Structure and orientation of collagen fibres in human mitral valve

The structure and distribution of collagen fibres in chordae tendineae, anterior leaflet and annulus fibrous of human mitral valve has been investigated using high and small angle X-ray diffraction. The molecular packing of collagen in native mitral valve components is very similar to that in native rat tail tendon. The distribution and orientation of collagen fibres in unstretched and stretched specimens has been deduced by the arcing of the high and small angle meridional reflections. Collagen fibres, which are aligned along the chordae tendineae, are preferentially distributed along the branchings of the chordae into the anterior leaflet and then course towards the annulus fibrous. However, in the anterior leaflet a considerable amount of collagen fibres are organized in a tridimensional isotropic network even after high deformation of the tissue.     

Low angle X-ray diffraction studies on stained rat tail tendons

Low angle X-ray diffraction patterns of rat tail tendons with heavy metal stains added were examined to help clarify the effects of fixation and staining on collagen fibrils. Fixing and staining of rat tail tendon fibers gives an X-ray pattern with an intensified 3.8 nm row and the preservation of most equatorial features found in the native pattern. The presence of the native pattern features suggests the value of fixation in preserving native structure before staining. Staining of rat tail tendon fibers without prior fixation led to the disappearance of the native equatorial features and the appearance of a new broad row line corresponding to a spacing of around 10.0--17.5 nm. This observation suggests that some alteration has taken place in the native structure and may be related to electron microscopic observations of units of 10.0--20.0 nm in collagen fibrils under some disruptive or developmental conditions.     

X-ray diffraction of reconstituted collagen fibers

The X-ray diffraction of fibers reconstituted from purified rat tail tendon collagen has been compared with that of native rat tail tendon. The axial structure is very similar in the two specimens, while the ordered lateral array found in the native state is only poorly reproduced in the reconstituted fiber. Thus, the axial order is determined by the collagen molecules alone, while the native lateral packing may depend, in part at least, on other tissue components.     

Phasing the meridional diffraction pattern of type I collagen using isomorphous derivatives

The meridional X-ray diffraction pattern of wet rat tail tendon contains information about the one-dimensional structure, or axial projection of electron density distribution of the type I collagen fibril. Using synchrotron radiation we have determined the intensities of the first 50 meridional X-ray diffraction reflections. The approach of isomorphous addition with reagents, selected using criteria of chemical reactivity, which label at fewer sites than the stains used in previous studies was applied to phase these 50 reflections to produce a one-dimensional electron density distribution map of a single D-repeat of the collagen fibril. This method is not model-dependent and thus constitutes the first unambiguous determination of the meridional phases of type I collagen.     

X-ray diffraction analysis of collagen fibers processed with glutaraldehyde and glycerol

Rat tail tendon collagen fixed with glutaraldehyde and treated with glycerol has been studied by X-ray diffraction technique. The evaluation of the distribution of the areas at higher and less density of molecular packing in the collagen fibrils has been carried out through the analysis of the intensity distribution of the low angle X-ray diffraction maxima. The results show that this treatment usually employed in the freeze-etching technique induces a modification of the degree of order in specific regions inside the axial period D.     

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.     

Structure and packing of microfibrils in collagen

X-ray diffraction, patterns suggest that the five-stranded microfibrils in the collagen of rat tail tendon are supercoiled and packed together on a square lattice with a statistical distribution of axial displacements between nearest neighbours.     

Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.     

Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation

Differential scanning calorimetry, high and small angle X-ray diffraction analyses have been carried out on air-dried and rehydrated rat tail tendon collagen in order to test the reversibility of collagen thermal denaturation. The mean enthalpy values calculated for the denaturation process of air-dried and rehydrated samples are ΔH_D = 9.0 ± 0.8 cal/g and ΔH_D = 11.9 ±0.7 cal/g respectively, while the denaturation temperatures are T_D = 112 ± 1°C and T_D = 51 ± 1°C. Partial reversibility of the coiled coil—random coil process can be obtained by storing the samples in air or more rapidly by equilibration in water. After denaturation air-dried collagen fibres recover not only their molecular structure but also their characteristic fibrillar structure. The latter does not greatly influence the mean experimental enthalpy values.     

Glutaraldehyde-induced changes in the axially projected fine structure of collagen fibrils

The fine structure of the collagen fibril, as seen in axial projection, is changed by treatment with glutaraldehyde. The changes are detectable in electron-optical staining patterns and in the intensities of the low-angle meridional X-ray diffraction maxima. Current knowledge of the amino acid sequence of collagen and of the axial arrangement of molecules in fibrils permits interpretation in terms of specific alterations to the axial distribution of electron density along the fibril. Analysis of fibril staining patterns from glutaraldehyde-treated calf skin collagen shows that uptake of staining ions in positive staining patterns is inhibited at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) and on other charged residues in the immediate neighbourhood of the glutaraldehyde-reactive residues. This can be seen as a "stain-exclusion effect" due to the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites. Such stain exclusion accounts for the drastic changes in the negative staining pattern following treatment with glutaraldehyde. The intensity changes observed in the low-angle meridional X-ray reflections from rat tail tendon, similarly treated, also can be explained by the presence of these bulky complexes. Existing data have been used to predict a model of the altered electron density profile indicating the axial distribution of glutaraldehyde along a D-period of moist tendon collagen.     

