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J.A. King, K. Papanikolopoulou, I. Mills, H. Inouye, A. Gross, D. Kirschner, S. Decatur; Formatin of Amyloid Fibers in vitro by Human gD–Crystallin and Its Isolated Domains . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2005.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether Human gD–crystallin can form amyloid fibrils in vitro. Amyloid fibrils are associated with a variety of human protein misfolding and protein deposition diseases. Bovine crystallins formed amyloid fibers under denaturing conditions (Meehan et al., J Biol Chem; 279(5):3413–3419, 2004) and amyloid fibers may form in the lens of mutant mice (Sandilands et al., EMBO J.; 21(22):6005–6014, 2002).
Human gD–crystallin expressed and purified from E.coli, was stable and soluble at 37oC pH 7, and refolded from the fully denatured state back to the native state under these conditions (Kosinski–Collins & King, Protein Sci.; 12:480–490, 2003). Fibrillar forms generated by incubation of the native protein at pH 3 were characterized by transmission electron microscopy, Conge Red binding, x–ray scattering, and FTIR spectroscopy.
Incubation of the protein at 37oC in 50 mM acetate buffer pH 3 at 50ug/ml – 5mg/ml for 2 days, led to formation of a viscous, gel–like solution. Examination of negatively stained samples by transmission electron microscopy revealed linear, non–branching fibrils of variable lengths, with diameters ranging from 15 to 35 nm. Incubation with the dye Congo Red generated the spectral red shift associated with dye binding to amyloid. The fibers were mounted and examined by low angle X–ray scattering. The resulting X–ray fiber diffraction patterns recorded from the samples were typical of amyloid fibers. A clear meridional reflection at 4.7 A and a more diffuse reflection on the equator between 10 and 11 A were present. FTIR was used to follow the evolution of the secondary structure of gD–Crys with time during incubation of the protein at pH 3. The native protein displayed a major band at 1640 cm–1 that converted during incubation at 37oC to a band at 1616 cm–1. An additional band at 1689 cm–1 also appeared with time. The presence of bands in the regions ∼ 1620 cm–1 and ∼1680 cm–1 has been attributed to the formation of intermolecular beta–sheet structure that characterizes the fibrillar amyloid motif. The isolated N–terminal 1–82 and C–terminal 86–174 domains of HgD–Crys were incubated and examined under the same conditions. These isolated domains also formed amyloid fibrils.
Human gD–crystallin as well as its isolated N–terminal and C–terminal Greek key domains can form amyloid fibrils in vitro.
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