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S. Yellamaraju, K.K. Sharma; Significance of Recognition Sequence–2 (Residues 60–71 in Alpha B Crystallin) in Alpha B–Alpha a Interactions . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3886.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously, using peptide scan method, we have identified that residues 42–57 and 60–71 in alpha B crystallin (tslspfylrppsflra, named as Recognition Sequence 1 or RS–1 and WFDTGLSEMRLE, named as Recognition Sequence 2 or RS–2) are involved in interaction with alpha A (IOVS 2001, 42(4) p.s292). The purpose of this study was to confirm and understand the significance of RS–2 region in subunit interactions between alpha A– and alpha B–crystallins by characterizing alpha B–crystallin mutants W60R, F61N and S66G employing spectroscopic methods and subunit exchange studies. Methods: Mutations were introduced using site–directed mutagenesis and the mutant proteins expressed in E. coli BL21(DE3)pLysS cells were purified by ion–exchange and gel filtration chromatography. The mutations were confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone–like activities of wild–type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between alpha A– and alpha B–crystallins was monitored by FRET (fluorescence resonance energy transfer). For this purpose, purified alpha B, alpha B W60R, alpha B F61N and alpha B S66G were labeled with Alexa fluor 350 and Alexa fluor 488 was used to label alpha A. Results: Both wild–type and mutant alpha B crystallins showed similar elution profiles on gel filtration chromatography. Tryptophan fluorescence intensity of alpha B S66G was 1.5–fold higher than that for wild–type. The intrinsic fluorescence of alpha B F61N was marginally lower than that of alpha B, where as the fluorescence intensity of alpha B W60R decreased by 40% when compared to alpha B. The fluorescence emission maximum was same for all the above proteins. The relative availability of hydrophobic sites in the mutants were in the order of alpha B S66G >>alpha B=alpha B F61N=alpha BW60R. The far–UV CD profiles for wild–type and αB–crystallin mutants indicated no significant changes in their secondary structures. Mutations did not alter the chaperone–like activity of these proteins. W60R mutation did not affect the subunit exchange rate between alpha B– and alpha A–crystallins. On the other hand, S66G mutation increased the subunit exchange rate by 2–fold, where as, F61N mutation decreased the subunit exchange rate between alpha B– and alpha A–crystallins by 35%. Conclusions: The results confirm our earlier observation that 60–71 region in alpha B–crystallin constitutes one of the alpha A–crystallin interacting sites.
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