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K.K. Sharma, S. Puttur; Deletion of Residues 54–61 (FLRAPSWF) in Alpha B–Crystallin Leads to Decreased Oligomeric Mass With Increased Chaperone–Like Activity . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3486.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Earlier we reported that in alpha B–crystallin 42–57, 60–71 and 88–123 sequences are involved in interaction with alpha A crystallin during the hetero–oligomer formation (IOVS 2001, 42(4) p.s292). The purpose of this study was to determine the role of alpha B–crystallin 54–61 region, comprising of the residues that separate the alpha–A interacting regions, in oligomerization, subunit–exchange and chaperone–like activity. Methods: Residues 54–61 in human alpha B crystallin were deleted by site–directed mutagenesis and the mutant protein was expressed in E.coli BL21(DE3)pLysS cells. The proteins were purified by ion–exchange and gel filtration chromatography. The purity was checked by SDS–PAGE and the mutation was confirmed by mass spectrometry. The structure and the degree of hydrophobicity were analyzed by spectroscopic methods. The chaperone–like activity of wild–type and mutant proteins was compared using citrate synthase (CS) as substrate. The interaction between 6HαA and αBΔ54–61 was tested using Alexa Flour labeled proteins employing FRET assay. Results: Deletion of residues 54–61 in αB crystallin significantly decreases the homo–oligomeric mass of the mutant protein. The average oligomeric mass of αB and αBΔ54–61, calculated using a Biosep–secC4000 column, were 650 and 475 kDa respectively. Deletion of a sequence that contains one of the two tryptophan residues in αB resulted in 50 percent decrease in intrinsic tryptophan fluorescence. Surprisingly, the αBΔ54–61 showed a two–fold increase in bis–ANS binding compared to the wild–type protein. Concomitant with increase in hydrophobicity, the deletion mutant showed a 2–3 fold increase in anti–aggregation activity during CS denaturation assay. Homo–oligomers of 6HαA (750 kDa) readily exchanged subunits with αBΔ54–61 homo–oligomers at 37oC, to form hetero–oligomers having an intermediate mass of 625 kDa. Conclusions: The 54–61 residues in αB crystallin do not contribute to the chaperone–like function of the protein but they are essential for higher order assembly of oligomers. The chaperone–like activity of αB–crystallin can be modulated by altering its oligomeric size and/or increasing the surface hydrophobicity.
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