Altered Protein Dynamics and Increased Aggregation of Human γs-Crystallin Due to Cataract-Associated Deamidations

Heather M. Forsythe, Calvin J. Vetter, Kayla Ann Jara, Patrick N. Reardon, Larry L. David, Elisar J. Barbar, Kirsten J. Lampi

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish (US)
Pages (from-to)4112-4124
Number of pages13
JournalBiochemistry
Volume58
Issue number40
DOIs
StatePublished - Oct 8 2019

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Crystallins
Cataract
Agglomeration
Lenses
Proteins
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Biomolecular Nuclear Magnetic Resonance
Denaturation
Glutathione Disulfide
Viperidae
Amides
Disulfides
Light scattering
Hydrogen
Cytoplasm
Solubility
Hot Temperature
Nuclear magnetic resonance
Light

ASJC Scopus subject areas

  • Biochemistry

Cite this

Altered Protein Dynamics and Increased Aggregation of Human γs-Crystallin Due to Cataract-Associated Deamidations. / Forsythe, Heather M.; Vetter, Calvin J.; Jara, Kayla Ann; Reardon, Patrick N.; David, Larry L.; Barbar, Elisar J.; Lampi, Kirsten J.

In: Biochemistry, Vol. 58, No. 40, 08.10.2019, p. 4112-4124.

Research output: Contribution to journalArticle

Forsythe, Heather M. ; Vetter, Calvin J. ; Jara, Kayla Ann ; Reardon, Patrick N. ; David, Larry L. ; Barbar, Elisar J. ; Lampi, Kirsten J. / Altered Protein Dynamics and Increased Aggregation of Human γs-Crystallin Due to Cataract-Associated Deamidations. In: Biochemistry. 2019 ; Vol. 58, No. 40. pp. 4112-4124.
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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 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.",
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AU - David, Larry L.

AU - Barbar, Elisar J.

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