DISULFIDE STRUCTURE OF BIOMEDICALLY IMPORTANT PROTEINS

二硫键是一些生物医学上重要的蛋白质形状和活性的关键决定因素。常见的出血性疾病，von Willebrand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，该缺陷扰乱了正常的血液凝血级联反应。人们对von Willebrand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对传统的蛋白质分解方法二硫键作图具有抵抗力。了解转化生长因子-β受体结合蛋白中的二硫键模式将有助于为开发治疗纤维化疾病(如Duchennes肌营养不良症)的药物(拮抗剂)提供重要的‘模板’；类似地，其他药物(激动剂)的开发可能通过促进对转化生长因子-β的负增殖反应而起到抗癌作用。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)的转化生长因子-β受体结合蛋白对传统的二硫键映射具有抵抗力。建立血管内皮生长因子同源二聚体的二硫键图谱分析方法，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依据。我们基于半胱氨酸残基的氰化和裂解的二硫键作图的新方法，为确定上述生物医学上重要的半胱氨酸基蛋白质的二硫键模式提供了新的希望，这些模式是传统方法所不能解决的。氰化对游离的巯基是有选择性的，可以在pH为3的条件下完成，这种条件可以抑制二硫键的扰乱问题。我们已经证明了氰化/裂解方法适用于含有相邻半胱氨酸的蛋白质，这一属性建议它成功地攻击本文描述的蛋白质构成的困难的分析挑战。在给定氨基酸序列和氰化/裂解产物的质谱图的情况下，将开发一种算法来指定二硫键中半胱氨酸的连接性。

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.