Abstract
Mg2+-induced polymerization of type III intermediate filament proteins vimentin and glial fibrillary acidic protein was studied by transient electric birefringence. In the absence of MgCl2 we found a net permanent dipole moment, approximately 45-nm-long dimers for vimentin, approximately 65-nm-long tetramers, hexamers, and possibly octamers for both proteins, and 100-nm aggregates for glial fibrillary acidic protein. Controlled oligomerization occurred after the addition of MgCl2. Although the solutions contained (small) aggregates of different sizes, more or less discrete steps in polymer formation were observed, and it was possible to discriminate between an increase in width and length. At the first stage of polymerization (in 0.3 mM MgCl2 for vimentin and 0.2 mM MgCl2 for glial fibrillary acidic protein), the permanent dipole moment disappeared without a change in length of the particles. At higher MgCl2 concentrations, structures of approximately 100 nm were formed, which strongly tended to laterally assemble into full-width intermediate filament structures consisting of about 32 monomers. This contrasts with previous models where first full-width (approximately 10-nm) aggregates are formed, which then increase in length. Subsequently, two discrete elongation steps of 35 nm are observed that increase the length to 135 and 170 nm, respectively. Possible structural models are suggested for the polymerization.
Highlights
Many eukaryotic cells contain cytoplasmic intermediate filaments (IFs),1 which are composed of proteins with a molecular mass of 40 –210 kDa [1, 2]
In a recent publication on vimentin IF structures, we have proven the occurrence of such dimers in low ionic strength solution [5]
For glial fibrillary acidic protein (GFAP) at pH 6.8, the effect of small amounts of MgCl2 (0.2 mM) on S1/S2 is comparable with that for vimentin, while NaCl did not result in such a decrease
Summary
Vimentin from Ehrlich ascites tumor cells and GFAP from bovine brain white matter, purified as described [27], lyophilized, and stored at Ϫ20 °C, was dissolved in 2 mM phosphate, pH 7.5, 6 mM 2-mercaptoethanol, 6 M urea. The vimentin and GFAP concentrations in the absence of MgCl2 were determined from the absorption using a. Theoretical Background—When dissolved optically anisotropic molecules are oriented by a square electric-field pulse, the solution becomes birefringent. 3) The field-free decay time of the birefringence signal, yielding information on size and flexibility of the molecules [29]. The Kerr constants were determined in solutions with a resistance of 200 ⍀ by dialysis of the proteins from the aqueous low salt buffers (0.7 mM sodium phosphate, pH 7.5, and 2 mM BisTris1⁄7HCl, pH 6.8) to buffers with different concentrations of MgCl2 (see Table I). The field-free decay of the birefringence was analyzed on a SUN4 workstation using the computer programs DISCRETE [34, 35] and CONTIN [36, 37]. Analyses were started 0.20 s (when the sample interval (SI) ϭ 0.1 or 0.2 s), 0.50 s (SI ϭ 0.5 s), or 1.0 s (SI ϭ 1.0 s) after the orienting field was turned off
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