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Increased hydrophobic surface exposure in the cataract-related G18V variant of human ?S-crystallin.

ABSTRACT: The objective of this study was to determine whether the cataract-related G18V variant of human ?S-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by ?B-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 molecules to the proteins, starting with the solution-state NMR structures of ?S-WT and ?S-G18V.?S-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 ?S-G18V that are not present in ?S-WT. Molecular docking predicts three binding sites that are specific to ?S-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 ?S-WT.Although both proteins display non-specific binding, more residues are involved in ANS binding to ?S-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site.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.

SUBMITTER: Khago D

PROVIDER: S-EPMC5942885 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin.

Khago Domarin D Wong Eric K EK Kingsley Carolyn N CN Freites J Alfredo JA Tobias Douglas J DJ Martin Rachel W RW

Biochimica et biophysica acta 20151014 1 Pt B

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 26459004

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Project description:Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (μs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Dataset Information

Increased hydrophobic surface exposure in the cataract-related G18V variant of human ?S-crystallin.

Publications

Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin.

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