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Kinetics Analyzer3

2009-01-29T04:26:00.000-08:00

The capacity of the recombinant collagen to form supra-molecular structures was studied using SLS crystallites, an artificialmode of molecular packing in which polyanions such as ATP cross-bridgepositively charged collagen molecules to induce lateral registeraggregation. SLS crystallites were obtained with recombinant collagenthat displayed a characteristic asymmetric cross-banding patternafter positive staining (Fig. 4). Their average length (261 ±11 nm) corresponded to that of single molecules and was consistentwith the lengths of SLS crystallites formed from bovine heterotrimerand homotrimer (271 ± 10 and 276 ± 7 nm, respectively). Moreover,alignment of all three forms of SLS crystallites revealed an overallsimilarity in the banding pattern indicating an identical distributionof polar residues (Fig. 4D).

Kinetics Analyzer2

2009-01-29T04:25:00.000-08:00

Fibril Formation Competence of rColl I-- Neutralizing the pH and increasing the temperature of purified collagen molecules in solution generally provokes the formationof a gel containing striated fibrils. Thus increasing the temperatureto 34-35 °C clearly favors fibril formation of native collagenmolecules. Because of the low T_m (30 °C) of the unhydroxylatedrecombinant collagen, fibril formation experiments could not becarried out at this temperature. Therefore, the capacity of theunhydroxylated recombinant collagen to form fibrils was exploredby performing experiments at 20 °C. Purified rColl I moleculeswere incubated in PBS, a physiological buffer that normally allowsfibril formation. As controls, fibril formation of bovine homotrimerand heterotrimer was carried out under the same conditions. Asexpected, a meshwork of fibrils exhibiting the typical D-periodicbanding pattern was observed when Het I and Hom I solutions wereincubated in PBS at 20 °C (Fig. 5, B and C). In contrast, negativestaining of rColl I aggregates formed in PBS showed thin filamentousstructures devoid of D-periodic striation (Fig. 5A). To checkwhether potential traces of plant contaminants might have preventedstriated fibril formation, bovine homotrimeric molecules weremixed with tobacco crude extract and then subjected to salt fractionation.Negative staining of the fibrils formed with this preparationshowed the presence of fibrils essentially identical to thoseobserved with reconstituted standard homotrimeric collagen I (datanot shown).

Kinetics Analyzer1

2009-01-29T04:23:00.001-08:00

Critical conditions allowing formation of striated fibrils from unhydroxylated recombinant collagen were identified by varyingthe ionic strength of the fibrillogenesis buffer. Under all otherconditions (variations of pH and temperature), no striated fibrilswere formed. Only when recombinant collagen was incubated in lowionic strength buffer (10 mM phosphate, pH 7) was striated fibrilassembly observed. Under these conditions, cross-banded fibrilswere predominant, and the measured periodicity was 65.9 ± 0.8nm which is close to the 67 nm expected value (Fig. 6, A-C). Underthe same conditions, Het I formed a meshwork of long and thinstriated fibrils (Fig. 6, D and E), whereas Hom I only aggregatedinto non-banded fibrils (Fig. 6F). The rColl I fibrils were particularlyheterogenous in size and morphology (Fig. 6, A-C). Fibril diametermeasurements confirmed that recombinant unhydroxylated fibrilspresented a broad distribution with a minimum at 15 nm and a maximumat about 500 nm. In contrast, Het I showed the highest frequencyof striated fibrils with a diameter of about 40 nm (Fig. 6G).

Kinetics Analyzer1

2009-01-29T04:23:00.000-08:00

Fibril formation in 10 mM phosphate was followed by monitoring turbidity at 313 nm as a function of time. The amplitude ofthe plateau value is a function of both the amount of reconstitutedfibrils and their final width. Fibrillogenesis kinetics underthese conditions (10 °C, 10 mM phosphate, pH 7) were instantaneouswhichever collagen was used. No significant difference was observedbetween the kinetics for Het I and rColl I (Fig. 7). The morphologyof the fibrils obtained after completion of turbidity measurementswas identical to that previously shown (Fig. 6). Thus, the formationof striated fibrils with unhydroxylated collagen, contrary tohydroxylated homotrimeric collagen I, is dependent on the lowionic strength of the buffer. As shown for native collagens, fibrilformation increased substantially the melting temperature of therecombinant collagen which reached 36 °C instead of 30 °C forthe isolated molecules (data not shown).

