Nanosecond fluctuations of the molecular backbone of collagen in hard and soft tissues: a carbon-13 nuclear magnetic resonance relaxation study

We have determined the amplitude of nanosecond fluctuations of the collagen azimuthal orientation in intact tissues and reconstituted fibers from an analysis of 13C NMR relaxation data. We have labeled intact rat calvaria and tibia collagen (mineralized and cross-linked), intact rat tail tendon and demineralized bone collagen (cross-linked), and reconstituted lathyritic (non-cross-linked) chick calvaria collagen with [2-13C]glycine. This label was chosen because one-third of the amino acid residues in collagen are glycine and because the 1H-13C dipolar coupling is the dominant relaxation mechanism. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancements were measured at 15.09 and 62.98 MHz at 22 and -35 degrees C. The measured NMR parameters have been analyzed by using a dynamic model in which the azimuthal orientation of the molecule fluctuates as a consequence of reorientation about the axis of the triple helix. We have shown that if root mean square fluctuations in the azimuthal orientations are small, gamma rms much less than 1 rad, the correlation function decays with a single correlation time tau and T1 depends only upon tau and gamma rms and not the detailed model of motion. Our analysis shows that, at 22 degrees C, tau is in the 1-5-ns range for all samples and gamma rms is 10 degrees, 9 degrees, and 5.5 degrees for the non-cross-linked, cross-linked, and mineralized samples, respectively. At -35 degrees C, gamma rms is less than 3 degrees for all samples. These results show that mineral and low temperature significantly restrict the amplitude of nanosecond motions of the collagen backbone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics of collagen side chains in hard and soft tissues. A multinuclear magnetic resonance study

We have prepared samples of (a) intact calvaria collagen (cross-linked and mineralized), (b) intact tendon collagen (cross-linked but not mineralized), and (c) reconstituted chick calvaria collagen (not cross-linked and not mineralized) containing [methyl-2H3]methionyl, [4,4-2H2]pyrrolidinyl, (4-fluorophenyl)alanyl, and [6-15N]lysyl residues. Using multinuclear magnetic resonance spectroscopy, we have investigated the molecular dynamics of the labeled amino acids. Guided by model compound studies, we reached the following conclusions regarding collagen side chain dynamics from our analysis of line shapes and relaxation rates. At 22 degrees C, imino residues in all samples have flexible rings with root mean square angular fluctuations in the 11-30 degree range. Nearly all labeled amino acid side chains reorient about at least two side chain single bonds. At temperatures below -30 degrees C, most of these side chain motions are absent in all the samples. Surprisingly, in contrast with results obtained for backbone motions, side chain motions are only marginally more hindered in mineralized samples as compared with nonmineralized samples, a result we discuss with reference to collagen-mineral interactions. We also discuss the possible relationship between collagen dynamics and function.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

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.     

13C/1H high power double magnetic resonance investigation of collagen backbone motion in fibrils and in solution

${}^{13}\text{C}{}^1\text{H}$ high power double magnetic resonance spectroscopy was used to investigate the mobility of the collagen peptide backbone. [1-¹³C]- and [2-¹³C]-glycine-labeled collagen samples (with >50% enrichment in ¹³C) were prepared via chick calvaria culture. ¹³C n.m.r.² spectra of labeled reconstituted collagen fibrils, of labeled helical collagen in solution, and of unlabeled bovine Achilles tendon collagen were obtained with scalar decoupling and with dipolar decoupling of protons. Proton-enhanced spectra were also obtained using cross-polarization techniques. n.m.r. parameters (linewidths, lineshapes, T₁ values, nuclear Overhauser enhancements, and cross polarization enhancements) were measured for the labeled samples and for collagen in natural abundance. Comparison of ¹³C n.m.r. parameters for bovine Achilles tendon fibrils and for reconstituted chick calvaria collagen fibrils established that chick calvaria collagen is a good model for the molecular dynamics of collagen in vivo.Spin-lattice relaxation times and nuclear Overhauser enhancements for [1-¹³C]- and [2-¹³C]glycine-labeled collagen indicated that R₁ ~2 × 10⁷s^?1 in solution, where R₁ is the diffusion constant for reorientation about the long axis of the molecule. A substantially smaller value for R₁ (2.6 × 10⁶s^?1) was calculated for an axially symmetric ellipsoid of revolution having dimensions appropriate to the collagen helix. The discrepancy between the rigid ellipsoid and n.m.r. values of R₁ suggests that the collagen molecule undergoes torsional reorientation, as well as rod-like reorientation, about its long axis.The T₁ and NOE values measured in the glycine-labeled fibrils show that rapid axial motion (R₁ ~ 10⁷s^?1) persists in the fibrillar state. In the collagen fibril the full width of the glycyl carbonyl powder pattern is 103 p.p.m. This value is substantially smaller than the rigid lattice value, 144 p.p.m., which provides further evidence for motion in the fibril. The observed powder pattern is axially asymmetric, which shows that certain azimuthal orientations are energetically preferred in the fibril. Taken together, the n.m.r. data provide strong evidence that rapid reorientation of the helix backbone occurs in the fibrils. This result shows that formation of a fibrillar structure does not require the existence of a unique set of intermolecular interactions at the helical surfaces.     

