DISULFIDE STRUCTURE OF BIOMEDICALLY IMPORTANT PROTEINS

生物医学重要蛋白质的二硫结构

• 批准号: 6498729
• 负责人: JACK T WATSON
• 金额: $ 22.32万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498729
• 关键词: acidity /alkalinity; chemical cleavage; chemical models; chemical reaction; cysteine; dimer; disulfide bond; growth factor receptors; intermolecular interaction; mass spectrometry; mathematics; method development; nuclear magnetic resonance spectroscopy; papain; protein sequence; protein structure; recombinant proteins; sulfhydration; sulfhydryl reagents; transforming growth factors; vascular endothelial growth factors; von Willebrand factor

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.

二硫键是形状的极其重要的决定因素，因此也是一些生物医学上重要的蛋白质的活性的决定因素。常见的出血性疾病冯维勒布兰德病似乎与特定蛋白质 (VWF) 的 18 个半胱氨酸（包括两对相邻半胱氨酸）之间的二硫键模式缺陷有关，该缺陷会破坏正常的凝血级联。人们对血管性血友病蛋白（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 对二硫键图谱的传统蛋白水解方法具有抵抗力。了解TGF-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（例如杜氏肌营养不良症）的药物（拮抗剂）提供重要的“模板”；同样，其他药物（激动剂）的开发可以通过促进对 TGF-β 的负增殖反应来充当抗癌剂。然而，高度打结、富含半胱氨酸（最多 12 个半胱氨酸，其中 3 个相邻）的 TGF-β 受体结合蛋白对传统的二硫键图谱具有抗性。开发 VEGF 同二聚体二硫键图谱的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。 我们基于半胱氨​​酸残基的氰基化和裂解的二硫键图谱新方法，为确定上述生物医学上重要的胱氨酸蛋白的二硫键模式提供了新的希望，而传统方法难以解决这些问题。氰基化对游离巯基具有选择性，并且可以在 pH 3 下完成，该条件可以抑制二硫键乱序问题。我们已经证明氰基化/裂解方法适用于含有相邻半胱氨酸的蛋白质，这一属性推荐它成功地解决本文描述的蛋白质带来的困难分析挑战。将开发一种算法来指定二硫键中半胱氨酸的连接性，输入氨基酸序列和氰基化/裂解产物的质谱。