Estimation of the binding force of the collagen molecule-decorin core protein complex in collagen fibril

Decorin belongs to the small leucine proteoglycans family and is considered to play an important role in extracellular matrix organization. Experimental studies suggest that decorin is required for the assembly of collagen fibrils, as well as for the development of proper tissue mechanical properties. In tendons, decorins tie adjoining collagen fibrils together and probably guarantee the mechanical coupling of fibrils. The decorin molecule consists of one core protein and one glycosaminoglycan chain covalently linked to a serine residue of the core protein. Several studies have indicated that each core protein binds to the surface of collagen fibrils every 67 nm, by interacting non-covalently to one collagen molecule of the fibril surface, while the decorin glycosaminoglycans extend from the core protein to connect to another decorin core protein laying on adjacent fibril surface. The present paper investigates the complex composed of one decorin core protein and one collagen molecule in order to obtain their binding force. For this purpose, molecular models of collagen molecules type I and decorin core protein were developed and their interaction energies were evaluated by means of the molecular mechanics approach. Results show that the complex is characterized by a maximum binding force of about 12.4 x 10(3) nN and a binding stiffness of 8.33 x 10(-8) N/nm; the attained binding force is greater than the glycosaminoglycan chain's ultimate strength, thus indicating that overloads are likely to damage the collagen fibre's mechanical integrity by disrupting the glycosaminoglycan chains rather than by causing decorin core protein detachment from the collagen fibril.     

The regulation of size and form in the assembly of collagen fibrils in vivo

A possible mechanism for regulating the lateral growth of collagen fibrils in vivo is considered. A growth inhibitor associated with a particular part of the long semiflexible collagen molecule restricts that part of the molecule to the surface of the growing assembly. Lateral accretion ceases when these inhibitors form a complete circumferential layer around the fibril surface. Cell-mediated removal of the inhibitors allows lateral growth to proceed to a second limiting layer, and so on to subsequent limiting layers. In this way, cycles of inhibitor removal and limited lateral accretion permit growth to be synchronized over large populations of fibrils. Observed diameter distributions in bundles of embryonic and neonatal fibrils are those expected from a mechanism of this kind. The mechanism depends on the existence of axial order (D-periodicity) in fibrils, but not on any specific lateral packing of molecules. Rather, contacts between newly assembled molecules are presumed to be partly fluid-like in lateral directions (except where covalent cross-links have formed). Some initial fluidity in lateral packing prior to cross-linking does not preclude the subsequent emergence of quasi-crystalline packing as cross-links form. The cylindrical shape of fibrils in vivo may also be attributable in part to fluidity of intermolecular contacts at the growing surface.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

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.     

New insight into the shortening of the collagen fibril D-period in human cornea

Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066–.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Collagen fibril structure is affected by collagen concentration and decorin

Collagen fibrils were obtained in vitro by aggregation from acid-soluble type I collagen at different initial concentrations and with the addition of decorin core or intact decorin. All specimens were observed by scanning electron microscopy and atomic force microscopy. In line with the findings of other authors, lacking decorin, collagen fibrils undergo an extensive lateral association leading to the formation of a continuous three-dimensional network. The addition of intact decorin or decorin core was equally effective in preventing lateral fusion and restoring the normal fibril appearance. In addition, the fibril diameter was clearly dependent on the initial collagen concentration but not on the presence/absence of proteoglycans. An unusual fibril structure was observed as a result of a very low initial collagen concentration, leading to the formation of huge, irregular superfibrils apparently formed by the lateral coalescence of lesser fibrils, and with a distinctive coil-structured surface. Spots of incomplete fibrillogenesis were occasionally found, where all fibrils appeared made of individual, interwined subfibrils, confirming the presence of a hierarchical association mechanism.     

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.     