Self-assembly of collagen type I molecules without telopeptides

The effect of temperature on the kinetics of formation of fibrils from rat tail collagen molecules devoid of telopeptides was studied. It was shown that the rats of fibril formation at 30 and 35 degrees C increases five- and eightfold, respectively, as compared with that at 25 degrees C. It was found that enthalpy of fibril denaturation at 30 degrees C is maximal for the collagen both with intact telopeptides and devoid of telopeptides. It was found that essential for the fibrilogenesis of type I collagen devoid of telopeptides are temperatures of 30 and 35 degrees C.     

Mechanism of in vitro collagen fibril assembly. Kinetic and morphological studies

The kinetics of in vitro fibril assembly of Type I collagen preparations that contain different amounts of covalently cross-linked oligomers was studied with turbidimetry. Fibril formation showed a lag phase with no solution turbidity and a growth phase with a sigmoidal increase in the solution turbidity. The length of the lag phase was inversely related to both the total collagen concentration and the amount of covalently cross-linked oligomers in the solution. Double logarithmic plots of t1/4, the amount of time it takes for 1/4 of the collagen to assemble into fibrils, versus the total collagen concentration were linear but the slope decreased from -0.84 to -2.3 with decreasing amounts of covalently cross-linked oligomers in the samples. Electron microscopy showed the formation of unbanded microfibrils with diameters in the range of 3-15 nm early in the lag phase and larger diameter banded fibrils coexisting with the microfibrils near the end of the lag phase. Centrifugation of the solution at the lag phase prolonged the lag time, presumably by removal of microfibrils, but subsequent growth of the fibrils was unaffected. The results suggest a cooperative nucleation-growth mechanism for the in vitro assembly of collagen fibrils which is consistent with the results of an equilibrium study of the fibril assembly reaction we reported earlier (Na, G. C., Butz, L. J., Bailey, D. G., and Carroll, R. J. (1986) Biochemistry 25, 958-966).     

Ageing changes in the tensile properties of tendons: influence of collagen fibril volume fraction

Connective tissues are biological composites comprising of collagen fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular matrices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, fibril diameter. However, quantitative attempts to correlate fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength (sigma) and stiffness (E) on the collagen fibril cross-sectional area fraction (rho), which is related to the fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6-35.3 months old were stretched to failure to determine sigma and E. Parallel measurements of rho as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of fibrils reinforcing a PG gel in tendons were used to investigate the influence of rho on ageing changes in sigma and E. The magnitudes of sigma, E, and rho increased rapidly from 1.6 months to 4.0 months (P-values <0.05) before reaching a constant (age independent) from 4.0 months to 29.0 months (P-values >0.05); this trend continued for E and rho (P-values >0.05) from 29.0 months to 35.3 months, but not for sigma, which decreased gradually (P-values <0.05). Linear regression analysis revealed that age-related changes in sigma and E correlated positively to rho (P-values <0.05). Collagen fibril cross-sectional area fraction rho is a significant predictor of ageing changes in sigma and E in the tail tendons of C57BL6 mice.     

Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.     

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

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

Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropy-driven process

Type I procollagen was purified from the medium of cultured human fibroblasts incubated with 14C-labeled amino acids, the NH2-terminal propeptides were cleaved with procollagen N-proteinase, and the resulting pC-collagen was isolated by gel filtration chromatography. pC-collagen did not assemble into fibrils or large aggregates even at concentrations of 0.5 mg.ml-1 at 34 degrees C in a physiological buffer. However, cleavage of pC-collagen to collagen with purified C-proteinase (Hojima, Y., (1985) J. Biol. Chem. 260, 15996-16003) generated fibrils that were visible by eye and that were large enough to be separated from solution by centrifugation at 13,000 x g for 4 min. With high concentrations of enzyme, the pC-collagen was completely cleaved in 1 h, and turbidity was near maximal in 3 h, but collagen continued to be incorporated in fibrils for over 10 h. Because the pC-collagen was uniformly labeled with 14C-aminoacids, the concentration of soluble collagen and, therefore, the critical concentration of polymerization were determined directly. The critical concentration was independent of the initial pC-collagen concentration and of the rate of cleavage. The critical concentration decreased with temperature between 29 and 41 degrees C and was 0.12 +/- 0.06 (S.E.) microgram.ml-1 at 41 degrees C. The thermodynamic parameters of assembly were essentially independent of temperature in the range 29 to 41 degrees C. The process was endothermic with a delta H value of +56 kcal.mol-1, but entropy driven with a delta S value of +220 cal.K-1.mol-1. The Gibbs energy change for polymerization was -13 kcal.mol-1 at 37 degrees C. The data demonstrate, for the first time, that type I collagen fibril formation de novo is a classical example of an entropy-driven self-assembly process similar to the polymerization of actin, flagella, and tobacco mosaic virus protein.     

