title: Structural analysis of the mutant protein D26G of human γS-crystallin, associated with Coppock cataract.
abstract: OBJECTIVE
  To analyze the protein structural features responsible for the aggregation properties of the mutant protein D26G human γS-crystallin (HGSC) associated with congenital Coppock-type cataract.
  
  
  METHODS
  cDNAs of wild-type (WT) and D26G mutant HGSC were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. Their secondary and tertiary structural features, aggregation tendencies, and structural stabilities were compared using spectroscopic (circular dichroism, intrinsic and extrinsic fluorescence), molecular modeling, and dynamics methods.
  
  
  RESULTS
  No difference was observed between the conformational (secondary and tertiary structural) features and aggregation properties between the WT and D26G proteins. The mutant, however, was structurally less stable; it denatured at a slightly lower concentration of the added chemical denaturant (at 2.05 M guanidinium chloride, cf. 2.20 M for the WT) and at a slightly lower temperature (at 70.8 °C, cf. 72.0 °C for the WT). The mutant also self-aggregated more readily (it turned turbid upon standing; at 65 °C, it started precipitating beyond 200 s, while the WT did not, even after 900 s). Molecular modeling showed that the Asp26-Arg84 contact (and the related Arg84-Asn54 interaction) was disturbed in the mutant, making the latter less compact around the mutation site.
  
  
  CONCLUSIONS
  The cataract-associated mutant D26G of HGSC is remarkably close to the WT molecule in structural features, with only a microenvironmental change in the packing around the mutation site. This alteration appears sufficient to promote self-aggregation, resulting in peripheral cataract.
authors.affiliation: Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.
authors.forename: Srinivasu
authors.initials: S
authors.lastname: Karri
authors.forename: Ramesh Babu
authors.initials: RB
authors.lastname: Kasetti
authors.forename: Venkata Pulla Rao
authors.initials: VP
authors.lastname: Vendra
authors.forename: Sushil
authors.initials: S
authors.lastname: Chandani
authors.forename: Dorairajan
authors.initials: D
authors.lastname: Balasubramanian
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D002386:Cataract / Q000151:congenital* / Q000235:genetics; D002942:Circular Dichroism; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D038222:gamma-Crystallins; C077648:CRYGS protein, human