PROTEIN BINDING SITES BY SOLIDS NMR

通过固体 NMR 测定蛋白质结合位点

• 批准号: 2180487
• 负责人: JACOB SCHAEFER
• 金额: $ 15.16万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180487
• 关键词: Bacillus subtilis; bacterial proteins; cell wall; chemical addition; chemical binding; computer simulation; covalent bond; electron nuclear double resonance spectroscopy; enzyme structure; fatty acid binding protein; inhibitor /antagonist; intestines; ionic bond; ligase; methanol; nuclear magnetic resonance spectroscopy; nuclease; peptidoglycan; protein structure; proteins; shikimate; thermolysin; tropomyosin

We propose to use rotational-echo, double- and triple-resonance solids NMR to measure interatomic distances between stable-isotope labels in amino-acid residues at or near binding sites of large proteins, and stable-isotope labels in substrate analogues or inhibitors bound to the proteins. These experiments can be performed on one multi-labeled site per 600 residues and will measure C-N, C-P, and C-F distances as great as 5, 8, and 12 A, respectively, with an accuracy of +/-0.5 A. Collaborations with five laboratories have been established to perform labeling of the proteins. The proteins to be examined include (1) rat intestinal fatty-acid binding protein, (2) methanol dehydrogenase, (3) 5-enolpyruvylshikimate-3-phosphate synthase, (4) plant glutamine synthetase, (5) thermolysin, and (6) tropomyosin. Analysis of these proteins and their complexes in both lyophilized and microcrystalline forms will be aided by continued development of new analytical techniques including (1) weak-pulse rotational-echo double resonance (REDOR), (2) many-cycle REDOR, (3) 17 O 13C REDOR, and (4) homonuclear REDOR. Computer simulations of rotational-echo triple-resonance (RETRO) NMR experiments will yield information about distances and orientations of clusters of three stable-isotope labels. RETRO NMR permits a general approach to the problem of identifying single sites in large, immobilized protein complexes with total [suppression of interferences from the natural-abundance background.

我们建议使用旋转回波，双重和三个共振 固体NMR测量稳定 - 同位素标签之间的原子间距离 在大蛋白的结合位点或附近的氨基酸残基中，以及 底物类似物或抑制剂中的稳定异位标记 蛋白质。 这些实验可以在一个多标签的站点上进行 每600个残留物，将测量C-N，C-P和C-F距离 5、8和12 a的精度分别为+/- 0.5。 已经建立了与五个实验室合作以执行 蛋白质的标记。 要检查的蛋白质包括（1）大鼠 肠道脂肪酸结合蛋白，（2）甲醇脱氢酶，（3） 5-烯醇硫化物-3-磷酸合酶，（4）植物谷氨酰胺 合成酶，（5）热溶酶素和（6）tropomyosin。 这些分析 蛋白质及其复合物在冻干和微晶 持续开发新的分析技术将有助于表格 包括（1）弱脉冲旋转 - 回声双共振（重复），（2） 多循环的重新多，（3）17 O 13C重新倍数和（4）同核重复。 旋转回波三谐（retro）NMR的计算机模拟 实验将产生有关距离和方向的信息 三个稳定同位标签的簇。 retro nmr允许一般 解决大型，固定的单个站点的问题 蛋白质复合物具有完全的[抑制来自 自然背景。