Christopher A. Miles

Polymer-in-a-Box Mechanism for the Thermal Stabilization of Collagen Molecules in Fibers

Collagen molecules in solution unfold close to the maximum body temperature of the species of animal from which the molecules are extracted. It is therefore vital that collagen is stabilized during fiber formation. In this paper, our concept that the collagen molecule is thermally stabilized by loss of configurational entropy of the molecule in the fiber lattice, is refined by examining the process theoretically. Combining an equation for the entropy of a polymer-in-a-box with our previously published rate theory analysis of collagen denaturation, we have derived a hyperbolic relationship between the denaturation temperature, Tm, and the volume fraction, epsilon, of water in the fiber. DSC data were consistent with the model for water volume fractions greater than 0.2. At a water volume fraction of about 0.2, there was an abrupt change in the slope of the linear relationship between 1/Tm and epsilon. This may have been caused by a collapse of the gap-overlap fiber structure at low hydrations. At more than 6 moles water per tripeptide, the enthalpy of denaturation on a dry tendon basis was independent of hydration at 58.55 +/- 0.59 J g-1. Between about 6 and 1 moles water per tripeptide, dehydration caused a substantial loss of enthalpy of denaturation, caused by a loss of water bridges from the hydration network surrounding the triple helix. At very low hydrations (less than 1 mole of water per tripeptide), where there was not enough water to form bridges and only sufficient to hydrogen bond to primary binding sites on the peptide chains, the enthalpy was approximately constant at 11.6 +/- 0.69 J g-1. This was assigned mainly to the breaking of the direct hydrogen bonds between the alpha chains.

The effects of hydrostatic pressure on ribosome conformation in Escherichia coli : an in vivo study using differential scanning calorimetry

Summary: Differential scanning calorimetry of whole Escherichia coli cells allowed the detection in vivo of changes in ribosome conformation. This enabled for the first time an analysis of the effects of high hydrostatic pressures on ribosomes in living cells. A correlation was observed between loss of cell viability and decrease in ribosome-associated enthalpy in cells subjected to pressures of 50-250 MPa for 20 min. Cell death and ribosome damage were therefore closely related phenomena. In pressure-treated cells, the thermogram peak temperatures decreased, suggesting that the remaining ribosomes had adopted a less stable conformation. During subsequent incubation of the cultures at 37°C, peak temperatures and enthalpies gradually increased over a period of 5 h. This change in ribosome conformation had no apparent effect on cell survival, as viability continued to decrease. The addition of 5 mM MgCl2 before pressure treatment of cells prevented the reduction in stability of surviving ribosomes but had no effect on the initial loss of enthalpy or on cell viability.

The role of the α2 chain in the stabilization of the collagen type I heterotrimer: A study of the type I homotrimer in oim mouse tissues

We have previously reported that the fragility of skin, tendon and bone from the oim mouse is related to a significant reduction in the intermolecular cross-linking. The oim mutation is unlikely to affect the efficacy of the lysyl oxidase, suggesting that the defect is in the molecule and fibre. We have therefore investigated the integrity of both the oim collagen molecules and the fibre by differential scanning calorimetry. The denaturation temperature of the oim molecule in solution and the fibre from tail tendon were found to be higher than the wild-type by 2.6deg.C and 1.9deg.C, respectively. With the loss of the alpha2 chain, the hydroxyproline content of the homotrimer is higher than the heterotrimer, which may account for the increase. There is a small decrease in the enthalpy of the oim fibres but it is not significant, suggesting that the amount of disorder of the triple-helical molecules and of the fibres is small and involves only a small part of the total bond energy holding the helical structure together. The difference in denaturation temperature of the skin collagen molecules (t(m)) and fibres (t(d)) is significantly lower for the oim tissues, 19.9deg.C against 23.1deg.C, indicating reduced molecular interactions and hence packing of the molecules in the fibre. Computation of the volume fraction of the water revealed that the interaxial separation of the oim fibres was indeed greater, increasing from 19.6A to 21.0A. This difference of 1.4A, equivalent to a C-C bond, would certainly decrease the ability of the telopeptide aldehyde to interact with the epsilon -amino group from an adjacent molecule and form a cross-link. We suggest, therefore, that the reduction of the cross-linking is due to increased water content of the fibre rather than a distortion of the molecular structure. The higher hydrophobicity of the alpha2 chain appears to play a role in the stabilisation of heterotrimeric type I collagen, possibly by increasing the hydrophobic interactions between the heterotrimeric molecules, thereby reducing the water content and increasing the binding of the molecules in the fibre.

