JOURNAL OF STRUCTURAL BIOLOGY 103, 212-224 (1990) Structure and Assembly of Calf Hoof Keratin Filaments ZEHRA SAYER&~ ANNE-MARIE MICHON,~ PILAR SICRE, AND MICHEL H. J. KOCH European Molecular Biology Laboratory, % DESY, Notkestrasse 85, D-2000 Hamburg 52, Federal Republic of Germany Received April 4, 1990, and in revised form June 26, 1990 Keratin filament polypeptides were purified from calf hoof stratum corneum with the aim of studying the in vitro assembly process and determining structural pa- rameters of reconstituted filaments. Anion exchange chromatography was used to obtain the most complete fractionation and identification of the acidic and basic components in the purified polypeptide mixture to date. The reassembly products of the fractionated components were investigated by electron microscopy. Fully reconsti- tuted filaments yield homogeneous solutions, and values of 9.8 nm for the filament diameter and 25 kDa/nm for the mass per unit length (M/L) were obtained by X-ray solu- tion scattering. The structures formed in solution at var- ious stages of filament assembly were not sufficiently ho- mogeneous to be studied by this technique. X-ray diffrac- tion patterns from native stratum corneum display strong maxima at 3.6 and 5.4 nm. Contrary to previous reports, these maxima do not appear to be due to lipids since they are also observed with delipidated rehydrated specimens. A series of weak maxima is also detected in the patterns of dry tissue. The absence of these features in the pat- terns of reconstituted filaments suggests that, in contrast to some electron microscopic observations, there are no prominent regularities in the structure of calf hoof ker- atin filaments. 0 1990 Academic Press, Inc. INTRODUCTION Keratin filaments are the intermediate filaments (IFS) observed in epithelia, epithelia-derived tissue, and various appendages such as hair, nail, and wool. Subunit proteins of keratin filaments, keratins or, “cytokeratins” in epithelia and epithelia-derived tis- sue and “hard d-keratins” in wool, hair, nail, claws, etc., constitute a most complex family of IF polypep- Abbreviations used: EDTA, ethylenediaminetetraacetic acid; FPLC, fast protein liquid chromatography; M/L, mass per unit length; NP-40, nonidet 40; PMSF, phenylmethylsulfonyl fluoride; Rx, radius of gyration of the cross section; SDS: sodium dodecyl sulfate. ’ To whom correspondence should be addressed. ’ Present address: Whitehead Institute for Biomedical Re- search, Nine Cambridge Centre, Cambridge, MA 02142. tides with more than 20 members in human and bovine tissues (Fuchs, 1988; Fraser et al., 1987; Os- born and Weber, 1986; Steiner-t and Parry, 1985). Keratins have been grouped into the acidic (p1 < 5.5) and basic (p1 > 6.0) subfamilies depending on differences in molecular weights, isoelectric points, antigenic determinants, and two-dimensional tryp- tic maps (Moll et al., 1982; Schiller et al., 1982; Sun et al., 1984; Franke et al., 1981; Cooper and Sun, 1986; Knapp et al., 1986). The acidic and basic sub- families are also called type I and type II, respec- tively, due to the strong similarities in amino acid sequences observed with type I and type II wool cx-keratins (Crewther et al., 1983; Hanukoglu and Fuchs, 1982). It was shown that two or more poly- peptides, always including at least one member from each subfamily, are expressed simultaneously in any tissue and that at least one member from each subfamily must be present for full filament forma- tion (Lee and Baden, 1976; Steiner-t et al., 1976; Franke et al., 1983; Hatzfeld and Franke, 1985; Quinlan et al., 1985; Fuchs, 1988). IF polypeptides range in molecular weight from about 44 to 200 kDa and all contain a central rod domain (about 310 residues) which consists of two a-helical domains, flanked by N- and C-terminal do- mains that are variable in size and chemical char- acter. Differences in the molecular weights of IF polypeptides and specificity of interactions in which different types of IFs are involved appear to be due to these variable end domains. Central rod domains of the constituent polypep- tides form the basic framework of all types of IFS and result in the observed common structural fea- tures (Geisler and Weber, 1982; Quinlan and Franke, 1983; Woods and Inglis, 1984; Quinlan et al., 1984; Ip et al., 1985; Quinlan et al., 1986). Different models have been proposed for higher levels of organization of subunits in the IF, i.e., end- to-end association and packing of tetramers. These models are based on X-ray diffraction data obtained mainly from hard d-keratin filaments (Fraser and MacRae, 1971; Fraser et al., 1986), calculations pre- 212 1047~8477/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.