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.     

Interpretation of the electron microscopical appearance of collagen fibrils from corneal stroma

The appearance in the electron microscope of mechanically-dispersed corneal collagen has been observed after positive staining with phosphotungstic acid and/or uranyl acetate and after negative staining with phosphotungstate ions. The distributions of positive stains (both cationic and anionic) were similar to those observed in other type I collagens (e.g. skin, tendon). A high correlation was found between charge density in the fibril and the distribution of charged amino acids predicted from the sequence of calf skin collagen. This correlation could be improved by including type III sequence data, suggesting the presence of 20% type III collagen within each fibril. Negative staining showed the usual collagen D-periodicity but without a clear gap/overlap structure. Detailed analysis revealed at least six sites where stain penetration was inhibited. Specific staining of glycosides using N,N,N′,N′-tetramethylethylenediamine(TEMED)-osmate suggested that these sites identify the covalent attachment of disaccharides to the collagen. Using synchroton X-ray diffraction from TEMED-osmate stained corneas we have determined the locations of the stain ions (and hence the glycosides) in the moist tissue. The results demonstrate that even though the detailed charge distribution and axial molecular packing in corneal collagen are similar to other type I collalgens, carbohydrate material, probably disaccharide, is attached at fairly regular intervals. This does not occur in other type I collagens. In particular, the presence of glycoside in the overlap region may play a role in producing the narrow uniform fibrils which are essential for the transparency of the cornea.     

Effects of pH and ionic strength on the structure of collagen fibrils

The roles of pH and ionic strength on the structure and stability of collagen fibrils have been investigated by means of x-ray and neutron diffraction techniques. High-angle x-ray diffraction shows that a salt concentration of 0.5M KCl is sufficient to reduce the osmotic swelling and related disordering in the pH range 1–3. The relative intensities of the low-angle meridional x-ray and neutron diffraction Bragg reflections vary with pH. Difference Fourier syntheses between pH 7 and 1.6 data indicate, for both x-ray and neutron diffraction, a reduced scattering contribution from the telopeptides at low pH. Lyotropic relaxation is a crucial step in the appearance at low pH of a doubling of the 668-Å axial periodicity (D) of collagen fibrils. These results suggest that electrostatic interactions are essential for the structural stability of the telopeptide regions and of the 1D and 3D intermolecular staggers between collagen molecules.     

Bovine corneal stroma contains a structural glycoprotein located in the gap region of the collagen fibrils

Treatment of bovine corneal stroma using SDS-containing extracting solutions removes a 135,000 MW glycoprotein from the main collagen framework of the tissue. Low-angle synchrotron X-ray diffraction patterns obtained from corneas extracted in this way indicate that the glycoprotein has been removed from the gap regions of the collagen fibrils and is thus an important structural component of the corneal stroma. The glycoprotein (GP 135) shares a number of properties with one of the subunits of type VI collagen, but tests have so far failed to establish their identity.     

Viscosity of chromatin solutions increases with increasing ionic strength

Increasing the ionic strength of rat liver chromatin solutions above 0.4 M causes increasing viscosity. This indicates transformation of the compact chromatin molecules to more elongated forms. In the range of 0.4–0.5 M ionic strength histone H1 is dissociating continuously from the chromatin and the quaternary structure chromatin unravels. At ionic strength higher than 0.5 M the viscosities of chromatin solutions are furthermore increasing due to structural deformation. Near 0.7 M ionic strength the core histones H2A and H2B begin to dissociate from the chromatin, and the opening of the nucleosome cores leads to increasing elongation of the chromatin molecules.     

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.     

The precipitation of toroidal collagen fibrils

The morphology of aggregates of calf-skin tropocollagen, precipitated by continuous injection into neutral phosphate buffers at 35 degrees , has been studied by electron microscopy. Although most of the collagen is precipitated as normal native fibrils, a small proportion forms closed toroidal structures having the usual native band-interband pattern. Theoretical considerations, based on elastic energies in a general microfibril model, predict that the toroids should have a simple super-helical structure, and this is not inconsistent with the observations. From the theoretical energies it was possible to estimate a crude lower limit of 3kcal./mole for the free energy of association of the tropocollagen macromolecules.     

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.     

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.     

Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma.

Ultrastructural data from x-ray diffraction studies of the cornea were used to estimate the refractive indices of the collagen fibrils and extrafibrillar material of human, ox, trout, and rabbit corneas. X-ray diffraction measurements of the size and spacing of the collagen fibrils and the separation between the constituent molecules of the fibrils were taken from a previous species study. The tissue volume fractions occupied by the stromal components were estimated and their refractive indices were calculated using the Gladstone-Dale law of mixtures. For the fibrils and extrafibrillar material, the refractive indices in the human cornea were 1.411 and 1.365; for the ox 1.413 and 1.357; for the rabbit 1.416 and 1.357; and for the trout 1.418 and 1.364, respectively. An alternative estimate based on the physical properties and chemical composition of bovine cornea, accounting for interfibrillar type VI collagen and cellular water, produced similar estimates of 1.416 and 1.356 for the fibrils and extrafibrillar material, respectively.     

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.     

