by Murugesan Raju, Puttur Santhoshkumar, K. Krishna Sharma
A substitution mutation in human aA-crystallin (aAG98R) is associated with autosomal dominant cataract. The recombinant mutant aAG98R 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 aA-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 aA-crystallin–derived mini-chaperone on the stability and chaperone activity of aAG98R-crystallin. Methodology/Principal Findings
Recombinant aAG98R 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 aAG98R. The rescuing of chaperone activity in mutanta-crystallin (aAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of aAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized aAG98R displayed chaperone activity comparable to that of wild-type aA-crystallin. The complexes formed between mini-aA–aAG98R complex and ADH were more stable than the complexes formed between aAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin. Conclusion/Significance
These results demonstrate that mini-chaperone stabilizes the mutant aA-crystallin and modulates the chaperone activity of aAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant aA-crystallins and preserve the chaperone function.