Effect of electrostatic interactions on the percolation concentration of fibrillar beta-lactoglobulin gels

The effect of electrostatic interactions on the critical percolation concentration (c(p)) of fibrillar beta-lactoglobulin gels at pH 2 was investigated using rheological measurements, transmission electron microscopy (TEM), and performing conversion experiments. A decreasing c(p) with increasing ionic strength was found. The fraction of nonaggregated beta-lactoglobulin was independent of ionic strength in the regime of 0.01-0.08 M. TEM experiments showed long fibrils (2-7 microm) for ionic strengths between 0.01 and 0.08 M. Since both the conversion of monomers and the contour length of the fibrils were independent of ionic strength (0.01-0.08M), the linear increase of c(p) with the Debye length can be attributed purely to an increase of electrostatic repulsion between the fibrils. This increase is explained in terms of an adjusted random contact model which takes into account the charge and semiflexibility of the fibrils.     

Mesostructure of fibrillar bovine serum albumin gels

The mesostructure of bovine serum albumin (BSA) at low pH was investigated. Rheological measurements were performed to determine the critical percolation concentration (c_p). A decreasing c_p with increasing ionic strength was found. Fibrils with a contour length of about 100–300 nm were found using transmission electron microscopy. The measured conversion of monomers into fibrils was independent of ionic strength (0.20–0.30 M). Dilution of BSA samples showed that the aggregation process is reversible and that there exists a critical concentration for the self-assembly of BSA. We explain the decreasing c_p with increasing ionic strength in terms of an adjusted random contact model.     

Strong impact of ionic strength on the kinetics of fibrilar aggregation of bovine beta-lactoglobulin

We investigate the effect of ionic strength on the kinetics of heat-induced fibrilar aggregation of bovine beta-lactoglobulin at pH 2.0. Using in situ light scattering we find an apparent critical protein concentration below which there is no significant fibril formation for all ionic strengths studied. This is an independent confirmation of our previous observation of an apparent critical concentration for 13 mM ionic strength by proton NMR spectroscopy. It is also the first report of such a critical concentration for the higher ionic strengths. The critical concentration decreases with increasing ionic strength. Below the critical concentration mainly "dead-end" species that cannot aggregate anymore are formed. We prove that for the lowest ionic strength this species consists of irreversibly denatured protein. Atomic force microscopy studies of the morphology of the fibrils formed at different ionic strengths show shorter and curvier fibrils at higher ionic strength. The fibril length distribution changes non-monotonically with increasing ionic strength. At all ionic strengths studied, the fibrils had similar thicknesses of about 3.5 nm and a periodic structure with a period of about 25 nm.     

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.     

Spatial distribution and orientation of dermatan sulfate in human medial collateral ligament

The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been suggested to contribute to the mechanical stability and material properties of connective tissues. The spatial distribution and orientation of DS within the tissue are relevant to these mechanical roles, but measurements of length and orientation from 2D transmission electron microscopy (TEM) are prone to errors from projection. The objectives of this study were to construct a 3D geometric model of DS GAGs and collagen fibrils, and to use the model to interpret TEM measurements of the spatial orientation and length of DS GAGs in the medial collateral ligament of the human knee. DS was distinguished from other sulfated GAGs by treating tissue with chondroitinase B, an enzyme that selectively degrades DS. An image processing pipeline was developed to analyze the TEM micrographs. The 3D model of collagen and GAGs quantified the projection error in the 2D TEM measurements. Model predictions of 3D GAG orientation were highly sensitive to the assumed GAG length distribution, with the baseline input distribution of 69+/-23 nm providing the best predictions of the angle measurements from TEM micrographs. The corresponding orientation distribution for DS GAGs was maximal at orientations orthogonal to the collagen fibrils, tapering to near zero with axial alignment. Sulfated GAGs that remained after chondroitinase B treatment were preferentially aligned along the collagen fibril. DS therefore appears more likely to bridge the interfibrillar gap than non-DS GAGs. In addition to providing quantitative data for DS GAG length and orientation in the human MCL, this study demonstrates how a 3D geometric model can be used to provide a priori information for interpretation of geometric measurements from 2D micrographs.     

