DISULFIDE STRUCTURE OF BIOMEDICALLY IMPORTANT PROTEINS

二硫键是一个至关重要的决定因素的形状，因此，一些生物医学上重要的蛋白质的活性。常见的出血性疾病，血管性血友病，似乎与破坏正常血液凝固级联的特定蛋白质（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。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对传统的二硫键定位的蛋白水解方法具有抗性。对TGF-β受体结合蛋白中二硫键模式的了解将有助于为开发用于治疗纤维化病症的药物（拮抗剂）提供重要的“模板”，杜兴氏肌营养不良症;类似地，其它药物（激动剂）的开发可通过促进对TGF-β的负增殖反应而用作抗癌剂。然而，高度打结的富含半胱氨酸（多达12个半胱氨酸，其中3个是相邻的）的TGF-β受体结合蛋白对常规二硫键图谱具有抗性。开发VEGF同源二聚体的二硫键作图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正确折叠提供基础。 我们的二硫键映射的新方法，基于半胱氨酸残基的氰基化和切割，为确定上述生物医学上重要的胱氨酸蛋白质的二硫键模式提供了新的希望，这些蛋白质是传统方法难以处理的。氰化对游离巯基是选择性的，并且可以在pH 3下完成，pH 3是抑制二硫键乱序问题的条件。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，这是推荐其成功攻击本文所述蛋白质所带来的困难分析挑战的属性。将开发一个a1 taxm来分配二硫键中半胱氨酸的连接性，给出氨基酸序列和氰基化/裂解产物的质谱的输入。

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.