John A. Chapman

The Staining Pattern of Collagen Fibrils: I. an Analysis of Electron Micrographs

Electron micrographs of calf-skin collagen in the reconstituted native-type fibril and the segmented long spacing (SLS) forms are matched with one another and with the distribution of charged residues in the known α1 chain sequence.It is shown that:a) the pattern of bands in SLS corresponds closely with the charge distribution in the sequenceb) most of the bands in the fibril pattern can be matched in position (but not in intensity) to bands in the SLS pattern, confirming a substantial measure of matching of charged regions with charged regions (and therefore of hydrophobic regions with hydrophobic regions) on assembly of molecules into fibrilsc) when the al chain sequences are regularly staggered by 233 residues (to simulate the postulated packing in a fibril) the greatest concentrations of charged residues occur in regions which correspond closely with the bands in positively stained fibrilsd) the positions of the abrupt contrast steps(the overlaplgap junctions) in the image of a negatively contrasted f...

Reconstitution of collagen fibrils in vitro; the assembly process depends on the initiating procedure

Electron microscopy and turbidimetry have been used to show that the reconstitution of native-type banded fibrils from a solution of acetic acid-soluble collagen (with intact telepeptides) can proceed by different assembly pathways, depending on the initiating procedure used to bring the collagen from a dissolved to a precipitating condition. Initiation requires raising the pH, temperature (T) and ionic strength (I) of the solution (here from pH 3.4, T=4°C, I=0.01 to pH 7.4, T=34°C, I=0.2). A widely used procedure is an initial increase in pH and I, followed by a rise in T (the ‘neutral start’ procedure); this leads, in the early stages, to the mesh of long thin non-banded filaments described by Gelman et al.9. When, however, the initiation steps are performed in reverse order, first raising T and then increasing pH and I (the ‘warm start’ procedure), non-banded filaments are not found as an abundant long-lived intermediate in the assembly process: instead, native-type banded fibrils constitute most of the precipitated material from the earliest stages onwards, and fibril formation takes place more rapidly. The simultaneous raising of T, pH and I (in a third procedure) not only yields banded fibrils throughout the course of precipitation (with no accumulation of filaments) but also results in a further increase in the rate of fibril formation. Filament formation appears therefore to be triggered by exposure to cold neutral solvent during initiation. When exposure to cold neutral solvent is avoided, an alternative and more efficient mode of assembly, involving the simultaneous lateral and axial growth of D-periodic fibrils, occurs.

The Staining Pattern of Collagen Fibrils: Ii. a Comparison With Patterns Computer-Generated From the Amino Acid Sequence

The known amino acid sequence of the α1 chain of collagen enables the distribution of electric charges along the molecule to be predicted (with the assumptions that the residues, including those in the telopeptides, are uniformly spaced and that the charge distribution in the α2 chain is similar to that in the α1). By summing this distribution for a set of molecules, mutually staggered by p residues, a histogram showing the predicted distribution of charge along a fibril can be generated. Histograms constructed in this way were compared with densitometric tracings taken from electron micrographs of band patterns from collagen fibrils doubly stained with metal-containing cations and anions. This comparison was made for various values of p, and it was extended to histograms artificially smoothed in an attempt to observe histograms and tracings at comparable resolutions. In this way p, which in the fibril is the number of residues per chain contained in the period D, can be deduced from the comparison yieldi...

Electron microscopy of the collagen fibril

Collagen is identified by those properties that stem from the predominantly triple-chain helical structure of its molecules. A prerequisite for the formation of this triple helix is a Gly-X-Y repeating tripeptide unit in the amino acid sequence of the three chains, where X and Y can be any amino acids but are often the imino acids proline and hydroxyproline. This sequence, with glycine in every third position and with an unusual abundance of hydroxyproline, forms the basis for the chemical identification of collagen (for review, see 1). An unambiguous physical identification is provided by X-ray diffraction; the helix parameters established by high-angle X-ray scattering are unique to collagen (2).