Kinetics Analyzer

2009-01-29T04:19:00.000-08:00

The study of collagen molecules that are correctly folded but lack hydroxyproline residues, as produced in transgenic plants,provides clues to understand how these residues contribute totriple-helical folding and, more strikingly, useful insights intothe molecular mechanisms that drivefibrillogenesis.

Role of Hydroxyproline in Collagen Folding-- Hydroxyproline plays an unquestionable role in the thermal stability of collagen molecules, where the number of hydroxyprolineresidues in the triple-helical domains is directly related tothe melting temperature. In collagen-like peptides that form triplehelices, the substitution of hydroxyproline for proline in theY-position of the repeating Gly-X-Y triplets increases the thermalstability by as much 15 °C (19, 20). As a result, the meltingtemperatures of recombinant unhydroxylated collagen I producedin plants and of fully hydroxylated parental collagen I homotrimershow distinct disparities (30 °C for the recombinant collagenversus 41 °C for the native bovine homotrimer). The role of hydroxyprolinein collagen folding is less clear. When hydroxylation is blockedby addition of ,'-dipyridyl, fibroblasts synthesize the unhydroxylatedform of procollagen I, known as protocollagen (21). Surprisingly,the data show that the folding of protocollagen is more efficientthan that of fully hydroxylated collagen. Apparently, becauseit lacks hydroxyproline, the stability of mismatched triple-helicalregions is decreased, and thus the protein is less likely to belocked into unfavorable conformations that impede the propagationstep. However, the folding of protocollagen I was found to beless efficient than the folding of collagen III. The latter formedfully aligned triple helices in vitro most likely because of thepresence of interchain disulfide bonds within the C-terminal endof the triple-helical domain (22). To avoid possible chainmisalignments that could disturb the folding rate, we chemicallycross-linked the collagen molecules before refolding the thermallydenatured protein. Identical fractions of folded triple heliceswere reached with fully hydroxylated collagen I homotrimer andrecombinant collagen indicating that, when cross-linked, the helix-to-coiltransition of both collagens was reversible. However, we observeda marked difference in the folding rate that was 5-fold fasterfor native homotrimeric collagen than for unhydroxylated recombinantcollagen.

The increased folding rate of collagen III in the presence of cis-trans-isomerase indicated that cis-trans-isomerization ofproline/hydroxyproline is a rate-limiting factor in collagen molecularassembly (23). In related work, substitution of (4R)-fluoroprolinefor (4R)-hydroxyproline in collagen-like peptides was shown toincrease the T_m of the triple helix (24). Because (4R)-fluoroprolinedid not provide a site for hydrogen bonding of water, these authorsproposed that an electron-withdrawing inductive effect of thehydroxyl or fluoro groups favors the trans-configuration of thepeptide bond required for formation of the triple helix. As aconsequence, the inductive effect of the hydroxyl group of hydroxyprolineenhances the stability of the triple helix by favoring the requisitetrans-conformation of the Hyp bond. In our results, the relativelyslow cis-trans-isomerization in the absence of hydroxyprolinethus likely became the rate-limiting factor for the propagationof the recombinant unhydroxylated collagen helix. In agreementwith this concept, the use of collagen-like peptides for refoldingstudies showed that all triplets of the form Gly-Xaa-Hyp promotedrapid folding, whereas Gly-Xaa-Pro triplets were less favorable(17).

Role of Hydroxyproline in Fibril Assembly-- Collagen self-assembly is an entropy-driven process that depends on ionic strength, pH, and temperature. Unlike hydroxylatedheterotrimers or homotrimers, we found that unhydroxylated homotrimersdid not assemble into striated fibrils under physiological bufferconditions. Instead, fibril formation of unhydroxylated collagenrequired both low ionic strength and temperature, the latter beingimposed by the relatively low thermal stability of themolecule.

