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Krishna Sharma, Murugesan Raju, Puttur Santhoshkumar; Stabilization of Matastable Cataract Causing αAG98R-Crystallin by αA-Mini-Chaperone. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3667.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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