Mica sandwich technique for preparing macromolecules for rotary shadowing

The sandwich technique, in which a drop of sample solution is spread into a thin layer between two pieces of freshly cleaved mica, is a simple-to-use alternative to spraying for depositing macromolecules onto mica. Test specimens of collagen molecules and actin filaments were found to suffer less shear-induced damage, they were more uniformly distributed, and only very small sample volumes were needed. Either drying from a glycerol solution (40-70% v/v) or freeze-drying can be employed. Glycerol-drying is simpler, but freeze-drying may offer better preservation of supra-molecular assemblies.

Pleomorphism in type I collagen fibrils produced by persistence of the procollagen N-propeptide

The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.

Vertebrate (chick) collagen fibrils formed in Vivo can exhibit a reversal in molecular polarity

A reversal in molecular polarity can occur in vertebrate collagen fibrils. This has been demonstrated using a method for isolating, from chick embryo tendon, entire collagen fibrils 2 to 14 microns in length and suitable for electron-optical examination. A polarity reversal is present in some, but not all, of these fibrils. Such fibrils have two N-ends. The transition region, occupying several D-periods in which the reversal occurs, is not restricted to a central location in a fibril. Analysis of the fibril banding pattern through the transition region shows that the relative axial alignment of antiparallel molecules brings oppositely-directed C-telopeptides into axial register. This could allow antiparallel molecules to be covalently linked via polymeric cross-links involving these C-telopeptides.

Isolation of the membrane-mesosome structures from micrococcus lysodeikticus

Micrococcus lysodeikticus cells possess a membranous organelle of about 2500 A diameter. This structure is similar to those found in other bacteria and the term “mesosome”, which was proposed by Fitz-James to describe such organelles, has been adopted here. The mesosomes of Micrococcus lysodeikticus have been isolated by differential centrifugation of lysed protoplasts and from lysates prepared by dissolution of the wall with lysozyme. The isolated mesosomes are composed of a series of concentric shells, each membrane being double and having an over-all thickness of about 75 A. There are probably four concentric membranes forming the mesosome particle which on isolation is frequently surrounded by the plasma membrane forming a fifth shell. The maximum number of concentric membranes observed in the isolated membrane-mesosome fractions was 5. The present procedure does not permit a differential separation of plasma membrane from mesosome and it is concluded that previous protoplast membrane preparations of this organism contained the mesosome structures.

A study of positive staining for electron microscopy using collagen as a model system—I. Staining by phosphotungstate and tungstate ions

Abstract Present knowledge of the amino acid sequences of the polypeptide chains of the collagen molecule and of the regular axial alignment of the molecules in fibrils makes possible a direct comparison of electron-optical staining data with chemical data. In this way, collagen can be used as a model system to study the effects of stains and other reagents on a protein. The first paper describes an analysis of the staining produced by phosphotungstate and tungstate ions at pH 3.2. A computer-aided correlation procedure was used to compare averaged electron-optical data from reconstituted fibrils of type I calf skin collagen with chemical sequence data. The results confirm that, under the conditions used, phosphotungstate and tungstate can be regarded as anionic stains which react with the positively-charged side-chains on collagen. A significantly higher correlation with the positive charge distribution can be achieved with tungstate. The prior removal by dialysis of phosphate ions (used to reconstitute the fibrils) enhances subsequent staining.

A study of positive staining for electron microscopy using collagen as a model system—II. Staining by uranyl ions

Abstract The staining behaviour of uranyl salts was studied using reconstituted periodic-banded collagen fibrils as a model system and comparing electron-optical data and collagen sequence data by a computer-aided correlation procedure. Because it is only weakly dissociated and a variety of ionic uranyl complexes (cationic, neutral and anionic) coexist in aqueous solution, uranyl acetate stains not only negatively-charged but also positively-charged side-chains on the collagen. The relative uptake on charged groups of opposite sign depends on several factors, including the concentration and pH of the staining solution and the presence of extraneous phosphate groups (from the buffer used to reconstitute the fibrils), but no conditions could be found which allowed uranyl acetate to stain negative charges only. When the stain was uranyl nitrate, known to dissociate strongly in aqueous solution, uptake of uranyl ions was predominantly (although not exclusively) on negative charges on the collagen, provided that extraneous phosphate had previously been removed by dialysis.