Subunit structure of the protofilament buidling blocks of intermediate filaments

The purposes of this study were to use chemical cross-linking reagents to elucidate the number of subunits in desmin and vimentin protofilaments, to elucidate conditions necessary for dissociation of the subunits of desmin protofilaments, and to understand the arrangement of the subunit chains of the protofilaments. Three chemical cross-linkers, namely ethylene glycolbis(succinimidyl succinate), bis 2-(succinimidooxy-carbonyloxy)ethyl sulfone, and glutaraldehyde, were used to examine the subunit composition of the approximately 2.5 nm diameter protofilaments that are present in pH 8.5, low ionic strength solutions of highly purified avian smooth muscle desmin or of mammalian smooth muscle vimentin. Analysis of cross-linked products by SDS-polyacrylamide gel electrophoresis demonstrated the presence of four species having molecular weights of approximately 56,000, 120,000, 170,000, and 220,000. These species corresponded to the monomer, dimer, trimer, and tetramer. These results were compatible with a tetrameric protofilament unit which probably consists of a dimer of a normal two-stranded coiled-coil. Dissociation of desmin protofilaments in the presence of urea or at elevated pH also was examined by cross-linking with glutaraldehyde and electrophoretic examination. In low ionic strength solutions, the tetrameric unit did not begin to dissociate into dimers until the pH reached about 12. The desmin was present as dimers at pH 12.5. The desmin protofilaments remained in the tetrameric form in the presence of 5 M urea at pH 8.5. Dissociation of the tetrameric form into dimers, plus some monomers, started to become evident in 6 M urea. Desmin was present primarily in the monomeric form in 8 M urea-containing solutions in the presence of 2-mercaptoethanol at pH 8.5. Analysis of products resulting from chemical cleavage at tryptophanyl residues of previously chemically cross-linked protofilaments is most compatible with an arrangement in which the two dimers in a tetrameric protofilament are arranged in an antiparallel fashion