Interactions of the neural cell adhesion molecule and the myelin- associated glycoprotein with collagen type I: involvement in fibrillogenesis

To gain insights into the functional role of the molecular association between neural adhesion molecules and extracellular matrix constituents, soluble forms of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM), representing most of the extracellular domains of the molecules, were investigated in their ability to modify fibrillogenesis of collagen type I. MAG and N-CAM retarded the rate of fibril formation, as measured by changes in turbidity, and increased the diameter of the fibrils formed, but did not change the banding pattern when compared to collagen type I in the absence of adhesion molecules. Scatchard plot analysis of the binding of MAG and N-CAM to the fibril-forming collagen types I, II, III, and V suggest one binding site for N-CAM and two binding sites for MAG. Binding of MAG, but not of N-CAM, to collagen type I was decreased during fibril formation, probably due to a reduced accessibility of one binding site for MAG during fibrillogenesis. These results indicate that the neural adhesion molecules can influence the configuration of extracellular matrix constituents, thus, implicating them in the modulation of cell-substrate interactions.     

Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration through mimicry of the bone extracellular matrix in vivo. The aim of this study was to compare the collagen types I, II, and III with regard to their ability to bind the small leucine-rich proteoglycans (SLRPs) decorin and biglycan during fibrillogenesis in vitro in phosphate buffer. In addition, the influence of SLRPs on the proportion of collagen molecules incorporated into fibrils during fibrillogenesis in vitro at high and low ionic strength was investigated, as were their effects on the morphology of collagen fibrils and the speed of fibrillogenesis. Considerably more biglycan than decorin was bound by all three collagen types. Collagen II bound significantly more SLRPs in fibrils than collagen I and III. Decorin and biglycan decreased the proportion of collagen molecules of all three collagen types incorporated into fibrils in similar fashion. Biglycan affected neither fibril diameter nor the speed of fibrillogenesis. Decorin reduced the fibril diameter of all three collagen types. The differences in SLRP-binding ability between collagen types could be of significance when selecting collagen type and/or SLRPs as scaffold materials for tissue engineering or implant coatings.     

Collagen I Self-Assembly: Revealing the Developing Structures that Generate Turbidity

Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.     

Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.     

The mammalian chitinase-like lectin, YKL-40, binds specifically to type I collagen and modulates the rate of type I collagen fibril formation

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 x 10(-3) to 4.50 x 10(-3) s(-1)). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.     

Turbidimetric and morphological studies of type I collagen fibre self assembly in vitro and the influence of fibronectin

Collagen undergoes several stages of self assembly including turbidimetric lag, growth and plateau steps. The later stages of type I collagen self assembly were studied by turbidity—time measurements, low angle laser light scattering and by determination of the birefringence retardation of collagen fibres formed in vitro. These studies were conducted in the presence and absence of fibronectin to evaluate the effect of fibronectin on the kinetics and extent of type I collagen fibrillogenesis. The results of these studies indicate that the collagen fibres observed at the end of the lag phase appear to be identical to fibres seen in the growth phase of turbidity—time curves based on fibre diameter and birefringence retardation measurements. Birefringence retardation measurements suggest that the diffracting unit may be the collagen fibril and that the volume fraction of fibrils in fibres is about 0.95 using a model developed for a series of parallel ellipsoids. Morphological observations suggest that the distribution of fibre diameters formed in vitro during the growth phase is narrow and appears to be independent of time with only the mass of collagen in fibres increasing during the growth phase. During the growth phase, layers of parallel fibres are formed with alternating layers appearing almost orthogonal. In the presence of fibronectin the mechanism of fibre formation appeared unchanged. It was concluded that fibronectin appeared to modify the kinetics of self assembly by preventing collisions between collagen molecules.     

Influence of monosaccharides on the fibrillogenesis of type I collagen

The in vitro self-assembly of Type I rat tail tendon collagen in the presence of monosaccharides was studied turbidimetrically. D(+)glucose and D(+)mannose delayed fibril formation significantly while D(-)fructose showed no variation. The effect of glucose was concentration dependent. Binding of glucose during fibrillogenesis was shown by the incorporation of [14C]glucose and also by SDS-polyacrylamide slab gel electrophoresis of the product. The results indicate that in vitro nonenzymatic glycosylation can take place in the short duration required for fibril formation.     

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.     

Fibrillogenesis in dense collagen solutions: a physicochemical study

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength = 150 mM, 20 °C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH ∼ 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.     

COMP acts as a catalyst in collagen fibrillogenesis

We have previously reported that COMP (cartilage oligomeric matrix protein) is prominent in cartilage but is also present in tendon and binds to collagens I and II with high affinity. Here we show that COMP influences the fibril formation of these collagens. Fibril formation in the presence of pentameric COMP was much faster, and the amount of collagen in fibrillar form was markedly increased. Monomeric COMP, lacking the N-terminal coiled-coil linker domain, decelerated fibrillogenesis. The data show that stimulation of collagen fibrillogenesis depends on the pentameric nature of COMP and not only on collagen binding. COMP interacts primarily with free collagen I and II molecules, bringing several molecules to close proximity, apparently promoting further assembly. These assemblies further join in discrete steps to a narrow distribution of completed fibril diameters of 149 +/- 16 nm with a banding pattern of 67 nm. COMP is not found associated with the mature fibril and dissociates from the collagen molecules or their early assemblies. However, a few COMP molecules are found bound to more loosely associated molecules at the tip/end of the growing fibril. Thus, COMP appears to catalyze the fibril formation by promoting early association of collagen molecules leading to increased rate of fibrillogenesis and more distinct organization of the fibrils.     

