Barth Dirk, Kyrieleis Otto, Frank Sabine, Renner Christian, Moroder Luis
Max-Planck-Institute für Biochemie, Am Klopferspitz 18 A, 82152 Martinsried, Germany.
Chemistry. 2003 Aug 4;9(15):3703-14. doi: 10.1002/chem.200304918.
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.
在成熟的III型胶原蛋白中，同三聚体通过由三个连接方式未知的二硫键构成的链间胱氨酸结在C端发生交联。这种在三条α链上每条都带有两个相邻半胱氨酸残基的胱氨酸结最近已被用于模型同三聚体的合成和表达。为了研究正确链间半胱氨酸配对的起源，合成了具有递增三联体数量且在C端和N端含有双半胱氨酰序列的(Pro-Hyp-Gly)(n)肽。其起源可能在热力学上与胶原蛋白三螺旋的形成相关联，就像蛋白质的氧化折叠过程那样，或者它可能代表一种折叠后事件。只有具有五个三联体（已知这是胶原肽自组装成三螺旋的最小数量）时，空气氧化才能以良好的产率（70%）产生同三聚体，其余的是链内氧化的单体。增加三联体的数量对产率没有影响，这表明形成了动力学捕获的中间体，它们不会被谷胱甘肽氧化还原缓冲液重新排列。在同三聚化步骤以及三螺旋稳定方面，胱氨酸结在N端的掺入效率显著较低。与由两个链间二硫键组成的人工C端胱氨酸结相比，III型胶原蛋白胱氨酸结产生的胶原同三聚体具有非常高的热稳定性，尽管与浓度无关的重折叠速率不受二硫键连接类型的影响。由于天然胱氨酸结能够方便地获得三螺旋热稳定性显著增强的同三聚体胶原肽，它很可能代表了一种制备类胶原创新生物材料的有前景的方法。相反，更费力地通过区域选择性形成的人工胱氨酸结仍然是制备异三聚体胶原肽的唯一合成策略。
