Abstract
Purpose: :
Mutant form of αA-crystallin, αAG98R, is an unstable protein with reduced chaperone activity. The mutant protein aggregates on storage and incubation at or near physiological temperature. Previous studies have shown that αA-Mini-chaperone, a peptide- DFVIFLDVKHFSPEDLTVK, representing the chaperone site in αA-crystallin functions like a molecular chaperone. The objective of the present study was to determine the effect of αA- mini-chaperone on the aggregation and chaperone activity of αAG98R-crystallin.
Methods: :
Mutant and wild type αA-crystallin were expressed in E.Coli and purified following the procedures described earlier. The aggregation of αAG98R in presence and absence of αA-mini-chaperone (supplied by GenScript) during incubation was followed by examining the samples under EM. In addition, gel filtration studies and DLS analysis of the samples in a multi-angle DAWN-EOS unit was performed. The binding of chaperone peptide to αAG98R was confirmed by HPLC analysis of the αAG98R-mini-chaperone complex and use of photoactive biotinylated mini-chaperone (biotin- DFVIFLDVKH (benzoylphenylalanine) SPEDLTVK) and MS analysis.
Results: :
Examination of aggregation prone αAG98R under EM showed that the oligomers begin interact with one another within 10 min of incubation at 40oC and to form initial aggregates consisting of 2 to 10 oligomers which coalesce to form larger aggregates of 10 -20 oligomers in 30 mins. We found that addition of mini-chaperone during αAG98R incubations prevented αAG98R from forming larger aggregates that precipitate with time. We also found that mini-chaperone stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystalin. Tryptic digestion and mass spectrometric analysis of αAG98R incubated with biotynylated photoactive mini-αA-crystallin showed that the mini-chaperone interacts at the 89-103 region of αAG98R.
Conclusions: :
These results demonstrate that mini-chaperone recognizes the structural changes in αAG98R due to mutation, stabilizes the mutant αA-crystallin and restores the lost chaperone activity. These findings should facilitate the design of peptide chaperones to stabilize and restore chaperone activity of mutant crystallins.
Keywords: crystallins • chaperones • mutations