Strain-induced reorientation of an intramuscular connective tissue network: implications for passive muscle elasticity

The most abundant intramuscular connective tissue component, the perimysium, of bovine M. sternomandibularis muscle was shown to be a crossed-ply arrangement of crimped collagen fibres which reorientate and decrimp on changing muscle fibre sarcomere length. Reorientation of perimysial strands was observed by light microscopy and identification of these strands as collagen fibres was confirmed by high-angle X-ray diffraction. Mean collagen fibre direction with respect to the muscle fibres ranged from approximately 80 degrees at sarcomere length = 1.1 micron to approximately 20 degrees at 3.9 microns. This behaviour was well described by a model of a crimped planar network surrounding a muscle fibre bundle of constant volume but varying length. Modelling of the mechanical properties of the perimysium at different sarcomere lengths produced a load-sarcomere length curve which was in good agreement with the passive elastic properties of the muscle, especially at long sarcomere lengths. It is concluded that the role of the perimysial collagen network is to prevent over-stretching of the muscle fibre bundles.     

Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration.

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.     

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.     

Vitrosin: a member of the collagen class

Vitrosin, a fibrous protein obtained from the vitreous humor of the eye in the form of an indefinitely long fibril about 100 to 150 A in diameter, has been identified as a member of the collagen class of proteins. It is characterized by the collagen wide-angle x-ray diffraction pattern, and axial periodicity of about 640 A determined by electron microscopy and small-angle x-ray diffraction, an amino acid pattern characteristic of collagen as determined by paper chromatography, and a hydroxyproline and glycine content also typical of collagen. The glycine-hydroxyproline ratio is somewhat lower than that for most vertebrate collagens.     

Cuticlin: a noncollagen structural protein from Ascaris cuticle

A structural protein was isolated from the cuticle of Ascaris lumbricoides and studied by chemical analyses, electron microscopy, and X-ray diffraction. It has high contents of proline and alanine and relatively low contents of glycine and basic amino acids. It does not give the characteristic wide-angle X-ray diffraction pattern of collagen. It is not susceptible to bacterial collagenase. In these respects, it is distinct from collagen. The name “cuticlin” is proposed for this protein.     

Structural study of the calcifying collagen in turkey leg tendons

The calcified turkey leg tendon represents a simple bone-like tissue that is ideally suited to analysis by diffraction methods. In this paper we report some structural studies of the tendon collagen in the uncalcified, fully calcified and partially calcified states. The low-angle meridional X-ray pattern from the uncalcified tendon is very similar to that of the rat tail tendon, and the resulting one-dimensional structure of the collagen fibril exhibits no feature that could be related to its eventual calcification. The structure of the fully calcified tendon, as determined by a combination of X-ray and neutron diffraction analyses, shows that the mineral is associated with the collagen at the level of the hole or gap region. In the calcifying tendon, increases in the amplitudes of the first and second X-ray meridional reflections are correlated with an increase in the mineral content of the collagen. On the basis of simple models, it is shown that this change in the pattern can be explained by a nucleation mechanism of calcification. It is concluded that when collagen becomes calcified the mineral penetrates throughout the fibril and is crystalline in the hole region but amorphous between the collagen molecules. The mechanism of calcification and the mechanical implications of the fully calcified structure are also discussed.     

A generalized packing model for type I collagen

Only tail tendon (TT) collagen has a sharp X-ray diffraction pattern, so that packing models for the equatorial arrangement of molecules in collagen fibrils have been developed primarily for TT collagen. A more general structure is developed applicable to all type I collagen tissues. Comparison of water content-equatorial diffraction spacing plots of several collagens shows all have essentially the same dry state diffraction spacing but differ as water content increases. TT collagen has the least spacing and the sharpest pattern. The interplanar spacing of the Hulmes-Miller quasi-hexagonal model for TT collagen was used to calculate the intermolecular spacing, which matched the observed diffraction spacing for bone matrix collagen. It is inferred that wet bone matrix collagen packs in a rectangular pattern because of the interaction between the many intermolecular crosslinks and the water absorbed on the collagen molecules. This argument also indicates that TT collagen packs into a quasi-hexagonal scheme because there are fewer intermolecular crosslinks than in bone matrix collagen.     

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.     

