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: 6298182
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6298182
• 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。这种更大的蛋白质(86个残基)折叠成三个 保留在蛋白水解性2-链中的天然二硫键 碎片，胰岛素。不幸的是，胰岛素原对 全条件结晶成功应用于胰岛素 它本身。缺乏结构性信息阻碍了以下努力 了解C-肽的作用和被切割的靶点 转化酶。因此，我们建议厘定 人胰岛素原的溶液结构。初步结果强劲 建议可以使用锌配位的六角体来实现这一点 在55×C和pH 7的条件下，其热稳定性和结构刚性 六角体提供将在这些条件下获得高分辨率光谱 条件。因为自旋系统的分辨率有限 连接肽与A、B结构域之间的连接 (通过转换酶进行生物识别的位置)以及因为 同位素标记因在大肠杆菌中的低效表达而受阻 寻求在最高场强下获得同核数据。