Collagenous peptides containing the collagenase cleavage site alpha 1(772-784) and alpha 2(772-784) of collagen type I were synthesized and assembled into heterotrimers via regioselective C-terminal interchain-disulfide bridging in a defined alpha 1 alpha 2 alpha 1＇ staggered register off the three peptide strands. Various approaches were attempted to induce and stabilize the collagen-characteristic triple-helical fold even in the sequence portion of the collagenase cleavage site with its weak triple-helix propensity. By N-terminal chain elongation with (Gly-Pro-Hyp), tripeptide repeats, particularly with n = 5, and in an even more pronounced manner, by incorporation of an additional tripeptide repeat adjacent to the cystine knot, a collagenous heterotrimer was obtained which was found to exhibit dichroic properties fully consistent with the triple-helical fold. Thermal denaturation revealed a remarkable stability with a melting temperature of 41 degrees C. Although the complex cystine knot of natural collagen was reduced in these synthetic heterotrimers to two interchain-disulfide bridges, it showed not only the expected entropic contribution to the refolding process by keeping the three chains assembled, but more importantly a triple-helix nucleation was induced. In fact, temperature jump experiments clearly revealed two-phase refolding kinetics very similar to those of the disulfide-bridged natural collagen fragment of Col 1-3, where refolding without nucleation difficulty was obtained followed by a slower process dominated by the cis --> trans isomerization for triple-helix propagation. These results would indicate that even the simplified artificial cystine knot is capable of aligning the three peptide chains in the defined alpha 1 alpha 2 alpha 1＇ one-residue shift register. Moreover, the synthetic heterotrimers were cleaved by interstitial collagenases in a single cut through all three chains without release of intermediates during the relatively slow enzymatic digestion process. This observation confirms that, with the de novo designed heterotrimers, functional collagen epitopes were mimicked in highly efficient manner; it also strongly suggests that the preselected alpha 1 alpha 2 alpha 1＇ register may indeed represent the correct staggered alignment of the a subunits at least in collagen type I.