Twisted plywood pattern of collagen fibrils in teleost scales: an X-ray diffraction investigation

The distribution and orientation of collagen fibrils, and apatite crystals, in the scales of a bony fish (Leuciscus cephalus) were investigated by X-ray diffraction. The small-angle diffraction patterns obtained with a microfocus scanning setup from most of the examined areas exhibit a distribution of intensity of the collagen reflections according to five preferential orientations, at 36 degrees from one another. It is suggested that the peculiar small-angle X-ray diffraction pattern is due to a plywood arrangement of collagen fibrils in successive layers parallel to the surface of the scale. The fibrils are strictly aligned in each layer and the alignment rotates by 36 degrees in successive layers, according to a discontinuous twist that generates a symmetric plywood pattern. The large spread of the wide-angle reflections does not allow one to distinguish the five directions of orientation in the intensity distribution of the 002 reflection of apatite. However, the patterns recorded from the less ordered regions of the scales display two different orientations of the 002 reflection and allow one to infer a preferential distribution of the apatite crystals with their c-axes parallel to the collagen fibrils. Although much electron microscopic evidence of plywood arrangements in calcified, as well as uncalcified, tissues has been reported, these are the very first diffraction data which unambiguously confirm the presence of these peculiar structures and suggest that this kind of investigation represents a powerful tool with which to study plywood arrangements in biological tissues.     

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.     

Synchrotron X-ray diffraction study of corneal stroma

Using a synchrotron X-ray source, it has been possible to record a low-angle diffraction pattern from fresh bovine corneal stroma.The pattern can be interpreted as arising from the short-range order packing of collagen fibrils in lamellae. Model calculations suggest that the positions of the fibrils remain correlated over distances corresponding to, at most, three fibril diameters (~ 120 nm). These results support theories of transparency of the cornea based on short-range order.Further, a study of the fibril spacing as a function of hydration confirms that water uptake occurs largely between the lamellae and in regions devoid of collagen fibrils, and shows that the fibril diameter increases with hydration.     

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.     

Short and long range order in basement membrane type IV collagen revealed by enzymic and chemical extraction

Oriented bovine lens capsules give X-ray diffraction patterns suggesting a considerable degree of order in the collagenous components, predominantly type IV collagen. Here we report the effects of preliminary treatment of lens capsules before orientation. Extraction with 4 guanidinium hydrochloride or with heparinase/hyaluronidase reveals the same collagenous diffraction patterns previously seen after extraction with 1 NaCl. There is a four-point pattern of d-spacing 3.9 nm, indicating liquid crystal cybotactic nematic organization, along with sharp streaked meridional reflections which index as orders of 21 nm. This suggests that the removal of basement membrane proteoglycans results in a reduction in diffuse scatter and clarification of the pattern. Extraction of the lens capsules with trypsin or dithiothreitol greatly reduces the intensity of the four-point pattern while leaving the meridional pattern unaffected. This strengthens the evidence that the 21 nm period has its origins in the collagen IV helix. Reduction in the four-point pattern could arise if disruption of non-helical NC1 domains or 7S overlap regions allows slippage of the collagen molecules on orientation, weakening the proposed 1 nm intermolecular stagger. Ultra-low angle diffraction patterns of extracted lens capsules show meridional reflections which index as a long-range axial repeat of approximately 95 nm. This is consistent with a model of microfibrils of type IV collagen in which the NC1 domains bind to the collagen helix at approximately 100 nm intervals, as has been previously suggested.     

X-ray diffraction studies on the structure of hydrated collagen

The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.     

Closest packing of two-stranded coiled-coils as a model for the collagen fibril.

We propose that in the collagen fibril, the triple-helical molecules form two-stranded coiled-coils of period 5 × 670A?. Coiled-coils are packed on a tetragonal lattice and are axially staggered with ten in the unit cell (observed side 55A?) so that it carries the 670A?periodicity of the fibril. When nearest neighbours have opposing supercoil hands, the observed tetragonal lattice represents closest packing of two-stranded coiled-coils. This proposal is consistent with the row line spacings measured from the low angle X-ray diffraction pattern of tendon and explains the systematic absences and the two undisputed equatorial reflections. Unlike explanations for the diffraction pattern which invoke a five-stranded microfibril, our interpretation is consistent with its equatorial intensity distribution.     

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.     

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae.Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found.A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.     

Molecular organization of type IV collagen: polymer liquid crystal-like aspects

A new X-ray diffraction pattern from type IV collagen is described, which can be interpreted on the basis of crystalline and liquid crystalline origins of the reflections. Bovine anterior lens capsules extracted with 1 NaCl and oriented by extension of 60% under constant load gave medium angle X-ray diffraction patterns showing many of the characteristics typical of liquid crystals. Prominent features, apart from those wide angle features attributable to the collagen triple helix, are (1) a four-point pattern of broad reflections at d-spacing 3.9 nm, and layer line spacing near 5 nm. (2) A broad intense equatorial peak centred at 1.24 nm, indicative of LIQUID=like lateral molecular associations (3) A set of five sharp, streaked meridional reflections (previously obscured by the broad peak near 5 nm in unextracted capsules). (4) A further six higher angle reflections of a diffuse, arced and broad appearance on the meridian. The sharp streaked meridional reflections emanate from a long-range periodicity of units 8–9 nm in diameter. These features form a self-consistent system if interpreted on the basis of staggered liquid crystal-like array of collagen molecules, in which case the first five meridionals and remaining broad reflections, sampled on the meridian, can all be indexed as orders of 21 nm.     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.