X-ray diffraction study into the effects of liming on the structure of collagen

The manufacture of parchment from animal skin involves processes that remove hair, fats, and other macromolecules. Although it is well understood that the collagen fibers "open up" during processing, this study uses small and wide-angle X-ray diffraction to measure quantitatively the changes induced at the nanoscopic and microscopic levels. The axial rise per residue distance within the collagen molecules is unaffected by salt and lime treatments. Salting of the hides appears to remove noncollagenous materials. The intermolecular lateral packing distance between the hydrated collagen molecules (1.4 nm) increases after salting ( approximately 1.5 nm) and liming ( approximately 1.55 nm); drying is responsible for a reduction to approximately 1.2 nm in all samples. The axial staggered array (d spacing) is reduced by 1 nm after liming and is unaffected by drying. The average fibril diameter increases from 103.2 to 114.5 nm following liming, and the fibril-to-fibril distance increases from 122.6 to 136.1 nm.     

The precision of lateral size control in the assembly of corneal collagen fibrils

Collagen fibrils in the corneal stroma have been recognised to have a high degree of uniformity of diameter and spatial arrangement compared with those in other mature connective tissues. The precision of this lateral size control has been determined in this study by mass per unit length measurements on fibrils isolated from adult bovine corneal stroma. At the molecular level, however, there are substantial variations in lateral size, both between fibrils and along individual fibrils. The mean mass per unit length was measured to be 304 kDa nm(-1), equivalent to 347 collagen molecules in transverse section and had a standard deviation of 8.3%. The variation of lateral size along individual fibrils was measured as a mass slope over approximately 7 microm lengths (100 D-periods) and had a mean mass slope equivalent to 0.56 molecules per D-period. Smoothly tapered tips of length approximately 7 microm were also observed with a mass slope of about approximately three molecules per D-period. The frequency of these tips was used to estimate a mean fibril length of approximately 940 microm in the sample tissue. Observations of molecular polarity within the fibril shafts and tips were used to consider possible models of fibril assembly.     

Mineralization of type I collagen

It was previously found that the lateral spacing of the collagen molecules in wet mineralized tissues is exactly proportional to the inverse wet density. Several properties were investigated and the same type of relationship was observed each time. A possible explanation is offered. It is hypothesized that mineral is deposited initially in the extrafibrillar space so as to isolate the fibrils. Further deposition reduces the net free fibril volume thereby decreasing the spacing between collagen molecules. The linear relationship is derived from density considerations together with limitations on the collagen packing structure described as the generalized packing model. Three experimental situations were studied: lateral spacing wet tissue versus density; lateral spacing dry tissue versus density; and lateral spacing versus water content. The observed variations of the spacing can be attributed to a structure where the mass of the tissue remains constant but the volume decreases linearly with increasing mineral content.     

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.     

The intermolecular space of reconstituted collagen fibrils

The extent, geometry and heterogeneity of the intermolecular space of hydrated, purified and reconstituted steer skin collagen fibrils has been characterized. The extent of the space has been assessed experimentally by an X-ray diffraction method and a new physical chemical technique, and found to be 1.14 ml per gram collagen. A theoretical model relating the intermolecular space to X-ray diffraction parameters has been presented, and this suggests that the geometry of the intermolecular space arises from a near-hexagonal packing of the collagen molecules. On the basis of an assumed microfibrillar packing model and a geometric construction of the shape of a collagen molecule, the distribution of the space within reconstituted collagen fibrils has been characterized as follows: 0.13 ml of the intermolecular space/g collagen can be attributed to the helical groove of the collagen molecules per se and 1.01 ml/g is interstitial; 0.66 ml/g is present in the form of “pores” (hexagonally-closed packed spaces), whereas 0.48 ml/g is present in the form of “holes” (hexagonal volume defects); 0.73 ml/g of the intermolecular space is associated with a region of the collagen fibrils where holes are localized and 0.41 ml/g is attributable to the regions of the fibril in which pores only are present.     

