bromochloroacetic-acid and disuccinimidyl-tartarate

The spatial distribution of the crosslinks that can be induced between lysine residues in trichocyte (alpha-) keratin intermediate filaments (IF) using disulfosuccinimidyl tartrate has been analyzed in detail and the results used to provide information about the three-dimensional (3-D) structure. The pattern of inter-molecular interactions derived from earlier studies is essentially two-dimensional in that it involves projection on to a cylinder followed by unwrapping to give a sheet. Crosslinks are observed between molecular strands four apart and it is shown that this can only occur if the paths of the molecular strands through the IF are systematically distorted. These crosslinks are clustered axially at intervals of around 15 nm, a value closely related to the pitch length of the constituent coiled-coil molecules in the rod domains. The number of crosslinks between adjacent molecular strands shows a striking difference depending on lateral direction and provides support for the concept of a head-to-tail stacking of tetramers defined by the A(CN) mode of packing to form protofilament substructures in the fully formed IF. Each protofilament would consist of a pair of oppositely directed molecular strands stabilized by A(11) and A(22) interactions identified in earlier work. A detailed model for the IF in the reduced state comprising a ring of eight protofilaments is suggested. When combined with earlier studies of crosslink formation in the oxidized state, the present findings lead to the conclusion that there is a major reorganization of the molecular packing within the protofilaments during keratinization in vivo. Taken in conjunction with existing X-ray data on the fully keratinized structures, the new evidence for a protofilament substructure also enables a detailed 3-D model for the mature IF to be suggested.

Topics: Animals; Cross-Linking Reagents; Fourier Analysis; Imaging, Three-Dimensional; Intermediate Filaments; Keratins; Lysine; Models, Structural; Oxidation-Reduction; Protein Structure, Tertiary; Succinimides; X-Ray Diffraction

One of the major obstacles to solving the full three-dimensional structure of keratin intermediate filaments (KIF) is the determination of the exact mode(s) of alignment of nearest-neighbor molecules; this in turn requires precise information of the lengths of the non-alpha-helical linker segments within the coiled-coil alpha-helical heterodimer molecule. In this study, we have induced lysine-lysine and cysteine-cysteine crosslinks between keratin intermediate filament molecules in small assembly-competent oligomers, isolated them and then characterized the natures and locations of the crosslinks. Of more than 100 found, 21 quantitatively major crosslinks were used to obtain the relative axial alignments of rod domain segments by least-squares fitting methods. Three dominant modes of alignment were found. In each case the molecules are antiparallel with the first involving molecules in approximate register (stagger = -0.2 nm), the second involving molecules staggered so as to bring the 1B segments into approximate alignment (stagger = -16.1 nm), and the third involving molecules staggered so as to bring the 2B segments into approximate alignment (stagger = 28.2 nm). In addition, the data enable quantitative estimates to be made for the first time of the lengths of the non-coiled-coil segments (L1 = 2.5 nm, L12 = 1.6 nm, L2 = 0.8 nm), and the total length of the rod domain (46.0 nm). Alignment of molecules according to these parameters permits construction of a two-dimensional surface lattice which displays a 1.6 nm (10 or 11 residue) overlap between similarly directed molecules. Together, the data predict six important overlapping sequence regions that recur about 16 times per 46 nm of filament length. Interestingly, synthetic peptides corresponding to these sequences, singly or in combination, significantly interfere with keratin filament structural integrity. These results thus represent the most significant set of structural constraints for KIF yet available and provide insights into how disease-causing mutations disrupt filaments and their organization in cells.

Topics: Amino Acid Sequence; Animals; Animals, Newborn; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Intermediate Filaments; Keratins; Mice; Mice, Inbred BALB C; Microscopy, Electron; Molecular Sequence Data; Peptide Fragments; Peptides; Skin; Succinimides; Trypsin

During development and differentiation, the intermediate filament component of the cytoskeleton of many cells and tissues is rebuilt by a dynamic exchange process in which one set of protein chains is replaced by another, without recourse to creation of a new network. One major example is the replacement of keratin 5/keratin 14 (K5/K14) keratin intermediate filaments (KIFs) by K1/K10 KIFs during terminal differentiation in the epidermis. The present work was undertaken to explore how this may occur. We have induced lysine-lysine cross-links with disulfosuccinimidyl tartrate in K5/K14 KIFs in order to determine the axial dimensions and relative axial alignments of the K5/K14 molecules. Many of the cross-links induced in subfilamentous oligomers containing one, two, or three molecules were also found in the intact KIF, indicating that the body of data thus generated provides physiologically relevant information on the structural organization in the KIF. A least-squares analysis using as data the positions of lysine residues involved in 23 induced cross-links has allowed the axial alignments of the various coiled-coil segments in the rod domain to be determined. Three modes of antiparallel alignment of two neighboring molecules were found: A11 (staggered by -16.7 nm), A22 (staggered by 28.8 nm), and A12 (almost in register; staggered by only 0.3 nm). Since the axial repeat length is about 1 nm less than the molecular length, the data require a fourth mode of molecule alignment, termed ACN, in which similarly directed molecules are overlapped by the equivalent of about 5-10 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Cell Differentiation; Chromatography, High Pressure Liquid; Conserved Sequence; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Epidermis; Humans; Intermediate Filaments; Keratins; Macromolecular Substances; Male; Microscopy, Electron; Models, Structural; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Succinimides; Trypsin