TitleAltered Protein Dynamics and Increased Aggregation of Human γS-Crystallin Due to Cataract-Associated Deamidations.
Publication TypeJournal Article
Year of Publication2019
AuthorsForsythe HM, Vetter CJ, Jara KAnn, Reardon PN, David LL, Barbar EJ, Lampi KJ
JournalBiochemistry
Volume58
Issue40
Pagination4112-4124
Date Published2019 Oct 08
ISSN1520-4995
Abstract

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 . However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed . 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 N 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 insolubility and aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

DOI10.1021/acs.biochem.9b00593
Alternate JournalBiochemistry
PubMed ID31490062
Grant ListP30 EY010572 / EY / NEI NIH HHS / United States
S10 OD018518 / OD / NIH HHS / United States
S10 OD012246 / OD / NIH HHS / United States
R01 EY027012 / EY / NEI NIH HHS / United States
R01 EY027768 / EY / NEI NIH HHS / United States