title: Mutations in human αA-crystallin/sHSP affect subunit exchange interaction with αB-crystallin.
abstract: BACKGROUND
  Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.
  
  
  RESULTS
  In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.
  
  
  CONCLUSIONS
  Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Ilangovan
authors.initials: I
authors.lastname: Raju
authors.forename: Lalita
authors.initials: L
authors.lastname: Oonthonpan
authors.forename: Edathara C
authors.initials: EC
authors.lastname: Abraham
mesh_terms: D002386:Cataract / Q000151:congenital / Q000209:etiology; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D021122:Protein Subunits / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000235:genetics*; D038203:alpha-Crystallin B Chain / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain