Kinetics Analyzer3

The capacity of the recombinant collagen to form supra-molecular structures was studied using SLS crystallites, an artificial mode of molecular packing in which polyanions such as ATP cross-bridge positively charged collagen molecules to induce lateral register aggregation. SLS crystallites were obtained with recombinant collagen that displayed a characteristic asymmetric cross-banding pattern after positive staining (Fig. 4). Their average length (261 ± 11 nm) corresponded to that of single molecules and was consistent with the lengths of SLS crystallites formed from bovine heterotrimer and homotrimer (271 ± 10 and 276 ± 7 nm, respectively). Moreover, alignment of all three forms of SLS crystallites revealed an overall similarity in the banding pattern indicating an identical distribution of polar residues (Fig. 4D).

Kinetics Analyzer2

Fibril Formation Competence of rColl I-- Neutralizing the pH and increasing the temperature of purified collagen molecules in solution generally provokes the formation of a gel containing striated fibrils. Thus increasing the temperature to 34-35 °C clearly favors fibril formation of native collagen molecules. Because of the low T_m (30 °C) of the unhydroxylated recombinant collagen, fibril formation experiments could not be carried out at this temperature. Therefore, the capacity of the unhydroxylated recombinant collagen to form fibrils was explored by performing experiments at 20 °C. Purified rColl I molecules were incubated in PBS, a physiological buffer that normally allows fibril formation. As controls, fibril formation of bovine homotrimer and heterotrimer was carried out under the same conditions. As expected, a meshwork of fibrils exhibiting the typical D-periodic banding pattern was observed when Het I and Hom I solutions were incubated in PBS at 20 °C (Fig. 5, B and C). In contrast, negative staining of rColl I aggregates formed in PBS showed thin filamentous structures devoid of D-periodic striation (Fig. 5A). To check whether potential traces of plant contaminants might have prevented striated fibril formation, bovine homotrimeric molecules were mixed with tobacco crude extract and then subjected to salt fractionation. Negative staining of the fibrils formed with this preparation showed the presence of fibrils essentially identical to those observed with reconstituted standard homotrimeric collagen I (data not shown).

Kinetics Analyzer1

Critical conditions allowing formation of striated fibrils from unhydroxylated recombinant collagen were identified by varying the ionic strength of the fibrillogenesis buffer. Under all other conditions (variations of pH and temperature), no striated fibrils were formed. Only when recombinant collagen was incubated in low ionic strength buffer (10 mM phosphate, pH 7) was striated fibril assembly observed. Under these conditions, cross-banded fibrils were predominant, and the measured periodicity was 65.9 ± 0.8 nm which is close to the 67 nm expected value (Fig. 6, A-C). Under the same conditions, Het I formed a meshwork of long and thin striated fibrils (Fig. 6, D and E), whereas Hom I only aggregated into non-banded fibrils (Fig. 6F). The rColl I fibrils were particularly heterogenous in size and morphology (Fig. 6, A-C). Fibril diameter measurements confirmed that recombinant unhydroxylated fibrils presented a broad distribution with a minimum at 15 nm and a maximum at about 500 nm. In contrast, Het I showed the highest frequency of striated fibrils with a diameter of about 40 nm (Fig. 6G).

Kinetics Analyzer1

Fibril formation in 10 mM phosphate was followed by monitoring turbidity at 313 nm as a function of time. The amplitude of the plateau value is a function of both the amount of reconstituted fibrils and their final width. Fibrillogenesis kinetics under these conditions (10 °C, 10 mM phosphate, pH 7) were instantaneous whichever collagen was used. No significant difference was observed between the kinetics for Het I and rColl I (Fig. 7). The morphology of the fibrils obtained after completion of turbidity measurements was identical to that previously shown (Fig. 6). Thus, the formation of striated fibrils with unhydroxylated collagen, contrary to hydroxylated homotrimeric collagen I, is dependent on the low ionic strength of the buffer. As shown for native collagens, fibril formation increased substantially the melting temperature of the recombinant collagen which reached 36 °C instead of 30 °C for the isolated molecules (data not shown).

