NMR INVESTIGATIONS OF BIOMOLECULAR STRUCTURE OF PROINSULIN IN SOLUTION

溶液中胰岛素原生物分子结构的核磁共振研究

• 批准号: 6309185
• 负责人: MICHAEL Aaron WEISS
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309185
• 关键词: biological products; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; structural biology

The biosynthesis of insulin proceeds through a precursor protein, proinsulin. This larger protein (86 residues) folds to form three native disulfide bonds, which are retained in the proteolytic 2-chain fragment, insulin. Unfortunately, proinsulin is refractory to crystallization under all conditions successfully applied to insulin itself. The absence of structural information has blocked efforts to understand the role of the C-peptide and the targets for cleavage by the converting enzymes. We therefore propose to determine the solution structure of human proinsulin. Preliminary results strongly suggest that this can be accomplished using the Zn-coordinated hexamer at 55xC and pH 7. The thermal stability and structural rigidity of the hexamer provide high-resolution spectra to be obtained under these conditions. Because of the limited resolution in spin systems at the junctions between the connecting peptide and the A- and B-domains (sites of biological recognition by converting enzymes) and because isotopic labeling is impeded by inefficient expression in E. coli, we seek to obtain homonuclear data at the highest field strength.

胰岛素的生物合成通过前体蛋白进行， 胰岛素原 这种较大的蛋白质（86个残基）折叠形成三个 天然二硫键，保留在蛋白水解2-链中 片段胰岛素 不幸的是，胰岛素原对 在所有条件下结晶成功应用于胰岛素 本身 由于缺乏结构信息， 了解C肽的作用和切割的靶点， 转化酶。 因此，我们建议确定 人胰岛素原的溶液结构。 初步结果显示， 建议这可以使用锌配位的六聚体来完成 在55 ° C和pH 7下。 热稳定性和结构刚性 六聚体提供了在这些条件下获得高分辨率光谱 条件 由于自旋系统的分辨率有限， 连接肽与A和B结构域之间的连接 （转化酶的生物识别位点），因为 同位素标记受到E.大肠杆菌，我们 设法在最高场强下获得人体数据。