In vivo studies of collagen molecular packing in tendon fibres of varying length and age studied via X-ray diffraction patterns obtained at a synchroton

X-ray diffraction studies were performed using a high brilliance synchrotron. The lateral packing of collagen molecules into fibrils was studied in fibre specimens of rat tail tendons. We investigated the packing scheme (a) at the lower and upper limits of the physiological range of length, and (b) in fibres from 40, 90 (sexual maturity) and 240 day animals. The results indicate that the R-positions of the Bragg reflections are independent of the fibre extension and animal age. Optimal structural order occurs at the lower limit of the physiological range of lengths and the disorder increases upon extension. The packing arrangement of the collagen molecules seems to remain unaltered within the age span studied, the fibril crystallinity does, however, incrase during maturity.     

Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

Multiple denaturational transitions in fibrillar collagen

The heat denaturation of pepsinized bovine nonfibrillar and fibrillar collagen was studied by differential scanning calorimetry. For fibrillar preparations that had been rapidly precipitated with stirring at low ionic strength, then resuspended at physiological ionic strength, multiple denaturational transitions were observed. At heating rates of 10°C/min, melting endotherms occurred at about 44, 50, 53, and 57°C. Fibrillar collagen that was slowly gelled without stirring at physiological ionic strength exhibited a similar series of endotherms, but the lower melting transitions were less conspicuous. In contrast, nonfibrillar bovine collagen in acidic solution showed only a single denaturational transition at 40°C. Nonfibrillar solutions at pH 7, to which inhibitors of fibrillogenesis were added, showed a major endotherm as high as 46°C. These results suggest that reconstituted fibrillar collagen contains a heterogeneous fibril population, possibly including molecules in a nonfibrillar state. It was proposed that the multiple melting endotherms of such preparations were due to sequential melting of molecular and fibril classes, each with a distinct melting temperature. The fibrillar classes may represent three or more types of banded and nonbanded species that differ from each other in packing order, collagen concentration, and possibly also in fibril width and level of cross-linking.     

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.     

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.     

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium ("weak-binding") state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.     

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.     

Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mM ionic strength, 15 degrees C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.     

The formation and thermal stability of in vitro assembled fibrils from acid-soluble and pepsin-treated collagens.

The role of the non-helical regions of the collagen molecule in fibrillogenesis has been investigated by comparing the kinetics of fibril formation of pepsin-treated acid-soluble collagen, acid-soluble collagen and mixtures of the two and by comparison of the thermal stabilities of the fibrils formed. The acid-soluble collagen was found to aggregate more rapidly than the pepsin-treated collagen under physiological conditions of pH and ionic strength. Variations in ionic strength, at physiological pH, were found to have differing effects on the aggregation of these two forms of soluble collagen. Fibrils formed from the pepsinized-collagen had a lower thermal stability tha n those formed from the intact collagen. The behavior observed with mixtures of acid-soluble and pepsin-treated collagens was found to be quantitatively consistent with the pepsinized collagen being able to utilize the nuclei formed by the acid-soluble collagen for subsequent growth. However, the use of the acid-soluble nuclei by the pepsinized collagen for growth did not enhance its rate of precipitation during the growth phase, nor did it enhance the thermal stability of the fibrils formed from the pepsinized collagen.     

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.     

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.     

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.     

Electrostatic interactions modulate the conformation of collagen I

The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl(2) solutions. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl(2) solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl(2) solutions are concluded to shift the IEP and influence the conformational stability of the protein.     

Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.     

Small angle scattering of cell nuclei

Neutron and X-ray small angle scattering techniques have been applied to study chromatin structure inside different types of cell nuclei. Scattering from genetically inactive chicken erythrocyte nuclei exhibits a maximum at Q = 0.1-0.15 nm-1 which cannot be observed by studying isolated chromatin derived from the same kind of cells. In highly active transcribing rat liver nuclei such a nuclear pattern is absent. The radius of gyration of isolated "superbeads" was determined. It is discussed whether the characteristic maximum of the nuclei originates from this superstructural organisation of chromatin. Rat liver nuclei were fractionated on sucrose gradients in order to determine whether the absence of the extra maximum in scattering profiles of these nuclei is due to overlapping effects of different chromatin organisation in the various cell types of the liver. As compared to unfractionated nuclei no strong deviations in the scattering profiles of the fractions could be observed. Erythrocyte nuclei were dialysed in buffers differing in the ionic strength of monovalent cations. The typical maximum from the nuclei is shifted from 60 nm (very low salt concentration) to about 35 nm (physiological ionic strength) and is linearly proportional to the decreasing radius of the nuclei. In conclusion, chromatin structure inside the nucleus has a scattering maximum due to an ordered packing of the fibres which is absent in nuclei with high genetic activity.     

