UC Irvine

The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin. We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecule to the solution-state NMR structures of γS-WT and γS-G18V. γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in $\gamma$S-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT. Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation.