The Role of Cystine Knots in Collagen Folding and Stability, Part I. Conformational Properties of (Pro‐Hyp‐Gly)₅ and (Pro‐(4S)‐FPro‐Gly)₅ Model Trimers with an Artificial Cystine Knot

Abstract

In analogy to the cystine knots present in natural collagens, a simplified disulfide cross-link was used to analyse the conformational effects of a C-terminal artificial cystine knot on the folding of collagenous peptides consisting of solely (Pro-Hyp-Gly) repeating units. Assembly of the alpha chains into a heterotrimer by previously applied regioselective disulfide-bridging strategies failed because of the high tendency of (Pro-Hyp-Gly)(5) peptides to self-associate and form homotrimers. Only when side-chain-protected peptides were used, for example in the Hyp(tBu) form, and a new protection scheme was adopted, selective interchain-disulfide cross-linking into the heterotrimer in organic solvents was successful. This unexpected strong effect of the conformational properties on the efficiency of well-established reactions was further supported by replacing the Hyp residues with (4S)-fluoroproline, which is known to destabilise triple-helical structures. With the related [Pro-(4S)-FPro-Gly](5) peptides, assembly of the heterotrimer in aqueous solution proceeded in a satisfactory manner. Both the intermediates and the final fluorinated heterotrimer are fully unfolded in aqueous solution even at 4 degrees C. Conversely, the disulfide-crossbridged (Pro-Hyp-Gly)(5) heterotrimer forms a very stable triple helix. The observation that thermal unfolding leads to scrambling of the disulfide bridges was unexpected. Although NMR experiments support an extension of the triple helix into the cystine knot, thermolysis is not associated with the unfolding process. In fact, the unstructured fluorinated trimer undergoes an equally facile thermodegradation associated with the intrinsic tendency of unsymmetrical disulfides to disproportionate into symmetrical disulfides under favourable conditions. The experimental results obtained with the model peptides fully support the role of triple-helix nucleation and stabilisation by the artificial cystine knot as previously suggested for the natural cystine knots in collagens.

The Role of Cystine Knots in Collagen Folding and Stability, Part II. Conformational Properties of (Pro‐Hyp‐Gly)n Model Trimers with N‐ and C‐Terminal Collagen Type III Cystine Knots

“…[6] We demonstrate that the monomeric species have to prefold into the Figure 1. Sequence composition of the homotrimers I ± V whose syntheses were attempted or carried out in the present study; heterotrimer VI with the C-terminal artificial cystine knot [6] is reported for comparison.…”

“…ESI-MS and, particularly, the isotope pattern of FT-ICR-MS confirmed the disulfide-cross-linked trimeric composition of the compounds as illustrated, for example, for trimer II, in Figure 2. Although even the NMR spectra were consistent with a single set of cystine connectiv- [6] for comparison. ities (see below), the unambiguous proof for the presence of a single cystine knot isomer can only be expected from X-ray crystallographic analysis.…”

“…Oxidation experiments: The single chains were synthesised on solid supports by optimised procedures [6] using S-tert-butylthio protection for the cysteine residues. The tripeptide derivative Fmoc-Pro-Hyp(tBu)-Gly-OH was used as the synthon for (Pro-Hyp-Gly) n chain elongations according to Fmoc/tBu chemistry.…”

“…This successful oxidative assembly of homotrimeric collagenous peptides compelled us to use synthetic (Pro-Hyp-Gly) n peptides of increasing chain length to compare the triple-helix inducing/stabilising effects of this natural cystine knot with the effects of the simplified artificial one used in the preceding article. [6] Moreover, oxidative refolding experiments with such peptides of increasing (Pro-Hyp-Gly) triplet numbers were expected to address the question as to what extent optimally matched self-associated trimers in equilibrium with nonstructured monomers and mismatched triple helices is affecting the yields of correctly oxidised trimers.…”

Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane

“…29,31,[53][54][55] Modeling and molecular dynamics (MD) simulations served to optimally fit the cystine knot into the triple-helix without steric clashes, 29 a fact that was recently confirmed by NMR structural analysis of a model peptide containing such disulfide connectivities. 56 In view of the positive experiences with these disulfide-cross-linked collagenous heterotrimers and also because of the presence of a cystine knot downstream from the ␣1␤1 adhesion epitope, the two heterotrimers A and B ( Figure 2) were selected as possible mimics of the ␣1␤1 adhesion epitope of collagen type IV. 55 These trimers contain the sequence portions 457-468 of the aligned ␣1 and ␣2 chains of collagen type IV which encompass the residues 461 directly involved in binding to ␣1␤1 integrin.…”