The variability in type I collagen helical pitch is reflected in the D periodic fibrillar structure

The variability in amino acid axial rise per residue of the collagen helix is a potentially important parameter that is missing in many structural models of fibrillar collagen to date. The significance of this variability has been supported by evidence from collagen axial structures determined by electron microscopy and X-ray diffraction, as well as studies of the local sequence-dependent conformation of the collagen helix. Here, sequence-dependent variation of the axial rise per residue was used to improve the fit between simulated diffraction patterns derived from model structures of the axially projected microfibrillar structure and the observed X-ray diffraction pattern from hydrated rat tail tendon. Structural models were adjusted using a genetic algorithm that allowed a wide range of structures to be tested efficiently. The results show that variation of the axial rise per residue could reduce the difference metric between model and observed data by up to 50%, indicating that such a variable is a necessary part of fibril model structure building. The variation in amino acid translation was also found to be influenced by the number of proline and hydroxyproline residues in the triple helix structure.     

Variations in collagen fibril structure in tendons

Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th–18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd–8th) and in the higher orders (29th–52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th–18th orders appear not to be related to features within the triple-helical domain of the molecule. Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify. Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.     

Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I.The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1α, 2α and 3α chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/ overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.     

The tensile properties and mode of fracture of elastoidin

The tensile properties and mode of fracture of elastoidin, a collagenous protein fibre from the fins of sharks, were compared with those of rat tail tendon fibres, considered to be a pure form of collagen. Elastoidin fibres were stronger than tendon in the dry state whereas the opposite was observed for fibres tested in the wet state. However, elastoidin was stiffer than tendon whether dry or wet. Scanning electron micrographs of the crosssections and fractured surfaces revealed that elastoidin fibres consisted of fibrils of varying diameter arranged in a lamellar fashion. From the nature of the fractured surfaces, it could be deduced that the primary failure mechanism for elastoidin was probably through a fissuring of the structure.     

Axial electron density of human scleral collagen. Location of proteoglycans by x-ray diffraction.

The low angle meridional x-ray diffraction pattern from fresh human sclera was analyzed to ascertain if collagen-bound proteoglycans affect the axially-projected electron density distribution to the same extent as appears to occur in the cornea. The results showed that, unlike cornea, the electron density of the sclera is similar to that seen in rat tail tendon collagen. The proteoglycans were specifically stained using either Cuprolinic blue or Cupromeronic blue, both under critical electrolyte conditions. The tissue was then examined by electron microscopy and by low angle x-ray diffraction. The electron-optical observations suggested that proteoglycans associate with collagen near the d/e staining bands in the gap zone. A difference Fourier analysis from the x-ray results confirmed that these observations were not e.m. preparative artefacts and allowed a quantitative estimate to be made of the axial extent of the proteglycans in the wet tissue.     

Torsion of tendon fibers upon hydration and temperature shifts

Hydration of an isolated rat tail tendon fiber was found to cause its torsion. A similar effect was observed upon changing the specimen temperature in the 12–38°C range. The direction and the angle of rotation of the distal end of the fiber did not depend on its length (12–80 mm). Rather, they depended on the prevalence of clockwise-or counterclockwise-driving collagen units, the distribution of which in the tendon fiber was apparently probabilistic. The phenomenon of collagen bundle rotation is considered in the context of the mechanism of mechanoreceptor stimulation by temperature shifts.     

Calculated X-ray diffraction pattern from a quasi-hexagonal model for the molecular arrangement in collagen

The near-equatorial region of the medium-angle X-ray diffraction pattern from native rat tail tendon contains sharp reflections which indicate that the collagen molecules are arranged in a crystalline manner within the fibrils. A successful indexing of these reflections would indicate that crystallographic unit cell in the fibrils while the intesities of the reflections are determined by the arrangement of the collagen molecules within a unit cell. It is shown that the quasi-hexagonal model proposed by Hulmes and Miller¹ with slight modifications accounts for the positions of the reflections [i.e. their (R, Z) values]. Previous models used mainly the R-values of the reflections published by Miller and Parry². This model gives a better account of the R-values of the reflections than previous models and, in addition, accounts for the Z-values and the intensities of the reflections. This represents the determination of the three-dimensional structure of the collagen in a native animal tissue, rat tail tendon, to 1 nm resolution.     

Structural modifications of air-dried tendon collagen on heating

The modification of collagen molecular packing as a function of the removal of bound and structural water have been investigated on air-dried rat tail tendon. Isothermal curves, dilatometric measurement, high and small angle X-ray diffraction patterns—recorded using conventional and synchrotron radiaiton sources respectively—have been obtained on samples heated in air at different temperatures up to 200°C. A shortening of collagen intermolecular distances and slight modifications of quaternary structure and fibre dimensions can be observed during the release of bound water. The removal of structural water is accompanied by disordering of the three polypeptide chains, a strong reduction of fibre length and d-axial spacing, and modifications of the electron density distribution inside the repeating period. The structural modifications observed during the removal of bound water and of most of the structural water, obtained on heating, are reversible. Release of the most lightly bound water, probably associated with the beginning of the depolymerization process, induces irreversible modification of the molecular packing.     

Collagen fibril morphology in developing chick metatarsal tendoms: 1. X-ray diffraction studies

The low angle equatorial X-ray diffraction ($\text{R ? 30 μ}\text{m}{}^{\mathrm{?1}}$) from hydrated embryonic chick metatarsal tendon contains minima and maxima that are not seen in mature tendons. This diffraction derives from the disordered array of parallel, cylindrical fibrils of collagen of small, uniform diameter that comprise the major part of this tissue. Comparison of the positions of the minima and maxima with those expected from an array of cylinders allows estimation of the mean diameter of the cylinders and the average centre-to-centre nearest neighbour separation. It was found that in the age range from 13 to 19 days fetal, the mean diameter increased from ～ 46 to ～ 58 nm, whereas the mean nearest neighbour separation remained constant at ～ 90 nm. Detailed analysis of the X-ray intensity profile of a 17 day fetal tendon indicated the presence of a paucidisperse distribution of fibril diameters with two or more discrete populations of preferred diameters separated by 10 to 12 nm.