Structure, Dynamics and Surface Hydrophobicity of the Cataract-Associated Mutant, Pro23Thr of Human Gamma D-crystallin: Molecular Basis of Cataract Formation

Abstract

The cataract-associated Pro23Thr (P23T) mutation in human gammaD-crystallin (HGD) is geographically widespread - thus there is considerable interest in determining the molecular basis of opacity. In an earlier study (1), we found that the mutant showed markedly lowered, retrograde solubility compared to wild-type HGD, leading to the conclusion that the aggregation of P23T was mediated by hydrophobic protein-protein interactions. Subsequently, using NMR (2) and a binding assay with the fluorescent dye Bis-ANS (3), we showed that, in fact, hydrophobic patches were generated on the surface of the mutant protein. Those studies (3) also suggested that the binding site for Bis-ANS on P23T may coincide with the self-association site of the mutant and result in its lowered, retrograde solubility. Here we present new NMR-evidence to identify the Bis-ANS binding sites, and using independent NMR dynamics studies, also show that there is a measurable reduction in the flexibility of the peptide backbone near the hydrophobic patches. These two factors, namely the creation of hydrophobic patches and the lowered peptide backbone flexibility, taken together make a compelling argument that the hydrophobic patches may indeed facilitate the nucleation of aggregates of P23T which are held together by net hydrophobic interactions. Such aggregation would explain the retrograde solubility as well as the lens opacity in vivo. 1. Pande et al (2005) Biochemistry, 44, 2491-2500. 2. Pande et al (2009) Biochem. Biophys. Res. Commun., 382, 196-199. 3. Pande et al (2010) Biochemistry,49, 6122-6129. Supported by NIH grants GM085006 to AS and EY010535 to JP.