High-resolution analysis of the modified quarter-stagger model of the collagen fibril

The location of transverse bands within the major repeating period of positively stained collagen fibrils was determined from electron micrographs by an optical averaging procedure. From these data and the published location of bands in SLS crystallites, we have prepared a two-dimensional representation, accurate to about 25 Å, of the modified quarter-stagger arrangement of molecules in the collagen fibril. With this information it is possible to demonstrate the relationship of loci on individual collagen molecules within the fibril. For example, the site where the collagen molecule is cleaved by tadpole collagenase, the site where a disaccharide unit is covalently bound to the α1-CB5 peptide, and the site of carboxyl-terminal intermolecular cross-linking all occur in the fibril near the amino-terminal edge of the “hole zone;” and the site of amino-terminal cross-linking occurs near the carboxyl-terminal edge of the “hole zone.”     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Time-dependent increase in the stability of collagen fibrils formed in vitro. I. Effects of pH and salt concentration on the dissolution of the fibrils.

The time-dependent increase in stability, as measured in terms of the rate of dissolution, of collagen fibrils formed in vitro from pepsin-treated collagen was significantly affected only by temperature, and not by either ionic strength or pH. This is in contrast with collagen fibril formation, a process which is greatly affected by ionic strength and pH. Within the range of temperature 29-37 degrees C, lower temperature caused slower fibril formation and faster fibril stabilization. These results suggest that the intermolecular interactions involved in stabilizing collagen fibrils are entirely different from those involved in fibril formation. Based on kinetic analysis of the dissolution and stabilization of the fibrils, it is proposed that collagen molecules first form unstable fibrils which become gradually stabilized on prolonged incubation, without necessarily introducing covalent cross-links.     

Fibrous long spacing type collagen fibrils have a hierarchical internal structure

Nanodissection of single fibrous long spacing (FLS) type collagen fibrils by atomic force microscopy (AFM) reveals hierarchical internal structure: Fibrillar subcomponents with diameters of approximately 10 to 20 nm were observed to be running parallel to the long axis of the fibril in which they are found. The fibrillar subcomponent displayed protrusions with characteristic approximately 270 nm periodicity, such that protrusions on neighboring subfibrils were aligned in register. Hence, the banding pattern of mature FLS-type collagen fibrils arises from the in-register alignment of these fibrillar subcomponents. This hierarchical organization observed in FLS-type collagen fibrils is different from that previously reported for native-type collagen fibrils, displaying no supercoiling at the level of organization observed.     

Echinoderm collagen fibrils grow by surface-nucleation-and-propagation from both centers and ends

Collagen fibrils from sea cucumber (class Holothuroidea) dermis were previously found to grow by coordinated monomer addition at both centers and ends. This analysis of sea urchin (class Echinoidea) collagen fibrils was undertaken to compare the growth characteristics of fibrils from two classes of echinoderms, and to determine whether a single growth model could account for the main features of fibrils from these two taxa. Native collagen fibrils (37-431 micrometer long) from the spine ligaments of the sea urchin Eucidaris tribuloides were studied by scanning transmission electron microscopy and image analysis. The analyses revealed the mass per unit length, and hence the number of molecules in cross-section, along the entire length of each fibril. The fibrils were symmetrically spindle shaped. The maximum mass per unit length occurred in the center of each fibril, where the fibril contains anti-parallel molecules in equal numbers. The two pointed tips of each fibril showed similar linear axial mass distributions, indicating that the two tips retain shape and size similarity throughout growth. The linear axial mass distributions showed that the tips were paraboloidal, similar to those of vertebrate and sea cucumber fibrils. The computed maximum diameters of the fibrils increased linearly with fibril length. The overall shapes of the fibrils showed that they retain geometric similarity throughout growth. Computer modeling showed that the simplest self-assembly mechanism that can account for the features of these fibrils, and of the sea cucumber fibrils that have been described, is one in which the fibril tips produce independent axial growth, while lateral growth takes place through a surface nucleation and propagation mechanism. This mechanism produces coordinated growth in length and diameter as well as geometric similarity, characteristic features of echinoderm collagen fibrils.     

Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

Nanomechanical Mapping of Hydrated Rat Tail Tendon Collagen I Fibrils

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 μm in length, were acquired at indentation speeds around 10⁵ nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10⁵ nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.