DISULFIDE STRUCTURE OF BIOMEDICALLY IMPORTANT PROTEINS

二硫键对某些生物医学上重要的蛋白质的形状和活性起着至关重要的决定作用。常见的出血性疾病，血管性血友病，似乎与一种特定蛋白质（VWF）的18个半胱氨酸（包括两对相邻的半胱氨酸）之间的二硫键模式缺陷有关，这种缺陷破坏了正常的血凝级联。关于血管性血友病蛋白（VWF）的半胱氨酸状态知之甚少，这些知识将有助于在分子水平上解释这种疾病的原因；然而，VWF对传统的蛋白水解方法进行二硫定位具有抗性。了解tgf - β受体结合蛋白中的二硫键模式将有助于为开发用于治疗纤维化疾病的药物（拮抗剂）提供重要的“模板”。，杜氏肌营养不良症；同样，其他药物（激动剂）的开发可能通过促进对tgf - β的负增殖反应来作为抗癌药物。然而，高度打结的富含半胱氨酸的tgf - β受体结合蛋白（多达12个半胱氨酸，其中3个相邻）对传统的二硫定位具有抗性。开发一种血管内皮生长因子二聚体的二硫定位方案将为设计和监测具有血管生成活性的相关药物蛋白的适当折叠提供基础。我们基于半胱氨酸残基氰化和切割的新方法，为确定上述生物医学上重要的半胱氨酸蛋白的二硫键模式提供了新的希望。氰基化对游离巯基是有选择性的，可以在pH为3的条件下完成，这种条件抑制了二硫化物乱序的问题。我们已经证明，氰基化/切割方法适用于含有相邻半胱氨酸的蛋白质，这一特性推荐它成功地攻击本文描述的蛋白质所带来的困难分析挑战。在输入氨基酸序列和氰化/切割产物的质谱的情况下，将开发一种算法来分配二硫键中半胱氨酸的连通性。

Disulfide bonds are a critically important determinant of the shape and, thus, the activity of some biomedically important proteins. The common bleeding disorder, von Willebrand Disease, appears to be related to a defect in the disulfide bonding pattern among 18 cysteines (including two pairs of adjacent cysteines) of a particular protein (VWF) that disrupts the normal blood clotting cascade. Very little is known about the cysteine status of the von Willebrand protein (VWF), and such knowledge will help explain the cause of this disorder at the molecular level; however, VWF is resistant to the conventional proteolytic approach to disulfide mapping. Knowledge of the disulfide bonding pattern in the receptor- binding proteins for TGF-beta will help provide an important 'template' for the development of drugs (antagonists) for treatment of fibrotic disorders ,e.g., Duchennes muscular dystrophy; similarly, the development of other drugs (agonists) may serve as anti-cancer agents by promoting the negative proliferative response to TGF-beta. However, the highly knotted, cysteine-rich (up to 12 cysteines, 3 of which are adjacent) receptor-binding proteins for TGF-beta are resistant to conventional disulfide mapping. Developing a protocol for the disulfide mapping of VEGF homodimer will provide the basis for designing and monitoring the proper folding of related pharmaceutical proteins with angiogenic activity. Our novel approach to disulfide mapping, based on cyanylation of and cleavage at cysteine residues, offers new hope for determining the disulfide bonding pattern of the biomedically important cystinyl proteins described above that are refractory to conventional methodology. Cyanylation is selective for free sulfhydryls and can be accomplished at pH 3, a condition that suppresses problems with disulfide scrambling. We have demonstrated that the cyanylation/cleavage approach is applicable to proteins containing adjacent cysteines, an attribute that recommends it for successfully attacking the difficult analytical challenges posed by the proteins described herein. An a1gorithm will be developed to assign the connectivity of cysteines in disulfide bonds given an input of amino acid sequence and mass spectra of cyanylation/cleavage products.