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.     

Type I collagen triplet duplication mutation in lethal osteogenesis imperfecta shifts register of alpha chains throughout the helix and disrupts incorporation of mutant helices into fibrils and extracellular matrix

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T(m) of 2 degrees C for helices with one mutant chain and a 6 degrees C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with proalpha1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences.     

Collagen fibril formation in vitro. The role of the nonhelical terminal regions.

We showed previously that fibril formation in vitro from rat tail tendon collagen requires a temperature-dependent initiation (Step 1) following which linear assembly to form thin filaments (Step 2) proceeds as rapidly at 4 degrees C as at 26 degrees C. Step 3, lateral assembly of filaments to form fibrils, is again temperature-dependent. We now find that Step 1 is complete in 6 min at 26 degrees C and the time is independent of collagen concentration in the range 0.08 to 0.39 mg/ml. Collagen treated with pepsin, which removes the nonhelical ends but leaves the triple helix intact, forms fibrils by a similar mechanism. However, Step 1 is altered or absent and early temperature changes produce a complex response consistent with an alternate, counterproductive pathway. Assembly is also much slower, particularly Step 2, and the fibrils formed are abnormal in that native banding is often absent and short tactoidal forms are common. These results suggest that in the assembly of fibrils from normal collagen the nonhelical ends are involved in an early conformational change and critically regulate later steps.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

(2)H and (15)N solid-state NMR spectroscopic techniques were used to investigate the membrane composition, orientation, and side-chain dynamics of the transmembrane segment of phospholamban (TM-PLB), a sarcoplasmic Ca(2+)-regulator protein. (2)H NMR spectra of (2)H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD(3)-Leu28, CD(3)-Leu39, and CD(3)-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the C(gamma)-C(delta) bond axis or by additional librational motion about the C(alpha)-C(beta) and C(beta)-C(gamma) bond axes. The (2)H NMR line shapes of all CD(3)-labeled leucines are very similar below 0 degrees C, indicating that all of the residues are located inside the lipid bilayer. At higher temperatures, all three labeled leucine residues undergo rapid reorientation about the C(alpha)-C(beta), C(beta)-C(gamma), and C(gamma)-C(delta) bond axes as indicated by (2)H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the (2)H NMR spectra indicated that the Leu51 side chain has less motion than Leu39 or Leu28, which is attributed to its incorporation in the pentameric PLB leucine zipper motif. The (15)N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for (15)N-labeled Leu TM-PLB were sigma(11) = 50.5 ppm, sigma(22) = 80.5 ppm, and sigma(33) = 229 ppm within +/-3 ppm experimental error. The (15)N chemical-shift value from the mechanically aligned spectrum of (15)N-labeled Leu39 PLB in DOPC/DOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal.     

Deuterium nuclear magnetic resonance of specifically labeled native collagen. Investigation of protein molecular dynamics using the quadrupolar echo technique.

Collagen was labeled with [3,3,3-d3]alanine and with [d10]leucine via tissue culture. 2H nuclear magnetic resonance (NMR) spectra were obtained of collagen in solution and as fibrils using the quadrupolar echo technique. The 2H NMR data for [3,3,3-d3]alanine-labeled collagen fibrils were analyzed in terms of a model for motion in which the molecule is considered to jump between two sites, separated azimuthally by an angle 2 delta, in a time which is rapid compared with the residence time in both sites. The data suggest that the molecule undergoes reorientation over an angle, 2 delta, of approximately 30 degrees in the fibrils, and that the average angle between the alanine C alpha--C beta bond axis and the long axis of the helix is approximately 75 degrees. Reorientation is possibly segmental. The T2 for [3,3,3-d3]alanine-labeled collagen fibrils was estimated to be 105 mus. The 2H NMR data for the methyl groups of [d10]leucine-labeled collagen were analyzed qualitatively. These data established that for collagen in solution and as fibrils, rotation occurs about the leucine side-chain bonds, in addition to threefold methyl rotation and reorientation of the peptide backbone. The T2 for the methyl groups of leucine-labeled collagen is estimated to be approximately 130 mus. Taken together, these data provide strong evidence that both polypeptide backbone reorientation and amino acid side-chain motion occur in collagen molecules in the fibrils. Stabilizing interactions that determine fibril structure must therefore depend upon at least two sets of contacts in any given local region.     

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

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

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.