Kinetics Analyzer

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 to triple-helical folding and, more strikingly, useful insights into the molecular mechanisms that drive fibrillogenesis.

Role of Hydroxyproline in Collagen Folding-- Hydroxyproline plays an unquestionable role in the thermal stability of collagen molecules, where the number of hydroxyproline residues in the triple-helical domains is directly related to the melting temperature. In collagen-like peptides that form triple helices, the substitution of hydroxyproline for proline in the Y-position of the repeating Gly-X-Y triplets increases the thermal stability by as much 15 °C (19, 20). As a result, the melting temperatures of recombinant unhydroxylated collagen I produced in plants and of fully hydroxylated parental collagen I homotrimer show distinct disparities (30 °C for the recombinant collagen versus 41 °C for the native bovine homotrimer). The role of hydroxyproline in collagen folding is less clear. When hydroxylation is blocked by addition of ,'-dipyridyl, fibroblasts synthesize the unhydroxylated form of procollagen I, known as protocollagen (21). Surprisingly, the data show that the folding of protocollagen is more efficient than that of fully hydroxylated collagen. Apparently, because it lacks hydroxyproline, the stability of mismatched triple-helical regions is decreased, and thus the protein is less likely to be locked into unfavorable conformations that impede the propagation step. However, the folding of protocollagen I was found to be less efficient than the folding of collagen III. The latter formed fully aligned triple helices in vitro most likely because of the presence of interchain disulfide bonds within the C-terminal end of the triple-helical domain (22). To avoid possible chain misalignments that could disturb the folding rate, we chemically cross-linked the collagen molecules before refolding the thermally denatured protein. Identical fractions of folded triple helices were reached with fully hydroxylated collagen I homotrimer and recombinant collagen indicating that, when cross-linked, the helix-to-coil transition of both collagens was reversible. However, we observed a marked difference in the folding rate that was 5-fold faster for native homotrimeric collagen than for unhydroxylated recombinant collagen.

The increased folding rate of collagen III in the presence of cis-trans-isomerase indicated that cis-trans-isomerization of proline/hydroxyproline is a rate-limiting factor in collagen molecular assembly (23). In related work, substitution of (4R)-fluoroproline for (4R)-hydroxyproline in collagen-like peptides was shown to increase the T_m of the triple helix (24). Because (4R)-fluoroproline did not provide a site for hydrogen bonding of water, these authors proposed that an electron-withdrawing inductive effect of the hydroxyl or fluoro groups favors the trans-configuration of the peptide bond required for formation of the triple helix. As a consequence, the inductive effect of the hydroxyl group of hydroxyproline enhances the stability of the triple helix by favoring the requisite trans-conformation of the Hyp bond. In our results, the relatively slow cis-trans-isomerization in the absence of hydroxyproline thus likely became the rate-limiting factor for the propagation of the recombinant unhydroxylated collagen helix. In agreement with this concept, the use of collagen-like peptides for refolding studies showed that all triplets of the form Gly-Xaa-Hyp promoted rapid 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 hydroxylated heterotrimers or homotrimers, we found that unhydroxylated homotrimers did not assemble into striated fibrils under physiological buffer conditions. Instead, fibril formation of unhydroxylated collagen required both low ionic strength and temperature, the latter being imposed by the relatively low thermal stability of the molecule.

To investigate further how the lack of hydroxylation might affect physical properties, we showed by dynamic light scattering that unhydroxylated collagen molecules appeared to be more compact/flexible than native collagen at temperatures between 11 and 25 °C. At 25 °C, the compactness/flexibility of the recombinant collagen was found to be comparable with that of the native heterotrimer at 35 °C, i.e. in both cases ~5 °C below their respective melting temperatures. Increased flexibility might actually favor fibril formation because molecules are likely to adopt a bent conformation within fibrils (25-27). Thus the flexibility of the recombinant molecule at 20-25 °C might be conducive to fibril formation. However, our data showed that at 20 °C unhydroxylated molecules did not form striated fibrils at physiological pH and ionic strength. In contrast, hydroxylated homotrimers and heterotrimers did assemble under these conditions, despite their reduced flexibility. Thus differences in molecular flexibility do not appear to be a major influence on the ability to form striated fibrils.

Low ionic strength conditions appear therefore to be the major requirement for fibril formation of unhydroxylated collagen homotrimers. Under the same conditions, hydroxylated bovine homotrimers form amorphous fibrils, whereas striated fibrils are formed at physiological ionic strength. We propose that the absence of hydroxylation would lead to an overall increase in hydrophobicity throughout the protein, thus masking specific hydrophobic interactions required for ordered self-assembly. The salting out effect at physiological salt concentrations would therefore be enhanced leading to amorphous precipitation of unhydroxylated collagen. Lowering the ionic strength would reduce the hydrophobic effect and lead to ordered self-assembly through both electrostatic and hydrophobic interactions. In contrast, amorphous precipitation of bovine homotrimers at low ionic strength might be due to the relative weakness of specific hydrophobic interactions under these conditions. Such an interpretation is favored by our observation that heterotrimers formed striated fibrils at low ionic strength, and by a previous report (5) that the greater hydrophobicity of the 2(I) chain compared with the 1(I) chain leads to improved fibril formation of heterotrimers. In addition to possible differences in hydrophobicity, the presence of hydroxyproline has been shown to enhance the hydration shell around collagen-like peptides in such a way as to influence directly the lateral packing of molecules in crystals (7-9). Direct Hyp-Hyp interactions between molecules have also been observed, particularly in 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 unhydroxylated collagen may differ from that in native collagen fibrils.

The integrity of the triple helix is a necessary prerequisite for correct fibril formation, and the telopeptides are known to play a catalytic role (28). In our experiments recombinant collagen was obtained in mature form, i.e. after removal of propeptides but without the use of pepsin, thereby leaving the telopeptides mostly intact at the N terminus and with 3-5 residues remaining at the C terminus (data not shown). In contrast, both the bovine heterotrimer and homotrimer were isolated following pepsin extraction. All molecules were indistinguishable by analysis of SLS crystallites formed in vitro. The observation that both bovine heterotrimers and homotrimers formed striated fibrils under physiological buffer conditions, whereas recombinant homotrimers with more intact telopeptides did not, further reinforces our conclusion that the differences in fibril formation were due to the lack of hydroxylation.

Fibril formation increases the thermal stability of native bovine collagen by as much as 10 °C (29). We showed that the melting temperature of recombinant collagen fibrils was also increased by about 6 °C, showing that thermal stability is raised once the unhydroxylated 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 previously thought to correct fibril formation under experimental conditions mimicking those that can occur physiologically. Understanding the molecular mechanisms that drive collagen self-assembly must therefore take account of the role of hydroxyproline.

Brimrose

Brimrose Corporation of America is the premier manufacturer of Electro-Optic solutions for the Optical Communication industry, Pharmaceutical and Chemical process-control fields, the NASA/Jet Propulsion Laboratory Spaceflight Center and for various Military DOD facilities.
We proudly carry the principles of world class quality and service throughout our organization.
For more information, download our Brimrose Capabilities PDF file and check it out for yourself...

AOTF NIR Miniature SPECTROMETER

Brimrose's new generation of solid-state AOTF-NIR Miniature "Hand-Held" Analyzers are designed for PORTABLE non-destructive contact/non-contact measurements of chemical and physical properties of powers, solids, liquids, gels, etc. in diffuse reflectance mode.
Miniature design helps to have more PORTABLE solutions. Additional probe attachments are available for analyzing liquid transmission measurements.