Tuning β-sheet peptide self-assembly and hydrogelation behavior by modification of sequence hydrophobicity and aromaticity

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.     

FTIRS in H2O demonstrates that collagen monomers undergo a conformational transition prior to thermal self-assembly in vitro

The assembly of type I collagen molecules into native fibrils can be accomplished in vitro in solutions at physiological ionic strength and pH by raising the temperature above 30 degrees C. The thermal self-assembly reaction exhibits a distinct lag phase. This lag phase has been proposed to be evidence for a conformational transition in the monomer. Fourier transform infrared spectroscopy (FTIRS) is a very sensitive probe of the H-bonded states within the triple helix. The carbonyl group spectrum (amide I, 1700-1600 cm-1) has been investigated in collagen/H2O solutions at 1 mg/mL under self-assembly conditions from 4 to 34 degrees C and, in the same range, at a higher ionic strength where self-assembly does not occur. The deconvoluted spectra show three very clear bands at approximately 1660, 1644, and 1630 cm-1. These bands vary in both frequency maxima and relative intensity over the temperature range examined. Spectra were also obtained in the amide II and III regions. Spectral changes were evident in the 22-26 degrees C range, under fibril-forming conditions, which lead to the hypothesis that the triple helix of the semiflexible collagen molecule is actually perfected during the lag phase, facilitating nucleation and intermolecular interaction. Further spectral changes after fibrils do form show that the molecules are once again distorted as they are bent to fit within the fibrils.     

Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism

In the search for the molecular mechanism of insulin fibrillation, the kinetics of insulin fibril formation were studied under different conditions using the fluorescent dye thioflavin T (ThT). The effect of insulin concentration, agitation, pH, ionic strength, anions, seeding, and addition of 1-anilinonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). The addition of seeds eliminated the lag phase. An increase in insulin concentration resulted in shorter lag times and faster growth of fibrils. Shorter lag times and faster growth of fibrils were seen at acidic pH versus neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation between the rate of fibril elongation and ionic strength. Agitation during fibril formation attenuated the effects of insulin concentration and ionic strength on both lag times and fibril growth. The addition of ANS increased the lag time and decreased the apparent growth rate for insulin fibril formation. The ANS-induced inhibition appears to reflect the formation of amorphous aggregates. The denaturant, urea, decreased the lag time, whereas the stabilizers, trimethylamine N-oxide dihydrate (TMAO) and sucrose, increased the lag times. The results indicated that both nucleation and fibril growth were controlled by hydrophobic and electrostatic interactions. A kinetic model, involving the association of monomeric partially folded intermediates, whose concentration is stimulated by the air-water interface, leading to formation of the critical nucleus and thence fibrils, is proposed.     

Shear Pulses Nucleate Fibril Aggregation

We have studied the effect of shear flow on the formation of amyloid fibrils of the whey protein β-lactoglobulin. β-Lactoglobulin aggregates into long, thin, and semiflexible fibrils upon heating at low pH and low ionic strength. Solutions with a protein concentration of 0.5% (w/w) were used, and the formation of fibrils was quantified with flow-induced birefringence, a proportional measure of the length concentration of the fibrils. From the decay of the birefringence after cessation of the flow, a length distribution could be fitted. Pulsed and continuous shear treatment of the samples resulted in a comparable enhancement of the fibrillar growth as compared to the fibrillar growth under quiescent conditions. This indicates that the onset of shear flow is the key parameter for the enhancement of fibrillar growth and not the continuous shear flow itself. This behavior is comparable to a nucleation-like process, during which preaggregates of the fibrils are induced during the onset of the flow and orthokinetic coagulation is absent. However, a difference was present in the length distribution between the pulsed and continuously sheared samples, which can be explained by the homogenizing effect of shear flow.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Iionization of tyrosyl groups of ovalbumin under native and denaturing conditions.

