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R. Chandrashekar, S. Rajan, E.C. Abraham; Hetero–Oligomeric Alpha Crystallins Formed From Reconstituted Alpha B Crystallin and Alpha A Crystallin Mutants . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1999.
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αA–Crystallin is known to undergo cleavage of C–terminal residues and most of such truncated αA–crystallins in their homo–oligomeric form have significantly decreased chaperone activity. However, it is quite possible they actually exist in the eye lens as hetero–aggregates formed with native αß–crystallin. The purpose of this study was to test the possibility that the reconstituted α–crystallins have different oligomeric size and chaperone activity from those of the constituent αA and αB subunits.
A site–directed mutagenesis kit from Stratagene was used to generate truncated rat αA–crystallin lacking 8 C–terminal residues (αA1–165) and R163G mutant in the same truncated protein. These mutants and rat αB–crystallin were expressed in E. coli and purified by Sephacryl S–300HR size – exclusion chromatography followed by molecular sieve HPLC. Reconsitution of theαA mutants and αA–wild type with αB was accomplished by previously published procedures of Bera and Abraham (Biochemistry 41: 297–305,2002). Purified protein solutions of a concentration of 4mg/ml were denatured with 4M GdnHCl for 6h at 4°C. Reconstitution was done with mixing αA–wt, αA1–165 or αA1–165(R163G) with αB–wt in a 1:1 ratio. This was followed by dialysis for 48h. Oligomeric size of the reconstituted proteins was determined by molecular sieve HPLC, chaperone assay was done with alcohol dehydrogenase as the target protein. Structural parameters were determined by CD measurements.
Truncated mutant (αA1–165) had 73% chaperone activity (%protection) as compared to αA–wt whereas reconstituted αB+αA1–165 had 100% protection as compared to αB+ αA–wt. Oligomeric size was similar in both. αA1–165(R163G) mutant had 60% chaperone activity and the reconstituted αB+αA1–165 (R163G) had 100% chaperone activity. Most remarkable was the observation that the oligomeric size of R1–165 (R163G) was 250kDa whereas that of αB+R1–165 (R163G) was 600 kDa which represents a 2.4 –fold increase in size to nearly normal level.
αA–crystallin mutants, when associated with native αB–crystallin shows substantial increase in the chaperone function and the oligomeric size. Thus, hetero–aggregate form of αA–crystallin mutants will be beneficial to the overall α–crystallin chaperone function.
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