Disulfide-bridged heterotrimeric collagen peptides containing the collagenase cleavage site of collagen type I. Synthesis and conformational properties

Collagenous peptides containing the collagenase cleavage site alpha 1(772-7 84) and alpha 2(772-784) of collagen type I were synthesized and assembled into heterotrimers via regioselective C-terminal interchain-disulfide bridg ing 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 portio n of the collagenase cleavage site with its weak triple-helix propensity. B y N-terminal chain elongation with (Gly-Pro-Hyp), tripeptide repeats, parti cularly with n = 5, and in an even more pronounced manner, by incorporation of an additional tripeptide repeat adjacent to the cystine knot, a collage nous heterotrimer was obtained which was found to exhibit dichroic properti es fully consistent with the triple-helical fold. Thermal denaturation reve aled a remarkable stability with a melting temperature of 41 degrees C. Alt hough the complex cystine knot of natural collagen was reduced in these syn thetic heterotrimers to two interchain-disulfide bridges, it showed not onl y the expected entropic contribution to the refolding process by keeping th e three chains assembled, but more importantly a triple-helix nucleation wa s 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 i somerization for triple-helix propagation. These results would indicate tha t even the simplified artificial cystine knot is capable of aligning the th ree peptide chains in the defined alpha 1 alpha 2 alpha 1' one-residue shif t register. Moreover, the synthetic heterotrimers were cleaved by interstit ial collagenases in a single cut through all three chains without release o f intermediates during the relatively slow enzymatic digestion process. Thi s observation confirms that, with the de novo designed heterotrimers, funct ional collagen epitopes were mimicked in highly efficient manner; it also s trongly suggests that the preselected alpha 1 alpha 2 alpha 1' register may indeed represent the correct staggered alignment of the a subunits at leas t in collagen type I.