Effects of decorin proteoglycan on fibrillogenesis,ultrastructure, and mechanics of type I collagen gels

The proteoglycan decorin is known to affect both the fibrillogenesis and the resulting ultrastructure of in vitro polymerized collagen gels. However, little is known about its effects on mechanical properties. In this study, 3D collagen gels were polymerized into tensile test specimens in the presence of decorin proteoglycan, decorin core protein, or dermatan sulfate (DS). Collagen fibrillogenesis, ultrastructure, and mechanical properties were then quantified using a turbidity assay, 2 forms of microscopy (SEM and confocal), and tensile testing. The presence of decorin proteoglycan or core protein decreased the rate and ultimate turbidity during fibrillogenesis and decreased the number of fibril aggregates (fibers) compared to control gels. The addition of decorin and core protein increased the linear modulus by a factor of 2 compared to controls, while the addition of DS reduced the linear modulus by a factor of 3. Adding decorin after fibrillogenesis had no effect, suggesting that decorin must be present during fibrillogenesis to increase the mechanical properties of the resulting gels. These results show that the inclusion of decorin proteoglycan during fibrillogenesis of type I collagen increases the modulus and tensile strength of resulting collagen gels. The increase in mechanical properties when polymerization occurs in the presence of the decorin proteoglycan is due to a reduction in the aggregation of fibrils into larger order structures such as fibers and fiber bundles.     

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1^+/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

The C-terminal extrahelical peptide of type I collagen and its role in fibrillogenesis in vitro: effects of ethylurea

Ethylurea was used to weaken hydrophobic interactions during collagen fibrillogenesis in vitro. Intact and enzyme-digested type I collagen was studied. In all preparations, ethylurea decreased the extent and rate of fibril formation, inhibition being greatest in the enzyme-digested collagens. With intact collagen (and probably also with carboxypeptidasedigested collagen), there was no evidence the ethylurea altered the mechanism of fibril growth; in pepsin-digested collagen, however, the growth mechanism was altered by ethylurea, possibly reflecting a conformational change of the “hydrophobic cluster” in the C-terminal peptide. Such a structural change could occur in a hydrophobic environment once the distal portion of the C-terminal peptide (presumed to be essential for its structural stability) is removed by pepsin. The results further emphasize the importance of hydrophobic interactions in collagen fibril nucleation and growth in vitro.     

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D=67nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.     

Segregation of type I collagen homo- and heterotrimers in fibrils

Normal type I collagen is a heterotrimer of two α1(I) and one α2(I) chains, but various genetic and environmental factors result in synthesis of homotrimers that consist of three α1(I) chains. The homotrimers completely replace the heterotrimers only in rare recessive disorders. In the general population, they may compose just a small fraction of type I collagen. Nevertheless, they may play a significant role in pathology; for example, synthesis of 10-15% homotrimers due to a polymorphism in the α1(I) gene may contribute to osteoporosis. Homotrimer triple helices have different stability and less efficient fibrillogenesis than heterotrimers. Their fibrils have different mechanical properties. However, very little is known about their molecular interactions and fibrillogenesis in mixtures with normal heterotrimers. Here we studied the kinetics and thermodynamics of fibril formation in such mixtures by combining traditional approaches with 3D confocal imaging of fibrils, in which homo- and heterotrimers were labeled with different fluorescent colors. In a mixture, following a temperature jump from 4 to 32 °C, we observed a rapid increase in turbidity most likely caused by formation of homotrimer aggregates. The aggregates promoted nucleation of homotrimer fibrils that served as seeds for mixed and heterotrimer fibrils. The separation of colors in confocal images indicated segregation of homo- and heterotrimers at a subfibrillar level throughout the process. The fibril color patterns continued to change slowly after the fibrillogenesis appeared to be complete, due to dissociation and reassociation of the pepsin-treated homo- and heterotrimers, but this remixing did not significantly reduce the segregation even after several days. Independent homo- and heterotrimer solubility measurements in mixtures confirmed that the subfibrillar segregation was an equilibrium property of intermolecular interactions and not just a kinetic phenomenon. We argue that the subfibrillar segregation may exacerbate effects of a small fraction of α1(I) homotrimers on formation, properties, and remodeling of collagen fibers.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

Type III Collagen,a Fibril Network Modifier in Articular Cartilage

The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an α1(II) and an α1(III) telopeptide to the α1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.     

Collagen fibrillogenesis in tendon development: current models and regulation of fibril assembly

Tendons are collagen-based fibrous tissues that connect and transmit forces from muscle to bone. These tissues, which are high in collagen type I content, have been studied extensively to understand collagen fibrillogenesis. Although the mechanisms have not been fully elucidated, our understanding has continued to progress. Here, we review two prevailing models of collagen fibrillogenesis and discuss the regulation of the process by candidate cellular and extracellular matrix molecules. Although numerous molecules have been implicated in the regulation of collagen fibrillogenesis, we focus on those that have been suggested to be particularly relevant to collagen type I fibril formation during tendon development, including members of the collagen and small leucine-rich proteoglycan families, as well as other molecules, including scleraxis, cartilage oligomeric matrix protein, and cytoskeletal proteins.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.