Background 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. Methods 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 molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. Results 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 γS-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. Conclusions Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site. General significance 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. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Bibliographical noteFunding Information:
The authors thank Dr. Philip Dennison and Dr. Dmitry Fishman for excellent management of the UCI Chemistry Department's NMR and Laser Spectroscopy Facilities, and Anne Diehl and Harmut Oschkinat for providing the α B-crystallin sample. The docking calculations were performed on the Extreme Science and Engineering Discovery Environment (XSEDE)  , which is supported by National Science Foundation grant ACI-1053575 . The computational work was supported by National Science Foundation grant DMR-1410415 to RWM and DJT, and the experimental work was supported by National Institutes of Health R01EY021514 to RWM.
© 2016 Elsevier B.V.
- ANS binding assay
- Chemical shift perturbation
- Protein aggregation
- Structural crystallin