Phenolic titration of ovalbumin was performed in the pH range 7-12 at 30 degrees C and at three ionic strengths viz. 0.033, 0.133 and 0.200. The conformational integrity of ovalbumin was studied by viscosity measurements at different pH values in the pH range 7-12.4. At ionic strength 0.133 two phenolic groups titrated reversibly with pKint = 10.31, and w = 0.032 up to pH 11.25 under native conditions. The value of w expectedly decreased with increase in ionic strength. Two additional phenolic groups became available for reversible titration between pH 11.25 and 11.95 after some conformational change. Above pH 12, the phenolic titration became irreversible and all of the nine tyrosine residues were titrated at pH 13.3 Exposure of ovalbumin to alkaline pH (12.4) caused considerable disruption of the native protein conformation. The reduced viscosity increased from 4.2 ml/g at pH 7.0 to 16.8 ml/g at pH 12.4 under identical conditions of the protein concentration. All of the nine tyrosyl groups of ovalbumin were titrated normally (pKint = 9.9) in a mixture of 5 M guanidine hydrochloride and 1.2 M urea. However, even in this mixture electrostatic interaction, as measured by w was not completely abolished.     

Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.     

Light scattering analysis of fibril growth from the amino-terminal fragment beta(1-28) of beta-amyloid peptide.

beta-Amyloid protein (beta-A/4) is the major protein component of Alzheimer disease-related senile plaques and has been postulated to be a significant contributing factor in the onset and/or progression of the disease. In the senile plaque, beta-A/4 appears as bundles of amyloid fibrils. The biological activity of beta-A/4 may be related to its state of aggregation. In this work, self-assembly, fibril formation, and interfibrillary aggregation of beta(1-28), a synthetic peptide homologous with the amino-terminal fragment of beta-A/4, were investigated. The predominant form of beta(1-28) detected by size-exclusion chromatography and polyacrylamide gel electrophoresis was apparently a tetramer which does not bind Congo red. Aggregates containing cross-beta sheet structures which bind Congo red and thioflavin T were observed at concentrations of approximately 0.3 mg/ml or greater. Concentrations of 0.5-1 mg/ml were necessary for aggregation into fibrils to be detectable by classical or quasielastic light scattering. Both fibril elongation and fibril-fibril aggregation occur over the time scale investigated. The kinetics of aggregation were much faster at physiological salt concentrations than at lower ionic strength. Ionic strength also appeared to influence the morphology of the fibril aggregates. The data indicate that sample preparation method and sample history influence fibril size and number density.     

Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro

The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed.     

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.     

Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro

Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.     

Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.     

A Kinetic Study of Ovalbumin Fibril Formation: The Importance of Fragmentation and End-Joining

The ability to control the morphologies of biomolecular aggregates is a central objective in the study of self-assembly processes. The development of predictive models offers the surest route for gaining such control. Under the right conditions, proteins will self-assemble into fibers that may rearrange themselves even further to form diverse structures, including the formation of closed loops. In this study, chicken egg white ovalbumin is used as a model for the study of fibril loops. By monitoring the kinetics of self-assembly, we demonstrate that loop formation is a consequence of end-to-end association between protein fibrils. A model of fibril formation kinetics, including end-joining, is developed and solved, showing that end-joining has a distinct effect on the growth of fibrillar mass density (which can be measured experimentally), establishing a link between self-assembly kinetics and the underlying growth mechanism. These results will enable experimentalists to infer fibrillar morphologies from an appropriate analysis of self-assembly kinetic data.     

X-ray diffraction evidence of collagen molecular packing and cross-linking in fibrils of rat tendon observed by synchrotron radiation.

X-ray diffraction patterns of fibres from 90 day (mature) rat-tail tendons were investigated using synchrotron radiation. The specimens were kept isometric at their corresponding in vitro rest length, and effects of pH and ionic strength were studied during short X-ray exposures. The results indicate that fibrils, equilibrated in physiological Ringer prior to exposure, have segregated lateral regions of well ordered collagen molecular packing. Lowering the ionic strength or the pH to 4.0 causes an order/disorder transition during which the fibril crystallinity decreases. At pH 3.5 a dramatic increase in the lateral swelling was observed. This effect was absent for fibres pretreated with sodium borohydride. The results are interpreted on the basis of cross-linking phenomena whereby the aldimine cross-link seems to be a controlling component of the lateral packing arrangement of collagen molecules.