stabilized by various post-translational oxidative modifications, such that high molecularweight disulfide complexes unique to cataract have been isolated and characterized. However, the molecular cause or denaturation process that elicits such complex disulfide formation is not known. Gamma-crystallin has been recently identified as a cytosol protein within these complexes. The tertiary structure of gamma-crystallin has been determined by x-ray analysis, and indicates a homologous two domain structure. The availability of the atomic coordinates has allowed various theoretical calculations to be performed including the electrostatic free energy stabilization for the separate N and C domains. These calculations have indicated a potential latent instability in the sulfhydryl rich N-domain. Such instabilities may suggest a defective protein structure. However, these results need experimental verification before its implication can be correlated with the cataractous process. Verification can be ascertained with high resolution Forier-transform nuclear magnetic resonance (FT-NMR) spectroscopy. This spectroscopic biophysical method is capable of detecting and monitoring specific single site nuclei within the protein due to the nature of the local magnetic micro-environment. After assignment of chemical shift positions associated with given nuclei of certain amino acids, subtle dynamic conformational perturbations caused by various defined biochemical agents in solution may be followed by relating the changes in chemical shift or relaxation of the respective nuclei behavior to various mathematical relationships. Such an approach with the various lens crystallins should aid in the discovery of the molecular cause of cataract and elicit the pathway of lenticular protein denaturation. Knowing the molecular basis of the lens protein denaturation can provide insights into the design of antagonists to reverse or retard such a pathological process. Furthermore, the intact lens metabolic changes induced by these cataract antagonists may be effectively followed and evaluated with FT-NMR, a non-invasive technique.