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E.C. Abraham, P. Thampi, S. Cao; The Hydrodynamic Properties of AlphaA–Crystallin and its C–terminal Truncated Mutants as Determined by Light Scattering. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3380.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Chaperone activity of an alphaA–crystallin mutant lacking 10 C–terminal residues (mutant 1) was only slightly affected whereas that of a mutant lacking 11 C–terminal residues (mutant 2) was severely affected. The purpose of this study was to show that the absolute masses, the hydrodynamic radii (Rh), and the cumulative weight fraction as a function of molar mass of these mutants and the alphaA wild type are the same or different. Methods: Recombinant alphaA–wild type and the two mutants were expressed in E coli and purified by size–exclusion chromatography on Sephacryl S 300 HR columns. Each protein sample was injected to a Shodex KW–803/804 molecular sieve HPLC column to which a Multi–Angle Light Scattering (MALS), Differential Refractive Index (DRI), and a Quasielastic Light Scattering (QELS) detectors were coupled. The MALS, DRI, and QELS chromatograms were analyzed by the ASTRA software. Molecular masses were computed from the MALS and DRI data and Rh from QELS data. Results: The molar mass for the wild type ranged from about 400 to 1000 kDa with a weight–averaged mass of 720 kDa. For the mutant 1, molar mass ranged from about 400 to 800 kDa with a weight–averaged mass of 564 kDa. The polydisperse nature of the wild type and this mutant was evident from these data and confirmed by commulative molar distribution (cmd) analysis. Strikingly, the mutant 2 was mostly monodisperse showing a nearly flat mass vs elution time plot. The cmd showed >80% in the same mass species. The calculated molecular mass was 88 kDa. Rh values were 7.2, 6.2, and 2.3 nm, respectively, for the wild type and the mutant 1 and mutant 2. Conclusions: Loss of 11 C–terminal residues including Arg–163 has a significant impact on the formation of fully oligomerized alphaA–crystallin because tetramers having 4 subunits rather than large oligomers were formed. This points to an important role for the C–terminal extension, Arg–163 in particular, and no significant role for the C–terminal flexible tail of nine amino acids in alphaA–crystallin oligomerization.
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