title: Structural and aggregation behavior of the human γD-crystallin mutant E107A, associated with congenital nuclear cataract.
abstract: OBJECTIVE
  To analyze the conformational features and aggregation properties of the mutant protein E107A human γD-crystallin (HGDC), associated with congenital nuclear cataract.
  
  
  METHODS
  cDNAs of wild type and E107A mutant were cloned and expressed in BL21 (DE3) pLysS cells and the proteins isolated and purified. The conformational properties and structural stability of the two proteins were compared using circular dichroism and fluorescence spectroscopic analysis. His-tagged cDNAs of the two proteins were transfected into HLE-3B human lens epithelial cells, and into HeLa cells and their in situ aggregation properties compared using immunofluorescence.
  
  
  RESULTS
  The mutant protein was found to be remarkably similar in its secondary and tertiary structural features to the wild type. Its structural stability, analyzed by guanidinium chloride-induced denaturation, was also found to be similar. Its solubility, however, was over hundred-fold less than that of the wild type, and it had the tendency to precipitate and form light scattering particles. That it had the tendency to self- aggregate was noticed by using bis-ANS and Nile Red as extrinsic fluorescent probes. Such aggregation was also seen in situ when transfected and expressed in HLE-3B and in HeLa cell lines.
  
  
  CONCLUSIONS
  E107A HGDC is yet another example of how a point mutation in the protein does not affect its conformation and stability but leads to substantial reduction in solubility and generation of light scattering aggregate particles in vitro and in situ when introduced into cell lines.
authors.affiliation: Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India.
authors.forename: Venkata Pulla Rao
authors.initials: VP
authors.lastname: Vendra
authors.forename: Dorairajan
authors.initials: D
authors.lastname: Balasubramanian
mesh_terms: D002386:Cataract / Q000151:congenital* / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007907:Lens Nucleus, Crystalline / Q000378:metabolism* / Q000473:pathology*; D018613:Microscopy, Confocal; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D014162:Transfection; D038222:gamma-Crystallins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C504903:CRYGD protein, human; D050505:Mutant Proteins; D038222:gamma-Crystallins