Identification of an Intermediate State in the Helix-Coil Degradation of Collagen by Ultraviolet Light*

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 °C following UV irradiation of collagen, compared with 39 °C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.

Thermal Stability of Collagen Fibers in Ethylene Glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.

Properties of collagen in OIM mouse tissues

The deletion of the f 2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the f 2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6° and 1.9°C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The f 2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.

Asymmetry in the triple helix of collagen-like heterotrimers confirms that external bonds stabilize collagen structure.

Heating and subsequent cooling mixtures of (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) peptides leads to formation of model heterotrimeric collagen helices that can be isolated by HPLC. These heterotrimeric collagen peptide helices are shown to be fundamentally unstable as denaturing then renaturing experiments result in heterotrimeric/homotrimeric mixtures. As the proportion of hydroxyproline-containing chains in the trimers increases, differential scanning calorimetry shows that the helix melting temperatures and denaturation enthalpies increasing non-linearly. Three types of Rich-Crick hydrogen bonds observed by NMR allow modelling of heterotrimeric structures based on published homotrimeric X-ray data. This revealed a small axial movement of (Pro-Hyp-Gly)(10) chains towards the C-terminal of the helix, demonstrating heterotrimeric asymmetry.

Thermal denaturation of collagen revisited

We have recently re-examined the characteristic sharp denaturation temperature of the collagen molecule and fibre. It has been generally accepted for many years that denaturation is an equilibrium process involving the rupture of hydrogen bonds. We have now proposed that the process is an irreversible rate process, in which uncoupling of thea-chains initially occurs in a thermally labile domain devoid of hydroxyproline. The domain is located near the C-terminal and following alignment of the molecules in the quarter-stagger-end-overlap arrangement is located in the gap region of the fibre. The domain appears to be conserved in type I of several animal species, and is present in types II and III. Collagen molecules that co-polymerise to form fibres, types V and XI, do not possess this labile domain.Ramachandran proposed that stabilisation of the triple helix occurred through hydrogen-bonded water-bridges involving the hydroxyl group of hydroxyproline. Recent studies have been equivocal, some questioning the role of water bridges and of hydroxyproline, whilst recent detailed X-ray studies of collagen-like peptides demonstrate the presence of a stabilising sheath of hydrogen-bonded water. Our findings support the proposal of hydrogen-bonded water-bridges stabilising the triple helix.

Factors affecting the ultrasonic properties of equine digital flexor tendons

The velocity, attenuation and apparent backscattering coefficient of 6-11-MHz ultrasound were measured in three orthogonal directions in equine deep digital flexor (DDF) and superficial digital flexor (SDF) tendons at 0 degree C. Ultrasonic measurements were examined for correlation with tendon water, collagen, DNA and glycosaminoglycans contents, determined by chemical analyses and with structure observed by scanning electron microscopy. The SDF tendon contained more water, more DNA (i.e., more cells), less collagen and less glycosaminoglycans and exhibited lower velocities and attenuations than the DDF tendon. Velocities were governed primarily by the adiabatic bulk modulus and density, perturbed by a highly direction-dependent rigidity. Ultrasound propagating across tendon generated frequency-independent backscattering which appeared to derive from the large interfaces between the fascicles, while along the fibres backscattering varied as f3.62 +/- 0.88 and appeared to derive from small structures such as collagen fibres. The mechanisms by which ultrasound is attenuated by tendon remain unknown.

Thermal stabilization of collagen in skin and decalcified bone

The state of collagen molecules in the fibres of tail tendon, skin and demineralized bone has been investigated in situ using differential scanning calorimetry (DSC). Hydroxyproline analysis and tissue digestion with bacterial collagenase and trypsin were used to confirm that the common cause of all the DSC endotherms was collagen denaturation. This occurred within a narrow temperature range in tendons, but over a wide temperature range in demineralized bone and old skin and demonstrated that in tendon and demineralized bone at least the same type I collagen molecule exists in different thermal states. Hypothesizing that this might be caused by different degrees of confinement within the fibre lattice, experiments were performed to measure the effect of changing the lattice dimensions by extracting the collagen into dilute solution with pepsin, swelling the lattice in acetic acid, and contracting the lattice by dehydration. A theoretical analysis was undertaken to predict the effect of dehydration. Results were consistent with the hypothesis, demonstrating that collagen molecules within the natural fibres of bone and old skin are located at different intermolecular spacings, revealing differences between molecules in the magnitude of either the attractive or repulsive forces controlling their separation. One potential cause of such variation is known differences in covalent cross-linking.