Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly.
The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D003599:Cytoskeleton / Q000648:ultrastructure*; D003956:Dialysis; D007633:Keratins; D007700:Kinetics; D008854:Microscopy, Electron; D011487:Protein Conformation; D011489:Protein Denaturation
D007633:Keratins; D002918:Chymotrypsin