| Glial fibrillary acidic protein (GFAP), a protein of 50 KD, is the sole subunit of astrocyte intermediate filaments (IF) in the central nervous system. This protein belongs to a family that includes vimentin, desmin, the keratins and the neurofilament proteins. Cytoskeletal pellets, which contained GFAP and neurofilaments, were obtained from bovine brain white matter by Triton extraction. GFAP was purified from the pellets by adsorption on hydroxylapatite.; X-ray diffraction of reassembled GFAP fibers, before autoclaving, showed a broad equatorial reflection at 0.99 nm and a meridional reflection at 0.51 nm consistent with a coiled-coil structure. The meridional reflection at 0.51 nm was lost after autoclaving and a strong equatorial reflection at 0.466 nm, developed showing the transformation from {dollar}alpha{dollar} to {dollar}beta{dollar} type structure. GFAP fibers showed a total of 8 maxima which can be indexed on a orthorhombic unit cell with dimensions of a = 1.048 nm, b = 1.007 nm and c = 2.96 nm (fiber axis). Positions and intensities of these maxima agree with those observed for beta-keratins.; Low angle light scattering of dilute solutions revealed that in 8 M urea, 10 mM NaH{dollar}sb2{dollar}PO{dollar}sb4{dollar}, pH 7.4, 1 mM DTT GFAP exists as a tetramer (225 KD). In Tris buffer, pH 8.0, 20 mM NaCl, 1 mM DTT, the average molecular weight was 2 {dollar}times{dollar} 10{dollar}sp6{dollar} D.; Circular dichroism (CD) measurements of GFAP in Tris buffer showed that the protein is about 60% alpha-helical, 20% beta structure and 20% random coil. The GFAP spectrum is relatively unaffected by urea at concentrations of up to 3-4 M but at 6 M or greater, the protein is largely random coil. Increasing concentrations of SDS from.01 to 1% in 10 mM NaH{dollar}sb2{dollar}PO{dollar}sb4{dollar}, pH 7.4 induced moderate changes in the secondary structure of GFAP, indicating a relatively compact structure in its native state. A fraction of GFAP, 0.3 to 0.5 mg/ml, was found to be soluble in water in undegraded form and CD measurements showed a value of 66% {dollar}alpha{dollar}-helix, consistent with theoretical calculations of the proposed structure for IF proteins. These studies provide experimental evidence for the alpha helical nature of native GFAP both in solution and as fibers as predicted from its amino acid sequence and that of other type III IF proteins. |
