In mature collagen type III the homotrimer is C-terminally cross-linked by an interchain cystine knot consisting of three disulfide bridges of unknown connectivity. This cystine knot with two adjacent cysteine residues on each of the three alpha chains has recently been used for the synthesis and expression of model homotrimers. To investigate the origin of correct interchain cysteine pairings, (Pro-Hyp-Gly)(n) peptides of increasing triplet number and containing the biscysteinyl sequence C- and N-terminally were synthesised. The possibilities were that this origin may be thermodynamically coupled to the formation of the collagen triple helix as happens in the oxidative folding of proteins, or it could represent a post-folding event. Only with five triplets, which is known to represent the minimum number for self-association of collagenous peptides into a triple helix, air-oxidation produces the homotrimer in good yields (70 %), the rest being intrachain oxidised monomers. Increasing the number of triplets has no effect on yield suggesting the formation of kinetically trapped intermediates, which are not reshuffled by the glutathione redox buffer. N-terminal incorporation of the cystine knot is significantly less efficient in the homotrimerisation step and also in terms of triple-helix stabilisation. Compared to an artificial C-terminal cystine knot consisting of two interchain disulfide bridges, the collagen type III cystine knot produces collagenous homotrimers of remarkably high thermostability, although the concentration-independent refolding rates are not affected by the type of disulfide bridging. Since the natural cystine knot allows ready access to homotrimeric collagenous peptides of significantly enhanced triple-helix thermostability it may well represent a promising approach for the preparation of collagen-like innovative biomaterials. Conversely, the more laborious regioselectively formed artificial cystine knot still represents the only synthetic strategy for heterotrimeric collagenous peptides.

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 triple-helical fold of collagen requires the presence of a glycine residue at every third position in the peptide sequence and is stabilized by proline and (4R)-4-hydroxyproline residues in positions Xaa and Yaa of the (Xaa-Yaa-Gly) triplets, respectively. Regular down/up puckering of these Xaa/Yaa residues is possibly responsible for the tight packing of the three peptide strands, which have a polyproline-II-like structure, into the supercoiled helix. (4R)-Configured electronegative substituents such as a hydroxy group or a fluorine substituent on the pyrrolidine ring of the residue in the Yaa position favor the up pucker and thus significantly stabilize the triple helix. A similar effect was expected from the corresponding (4S)-isomers in the Xaa positions, but the opposite effect has been observed with (4S)-hydroxyproline, a result that has been speculatively attributed to steric effects. In this study, (4R)- and (4S)-fluoroproline residues were introduced into the Xaa position and potential steric effects were thus avoided. Contrary to expectations, (4S)-fluoroproline prevents triple-helix formation, whereas (4R)-fluoroproline stabilizes the polyPro II conformation, but without supercoiling of the three strands. The latter observation suggests that folding of the single chains into a polyproline II helix is not directly associated with triple helix formation and that fine tuning of van der Waals contacts, electrostatic interactions, and stereoelectronic effects is required for optimal packing into a triple helix.

The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultrastructural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteinerich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.The nematocyst is a unique subcellular organelle produced during a highly ordered secretion and assembly process of proteins into a giant post-Golgi vesicle (1). Nematocysts are of varying morphology and serve defensive or locomotory functions in cnidarians including hydroids, jellyfish, sea anemones, and corals. Nematocyst discharge, triggered by mechanical or chemical stimulation, is one of the fastest processes in biology driven by the extreme internal pressure of the capsule (15 MPa) and the gain of volume during exocytosis of the inverted tubule (2). The wall of the nematocyst, as an adaptation to this mechanical stress, is expected to combine both high resistance and flexibility. Investigation of the wall surface by atomic force microscopy (AFM) 1 and field emission scanning electron microscopy (FESEM) revealed a dense globular structure that could be removed either mechanically by the cantilever tip or chemically by dithiothreitol treatment exposing a smooth layer with a fibrous, collagen-like appearance (3). A preliminary structural model was proposed in which collagen molecules are assembled to fibers forming the inner wall covered by an outer layer of globular particles (3, 4).A major constituent of the capsule wall is a family of unusually short collagens, termed minicollagens (5). They comprise a central collagen triple helix with 12-16 Gly-X-Y repeats flanked by polyproline stretches and terminal cysteine-rich domains (MCRDs) with a conserved pattern of six closely set cysteines. Minicollagens are trimeric molecules that are expressed as soluble precursors, which during nematocyst maturation polymerize by a switch in the disulfide linkage from intramolecular to intermolecular connections (1, 6). This process is accompanied by a loss of minicollagen antibody reactivity in the head and tentacles regions of Hydra in mature nematocysts (1).NOWA is a 90-kDa glycoprotein that has been described to be associated with the globular structure of the nematocyst outer surface (4, 5). Interestingly, the molecular architecture of NOWA comprises a C-terminal octarepeat of the minicollagen cysteine-rich domain, suggesting a p...

Conversion of single-chain or disulfide-bridged dimeric collagenous peptides into Cys(Npys) derivatives as activated species for subsequent regioselective thiol/disulfide exchange reactions leads to side products whose origin and nature was determined by HPLC and ESI-MS. In both cases the high tendency of the educts to self-associate into triple-helical homotrimers, as assessed by their dichroic properties in the reaction media, is responsible for the failure of this well established cysteine chemistry. Only by optimizing the synthetic strategy or by exploiting a kinetic control of the reaction, could these conformation-dependent limitations be more or less efficiently bypassed for the regioselective assembly of heterotrimeric collagen model peptides crosslinked with artificial cystine knots.