Journal
PROTEIN SCIENCE
Volume 29, Issue 9, Pages 1945-1963Publisher
WILEY
DOI: 10.1002/pro.3915
Keywords
cataracts; crystallins; deamidation; dynamic and static light scattering; hydrogen-deuterium exchange; mass spectrometry; oxidation; protein aggregation; protein unfolding
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Funding
- NEI NIH HHS [P30 EY010572, EY027012, EY027768, R01 EY027012, EY010572, R01 EY027768] Funding Source: Medline
- NIH HHS [OD012246, S10 OD012246] Funding Source: Medline
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Age-related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long-lived. A major crystallin, gamma S, is heavily modified by deamidation, in particular at surface-exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated gamma S was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of gamma S was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen-deuterium exchange, and susceptibility to disulfide cross-linking. Compared with wild type gamma S, a small population of each deamidated mutant aggregated rapidly into large, light-scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of gamma S. We suggest that these perturbations disrupt the native disulfide arrangement of gamma S and promote the formation of disulfide-linked aggregates. The lens-specific chaperone alpha A-crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize gamma S-crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.
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