Photoinduced fibrils formation of chicken egg white lysozyme under native conditions

Recent findings showed that transiently accessing structurally native‐like yet energetically higher conformational states is sufficient to trigger the formation of protein fibrils. Typically, these conformational states are made available through changing solvent conditions or introducing mutations. Here we show a novel way to initialize fibril formation for Chicken egg white lysozyme (CEWL) under native conditions via controlled UV illumination. Through a cassette of tryptophan‐based photochemistry, the two terminal disulfide bonds in CEWL can be selectively reduced. The reduced CEWL is then converted to conformational states with the C‐terminal fragment floppy upon thermal fluctuation. These states serve as precursors for the fibrillar aggregation. Intriguingly, the CEWL fibrils are stabilized by intermolecular disulfide bonds instead of noncovalent β‐sheet structures, distinct from the amyloid‐like lysozyme fibrils reported before. Based on the experimental evidences and all‐atom molecular dynamics simulation, we proposed a “runaway domain‐swapping” model for the structure of the CEWL fibrils, in which each CEWL molecule swaps the C‐terminal fragment into the complementary position of the adjacent molecule along the fibrils. We anticipate that this fibrillation mechanism can be extended to many other disulfide‐containing proteins. Our study stands for the first example of formation of protein fibrils under native conditions upon UV illumination and poses the potential danger of low UV dose to organisms at the protein level. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

Rearrangement of chromatin structure induced by increasing ionic strength and temperature.

Native rat liver chromatin fragments exposed to 600 mM NaCl at 37 degrees C for 45 min exhibit substantial modification of their original (approximately 200 base pairs) repeating subunit structure: a new repeat of 140 base pairs, superimposed on a high background, is observed after micrococcal nuclease digestion. The same material appears, in the electron microscope, as clusters of tightly packed beads connected by stretches of 'free' DNA. These modifications are not observed when the native chromatin is incubated at 37 degrees C at NaCl concentrations up to 400 mM. When native rat liver chromatin depleted of histone H1 by tRNA extraction is exposed to ionic strengths up to 600 mM NaCl at 4 degrees C, almost no modifications of the original native repeating structure are observed. However, when the incubation is carried out at 37 degrees C in 150, 300 or 400 mM NaCl, rearrangements of the native structure occur as indicated by micrococcal nuclease digestion and electron microscopic studies. Incubation of H1-depleted chromatin at 600 mM NaCl for 45 min at 37 degrees C induces, as for the native chromatin, a complete rearrangement characterized by the appearance of a 140-base-pair repeat superimposed on a high background upon digestion by micrococcal nuclease. It is suggested that these rearrangements are mediated by hydrophobic interactions between the histone cores and are prevented at ionic strengths lower than 500 mM by the presence of histone H1.     

The crystalline structure of collagen fibrils in tendon.

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with $\text{b = 75.5}\text{A}\text{?}$, β = 93 °, $\text{a =}\text{b}\text{sin}\text{β}$, a = 90 °, $\text{c = n × 668}\text{A}\text{?}$, where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.     

A new variety of spondyloepiphyseal dysplasia characterized by punctate corneal dystrophy and abnormal dermal collagen fibrils

Summary Several individuals from one family are described with a unique form of spondyloepiphyseal dysplasia. Characteristic features include shorttrunked short stature, punctate corneal dystrophy and marked disorganization of dermal collagen fibrils when examined by transmission electron microscopy. Inheritance is compatible with either dominance and a variable expression or X-linkage. Although the basic defect has not been determined, the tissue distribution is consistent with a defect in a non-collagenous component that affects collagen fibril formation or stability.     

Structural transformation of collagen fibrils in corneal stroma during drying. An x-ray scattering study.

X-ray scattering experiments were performed on human corneas during drying. In a first stage the collagen interfibrillar distance decreased considerably. Then, at a critical point of dehydration, a structural transformation of the collagen fibrils was observed. This finding leads to a two-stage drying model, which explains the discrepancy between the collagen fibril diameters determined by x-ray scattering and by electron microscopy. Our results strongly suggest that the collagen fibrils in the corneal stroma are surrounded by a cylindrical coating made mainly of proteoglycans. The coating appears as a three-dimensional fractal network with fractal dimension of 2.7 +/- 0.1.     

The staining pattern of collagen fibrils. Improved correlation with sequence data.

The periodic banding pattern of stained collagen fibrils observed in the electron microscopic can be correlated with the charge distribution deduced from the amino acid sequence. Earlier work used alpha 1 chain sequence data only. The present study incorporates alpha 2 as well as alpha 1 sequence data, so that the complete distribution of charged residues is used. Correlation is improved if it is supposed that the extrahelical terminal regions are contracted. The optimal value of the periodicity, D, (previously 232.3 +/- 0.5 residues using alpha 1 data only), is now 234.2 +/- 0.5 residues, assuming uniform spacing of residues in the helical body of the molecule. This value agrees better with values obtained by others from analyses of interactions between molecules, using sequence data alone. Using the improved value of D, the relative axial locations of the charged residues in the fibril are displayed. In this way, the charged residues contributing to each band in the fibril staining pattern can be identified.