To investigate further how the lack of hydroxylation might affect physical properties, we showed by dynamic light scatteringthat unhydroxylated collagen molecules appeared to be more compact/flexiblethan native collagen at temperatures between 11 and 25 °C. At25 °C, the compactness/flexibility of the recombinant collagenwas found to be comparable with that of the native heterotrimerat 35 °C, i.e. in both cases ~5 °C below their respective meltingtemperatures. Increased flexibility might actually favor fibrilformation because molecules are likely to adopt a bent conformationwithin fibrils (25-27). Thus the flexibility of the recombinantmolecule at 20-25 °C might be conducive to fibril formation. However,our data showed that at 20 °C unhydroxylated molecules did notform striated fibrils at physiological pH and ionic strength.In contrast, hydroxylated homotrimers and heterotrimers did assembleunder these conditions, despite their reduced flexibility. Thusdifferences in molecular flexibility do not appear to be a majorinfluence on the ability to form striatedfibrils.

Low ionic strength conditions appear therefore to be the major requirement for fibril formation of unhydroxylated collagenhomotrimers. Under the same conditions, hydroxylated bovine homotrimersform amorphous fibrils, whereas striated fibrils are formed atphysiological ionic strength. We propose that the absence of hydroxylationwould lead to an overall increase in hydrophobicity throughoutthe protein, thus masking specific hydrophobic interactions requiredfor ordered self-assembly. The salting out effect at physiologicalsalt concentrations would therefore be enhanced leading to amorphousprecipitation of unhydroxylated collagen. Lowering the ionic strengthwould reduce the hydrophobic effect and lead to ordered self-assemblythrough both electrostatic and hydrophobic interactions. In contrast,amorphous precipitation of bovine homotrimers at low ionic strengthmight be due to the relative weakness of specific hydrophobicinteractions under these conditions. Such an interpretation isfavored by our observation that heterotrimers formed striatedfibrils at low ionic strength, and by a previous report (5)that the greater hydrophobicity of the 2(I) chain compared withthe 1(I) chain leads to improved fibril formation of heterotrimers.In addition to possible differences in hydrophobicity, the presenceof hydroxyproline has been shown to enhance the hydration shellaround collagen-like peptides in such a way as to influence directlythe lateral packing of molecules in crystals (7-9). Direct Hyp-Hypinteractions between molecules have also been observed, particularlyin the structure of the triple-helical peptide (Pro-Hyp-Gly)₄-Glu-Lys-Gly-(Pro-Hyp-Gly)₅(11). Thus the lateral packing in fibrils of unhydroxylatedcollagen may differ from that in native collagenfibrils.

The integrity of the triple helix is a necessary prerequisite for correct fibril formation, and the telopeptides are knownto play a catalytic role (28). In our experiments recombinantcollagen was obtained in mature form, i.e. after removal of propeptidesbut without the use of pepsin, thereby leaving the telopeptidesmostly intact at the N terminus and with 3-5 residues remainingat the C terminus (data not shown). In contrast, both the bovineheterotrimer and homotrimer were isolated following pepsin extraction.All molecules were indistinguishable by analysis of SLS crystallitesformed in vitro. The observation that both bovine heterotrimersand homotrimers formed striated fibrils under physiological bufferconditions, whereas recombinant homotrimers with more intact telopeptidesdid not, further reinforces our conclusion that the differencesin fibril formation were due to the lack ofhydroxylation.

Fibril formation increases the thermal stability of native bovine collagen by as much as 10 °C (29). We showed that themelting temperature of recombinant collagen fibrils was also increasedby about 6 °C, showing that thermal stability is raised once theunhydroxylated molecules are stabilized by the fibril structure.This result might be of interest for its use as biomaterials.We conclude that hydroxyproline might contribute more than previouslythought to correct fibril formation under experimental conditionsmimicking those that can occur physiologically. Understandingthe molecular mechanisms that drive collagen self-assembly musttherefore take account of the role ofhydroxyproline.

Brimrose

2009-01-27T16:39:00.000-08:00

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2009-01-27T16:37:00.000-08:00

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