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.     

Swelling studies on the cornea and sclera: the effects of pH and ionic strength.

The biophysical properties of the cornea and sclera depend on the precise maintenance of tissue hydration. We have studied the swelling of the tissues as a function of pH and ionic strength of the bathing medium, using an equilibration technique that prevents the loss of proteoglycans during swelling. Synchrotron x-ray diffraction was used to measure the average intermolecular and interfibrillar spacings, the fibril diameters, and the collagen D-periodicity. We found that both tissues swelled least near pH 4, that higher hydrations were achieved at lower ionic strengths, and that sclera swelled about one-third as much as cornea under most conditions. In the corneal stroma, the interfibrillar spacing increased most with hydration at pH values near 7. Fibril diameters and D-periodicity were independent of tissue hydration and pH at hydrations above 1. Intermolecular spacings in both tissues decreased as the ionic strength was increased, and there was a significant difference between cornea and sclera. Finally, we observed that corneas swollen near pH 7 transmitted significantly more light than those swollen at lower pH levels. The results indicate that the isoelectric points of both tissues are close to pH 4. The effects of ionic strength can be explained in terms of chloride binding within the tissues. The higher light transmission achieved in corneas swollen at neutral pH may be related to the fact that the interfibrillar fluid is more evenly distributed under these conditions.     

Morphological characterization of tendon development during chick embryogenesis: measurement of birefringence retardation

Morphological observations and physical measurement of (I) birefringence retardation, (2) mean fibre profile width, and (3) cell volume fraction were used to characterize chick hind limb extensor tendon development. Observations were made at days 7, 10, 14 and 17 embryologic and 1-1.5 post-hatching. Microanatomical observations illustrated a sequential development of tendon microanatomy consisting of (1) a uniaxial cellular framework with discontinuous collagen fibril bundles present in day 7 embryos; (2) a continuous network of birefringent collagen fibres, and early evidence of tendon fasciculation and crimp development by embryonic day 10; and (3) completion of the basic cytoarchitecture of tendon observed at day 14 of embryogenesis. These observations suggest that collagen deposition in tendon involves first a longitudinal and then a lateral organization of tendon fibroblasts. Associated with the progressive anatomical development of tendon was an increase in birefringence retardation, mean collagen fibre profile width, and a decrease in the cell volume fraction. Birefringence retardation per unit thickness, however, did not change. This suggested that the fibril packing density of the fibres remained constant, although the fibres were observed to increase in size. These results indicate that collagen fibrillogenesis in vivo can be quantitatively studied by measurement of the birefringence retardation using polarized light.     

The self-assembly of collagen molecules

The aggregation of native acid-soluble collagen (N-ASC) and of pronase-treated acid soluble collagen (P-ASC) was examined in solution under conditions which varied from those of minimum collagen-collagen interaction to those leading to incipient fiber formation. Molecular weights and weight distributions were determined in the analytical ultracentrifuge using the Yphantis high speed sedimentation equilibrium and Aarchiblad approach-to-equilibrim techniques. The aggregation was pH and ionic strength dependent in each case. Under conditions of minimum aggregation (low pH, low ionic strength), N-ASC showed the presence of permant aggregates. At higher pH and ionic strength, a higher fraction of aggregate was formed but these were of the same charcter and molecular weight as the permanent aggregates. The aggregates were of a single molecular size, with a weight of 1.5 × 10⁶ daltons, compared with a monomer collagen weight of 3.1 × 10⁵ daltons. The P-ASC formed aggregates also but to a much lower extent and the maximum aggregate size corresponded to dimers in molecular weight. These data show the major importance of molecular end-regions in collagen aggregation to form native type fibers and, by virtue of the discrete size of the N-ASC aggregates, support the microfibrillar hypothesis for the assembly of collagen fibrills.     

A synchrotron radiation X-ray diffraction study of the crystallinity of tendon fibres

The equatorial diffraction pattern of tendon collagen fibres was measured during short successive exposures at different lengths using a double focusing X-ray synchrotron radiation camera with film and with an area detector. Similarly, patterns from thin fibres from premature rats were recorded. The patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that in the initial part of the linear region of the stiffness-versus-length curve, the collagen fibres are characterized by a quasihexagonal arrangement of collagen molecules, whereas at the end of this region, the molecular arrangement becomes hexagonal.