Recently, the spatial distribution of the crosslinks induced between lysine residues in trichocyte (alpha-) keratin intermediate filaments (IF) using disulfosuccinimidyl tartrate was analyzed in detail and the results used to provide information about the three-dimensional structure of the IF [Fraser, R.D.B., Parry, D.A.D., 2005. The three-dimensional structure of trichocyte (hard alpha-) keratin intermediate filaments: features of the molecular packing deduced from the sites of induced crosslinks. J. Struct. Biol. 151, 171-181.] The presence of small amounts of 0--> +/-4 crosslinkages between molecular strands four distant in the network implied that the three-dimensional network of interacting molecules must be deeply puckered, but no specific suggestions were made about the nature of the puckering. Whilst it was recognized that there may be more than one type of molecular environment in the structural repeat the initial analysis was confined to the simplest case in which all molecules had the same environment, that is to say the asymmetric unit comprised a single molecule. Further studies reported here suggest that it is likely that the asymmetric unit consists of at least two and possibly as many as four molecules and the implications of this for modeling the structure of trichocyte IF are discussed.

Previous studies of the X-ray diffraction pattern of the crystalline regions of type I collagen fibrils yielded information on the unit cell parameters and also the orientation of the pseudo-hexagonally packed molecular segments in the overlap region. The absence of Bragg reflections at high angles attributable to the molecular segments in the gap region led to the suggestion that these segments were more mobile than those in the overlap region. We report a study of the low-angle Bragg reflections in a search for information about the nature of the orientation and packing of the molecular segments in the gap region. We conclude that the (m = 0, n = 0) helix layer plane of the molecular segments in the overlap region makes little or no contribution to the Bragg reflections at low angles, and identify three possible origins for the observed low-angle reflections in the electron density contrast associated with: (1) the "hole" created by the missing molecular segment in the gap region; (2) the telopeptides; or (3) the axial regularities in amino acid residues of a particular type, with periodicities of D/5 or D/6. Sufficient information is available to investigate the first two of these possibilities, and the results obtained suggest specific arrangements for the molecular segments in the overlap and gap regions, and specific connectivities between the molecular segments in successive overlap regions. In addition, we have examined the amino acid sequence and identified features related to the mobility of the molecular segments in the gap region and to the regions where it is thought that molecules are kinked.

Recent studies of the structure of Type I collagen fibrils (Piez and Trus,Biosci. Rep.1:801–810, 1981; Fraser, MacRae, Miller and Suzuki,J. Mol. Biol.167:497–521, 1983) suggest that the segments of the collagen molecule which comprise the gap region are more mobile than those which comprise the overlap region. We have analyzed the distribution of amino acid residues and triplet types between the two regions, and find significantly non-uniform distributions for Ala, Gln, His, Hyp, Leu, Phe, and Tyr, and for triplets containing two imino acid residues. Taken together with the lower packing density in the gap region these observations provide a basis for understanding the greater mobility of the molecular segments in the gap region. In addition, we have examined the linear distribution of residue types in the two regions and also the hydropathy profile (Kyte and Doolittle,J. Mol. Biol.157: 105–113, 1982). These reveal a segment of the gap region comprising helical residues 165–173, 399–407, 633–641 and 867–975 which has the highest hydropathy index, is devoid of charged residues, and contains very high proportions of Ala, Hyp and Phe.

Molecules of intermediate filament (IF) proteins contain a central rod domain in which the two constituent chains have a predominantly α-helical conformation and are coiled around one another to form segments of two-strand rope. Possible interactions between the two long segments, termed 1B and 2 were investigated by a technique successfully employed in studies of the modes of association of collagen molecules by Miller and coworkers. Prominent maxima were found in all of the six possible modes of association between the rod domain segments in individual IF proteins and certain maxima were found to be common to all IF. The surface lattice of the IF from α-keratin has been determined and possible bonding arrangements between the rod-domain segments are catalogued. A systematic search was carried out for combinations of interaction maxima which were consistent with the dimensions of the surface lattice. By the further application of stereochemical constraints, models for the topological arrangement of the rod-domain segments on the surface lattice were derived and these are illustrated and discussed.

In a previous communication (Biosci. Rep. 3, 517–525, 1993) we described quantitative X-ray diffraction studies of α-keratin which were shown to be consistent with the presence of finite arrays of repeating units, successive arrays being set down at axial intervals of 470 Å. In addition the axial interval between repeating units in an array was shown to be 197.9 Å. It was suggested that this could most readily be explained by supposing that a surfacelattice was present which contained a dislocation along a helical path with unit height h = 470 Å and unit twist |t| = 49.1°. The number of repeating units was shown to be in the range 7–9. With 7 repeats the mismatch of the lattice along the dislocation is small and this choice was used to develop a detailed model for the filament. Subsequent studies of molecular interactions have shown however that the coiled-coil rope segments in the rod domain of the molecule are most probably oriented parallel to the dislocation, and so minimization of lattice mismatch may be less important than originally supposed. In the present communication it is shown that the choice of 8, rather than 7, for the number of repeating units yields a model which is more compatible with estimates of the linear density and also provides the basis for a general model for polymorphism in intermediate filament lattices.

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with b = 75.5 Å, β = 93 °, a = bsinβ, a = 90 °, c = n × 668 Å, where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.

Two amino acid sequences from potentially helical fragments of low-sulphur proteins from α-keratin have been analysed computationally and periods 9.4 and 28 residues long noted in the axial disposition of charged residues. Ionic interactions between chains have also been calculated and these indicate a preference for the helical fragments to aggregate in parallel with zero shift between chains in a manner essentially identical to that found for α-tropomyosin.

Quantitative measurements of the intensity of the meridional reflections in the X-ray-diffraction pattern of α -keratin are shown to be consistent with a microfibril structure in which a surface lattice with an axially projected period around 200 Å is subject to a periodic interruption with an axially projected period of 470 Å. Taken in conjunction with recent evidence on the chemical structure of α-keratin and other inter-mediate filaments this finding enables an elaboration to be made of a model proposed earlier by RDB Fraser, TP MacRae, & E Suzuki (3. Mol. Biol.108, 435–452, 1976.) for the α-helical framework of the microfibrii. The disposition and connectivity of the helical segments suggested here provides a straightforward explanation of a number of recent physicochemical and electron-microscopical observations on intermediate filaments and provides a starting point for the development of models for the framework of other intermediate filaments.

High-resolution low-angle meridional and near-meridional X-ray diffraction data have been collected from specimens of α-keratin and various heavy-atom derivatives of α-keratin. The observed reciprocal spacings of the layer lines satisfy the selection rule Z=m/h+n/P+s/Pd