title: αA-Crystallin-derived mini-chaperone modulates stability and function of cataract causing αAG98R-crystallin.
abstract: BACKGROUND
  A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37 °C. Our previous studies have shown that αA-crystallin-derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin-derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin.
  
  
  RESULTS
  Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA-αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.
  
  
  CONCLUSIONS
  These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function.
authors.affiliation: Department of Ophthalmology, University of Missouri-Columbia School of Medicine, Columbia, Missouri, United States of America.
authors.forename: Murugesan
authors.initials: M
authors.lastname: Raju
authors.forename: Puttur
authors.initials: P
authors.lastname: Santhoshkumar
authors.forename: K Krishna
authors.initials: KK
authors.lastname: Sharma
mesh_terms: D000595:Amino Acid Sequence; D002386:Cataract / Q000235:genetics*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain