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Abstract

Vimentin, a major cytoskeletal protein of many cells of mesenchymal tissues as well as of cultured cells of various kinds, is the constituent protein of a special class of intermediate-sized (7 to 11 nm) filaments. These are insoluble at high and low ionic strength over the physiologically relevant pH range (5·5 to 7·5). Vimentin from cytoskeletons of various cells (porcine eye lens tissue, mouse 3T3, rat RVF-SMC, and hamster BHK-21 cells) was purified and solubilized in urea or guanidinium hydrochloride. When solutions of denatured, monomeric vimentin were dialyzed against buffers of various ionic strengths containing 2-mercaptoethanol, vimentin renatured and long (up to 3·5 μm) intermediate-sized filaments were formed. The reconstituted filaments were indistinguishable from native vimentin filaments by electron microscopy and X-ray diffraction. Native and reconstituted intermediate-sized filaments containing other proteins (prekeratin, desmin) were examined in parallel. The high degree of molecular order in reconstituted filaments was demonstrated by meridional reflections at 0·15 and 0·51 nm and equatorial reflections at 0·98 and 6·3 nm, indicating coiled-coil arrangements of α-helices of vimentin.

The results show that vimentin spontaneously refolds into α-helical arrangements and assembles, over a broad range of salt concentrations, into long intermediate-sized filaments. The process does not involve disulfide bond formation or the presence of other proteins. The similar reconstitution properties of various intermediate filament proteins suggest that such proteins, albeit different in polypeptide composition and amino acid sequence, contain regions with similar principles of sequence arrangement that define α-helical domains and are responsible for the assembly into morphologically similar intermediate-sized filaments.