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I.A. Mills, S.L. Flaugh, M.S. Kosinski–Collins, J.A. King; Engineering Protein Chimeras of Human D and Human S Crystallin to Identify Sequence Components Involved in vitro Aggregation of Human D Crystallin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3892.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Analysis of cataractous lens tissue reveals an abundance of protein aggregates consisting of multiple lens protein species. A major component of these aggregates is the lens crystallin proteins. The γ–crystallins are monomeric proteins that consist of two domains containing anti–parallel ß–sheet Greek key motifs. Two γ–crystallins, Human γD Crystallin (HγD–Crys) and Human γS Crystallin (HγS–Crys) are abundant within the lens where HγD–Crys is localized in the lens nucleus while HγS–Crys is localized primarily in the cortex of the lens. Previously, in vitro productive refolding studies identified an aggregation pathway for HγD–Crys upon dilution out of denaturant. Surprisingly, off pathway aggregation has not been observed for HγS–Crys. Our approach is to engineer chimeras of these two proteins in order to decipher the particular sequence(s) of HγD–Crys involved in aggregation. Methods: In vitro studies have been performed on the unfolding, refolding, and aggregation pathways of both HγD–Crys and HγS–Crys proteins using fluorescence spectroscopy and solution turbidity measurements. To determine the region(s) of HγD–Crys involved in the aggregation pathway, the N terminal and C terminal sequences of each protein were cloned and chimeras constructed with each domain exchanged; for example, HγD–Crys N terminal domain linked with HγS–Crys C terminal domain. Results: Equilibrium refolding studies and solution turbidity measurements on the HγD–Crys N terminal domain indicates that the N terminal does not aggregate. This result indicates the possibility of the HγD C terminal domain’s involvement in the aggregation pathway. A similar analysis of the C terminal domain and chimera engineered proteins are underway. Conclusions: By engineering and analyzing different chimera protein species, we may be able to determine the region(s) of HγD–Crys that is necessary and sufficient for in vitro aggregation pathway.
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