title: Deamidation of N76 in human γS-crystallin promotes dimer formation.
abstract: BACKGROUND
  Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554-3561). In the current work, this modification has been extensively characterised in vitro.
  
  
  METHODS
  Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC-MALLS and analytical ultracentrifugation methods.
  
  
  RESULTS
  Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer.
  
  
  CONCLUSIONS
  Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation.
  
  
  CONCLUSIONS
  Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
authors: Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia.
authors: Nicholas J
authors: NJ
authors: Ray
authors: Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia.
authors: Damien
authors: D
authors: Hall
authors: Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia. Electronic address: john.carver@anu.edu.au.
authors: John A
authors: JA
authors: Carver
mesh_terms: D000577:Amides / Q000737:chemistry*; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D013696:Temperature; D038222:gamma-Crystallins / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D000577:Amides; D038222:gamma-Crystallins
keywords: Ageing; Cataract; Crystallin; Lens; Post-translational modification; Small heat-shock protein