Structural features of epidermal keratin filaments reassembled in vitro.

We have studied the structure of epidermal keratin filaments polymerized in vitro, addressing two different levels of organization. First, we have determined the amino acid sequence of a mouse epidermal keratin subunit from the nucleotide sequence of a cDNA clone. The subunit contains a large central region, representing about 50 percent, whose sequence strongly suggests that it assumes a coiled-coil alpha-helical conformation. This is flanked on the amino and carboxyl terminals by long glycine-rich sequences. Second, we have used scanning transmission electron microscopy to study the structure of frozen, unstained filaments. Analyses of such images provides information on the mass per unit length and on the distribution of mass within the filament. These data impose rigorous constraints on possible models for the packing of protofilaments within the filament. Epidermal keratin filaments assembled in vitro are polymorphic; however, the majority of bovine filaments weigh about 37 kD/nm, but most human filaments have masses of only about 27 kD/nm. The filament width is at least 15 nm, substantially more than the generally accepted value of 8 to 10 nm, owing to the existence of low-density mass at the periphery that has not been visualized by conventional microscopic methods. We currently postulate that the alpha-helical regions of the subunits comprise the structural core or backbone of the filament from which at least some of the glycine-rich sequences protrude.