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M.P. Chan, Y.V. Sergeev, P.T. Wingfield, J.F. Hejtmancik; Heat–Induced Aggregation and Binding to –Crystallin of Amino–Arm Truncated ßA3–Crystallin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2008.
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© ARVO (1962-2015); The Authors (2016-present)
ß–Crystallins undergo proteolytic cleavage of terminal extensions upon aging. In particular, truncation of the amino–arm of ßA3–crystallin results in increased UV–induced aggregation and alters its affinity for dimerization. This study further characterizes this protein with respect to dimerization and its susceptibility to heat–induced unfolding, aggregation and binding to α–crystallin.
Recombinant ßA3–crystallin with truncated amino–terminus (rßA3tr) was expressed in E. coli and purified by chromatography. Aggregation was induced at 55°C with and without presence of bovine α–crystallin, and detected by absorption at 360nm. Samples were analyzed by size–exclusion chromatography (SEC, Superose–6), centrifugation followed by SDS–PAGE and Western blots of the pellets and supernatant. Sedimentation coefficient and apparent molecular mass of rßA3tr at 1mg/ml were determined by sedimentation velocity analysis.
Absorption at 360nm shows that aggregation of rßA3tr was suppressed in presence of α–crystallin. This is also consistent with the significantly reduced level of insoluble rßA3tr on SDS–PAGE and Western blots and co–migration of soluble rßA3tr and α–crystallin on SEC. Sedimentation analysis of rßA3tr gives a sedimentation coefficient of 3.25s, and apparent molecular masses of 42,619Da and 42,809Da at 15°C and 20°C, respectively.
Recombinant ßA3–crystallin denatures upon heating and is partially rescued by α–crystallin, which binds and solubilizes rßA3tr. The average estimated molecular mass suggests that at 1 mg/ml rßA3tr exists predominantly as a dimer, consistent with data from analytical ultracentrifugation studies. Changes in molecular mass with temperature, although small, are consistent with dimerization being driven by entropic forces, once again consistent with data from analytical ultracentrifugation studies.
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