The fibrillar substructure of keratin filaments unraveled.
We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four "4.5-nm protofibrils" can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into "2-nm protofilaments." In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils.
D002413:Cations, Divalent / Q000494:pharmacology; D003599:Cytoskeleton / Q000032:analysis* / Q000648:ultrastructure; D004817:Epidermis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007633:Keratins / Q000032:analysis*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008954:Models, Biological; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D014357:Trypsin
D002413:Cations, Divalent; D046911:Macromolecular Substances; D010710:Phosphates; D007633:Keratins; D014357:Trypsin