STRUCTURE OF PROTEINS IN CELL WALLS BY REDOR NMR

通过 REDOR NMR 分析细胞壁中的蛋白质结构

• 批准号: 6019027
• 负责人: JACOB SCHAEFER
• 金额: $ 21.05万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019027
• 关键词: G protein; Staphylococcus aureus; amphiphilicity; amphotericin B; antifungal antibiotics; cell wall; conformation; membrane proteins; membrane structure; nuclear magnetic resonance spectroscopy; peptidoglycan; pore forming protein; protein structure function; radiotracer; rhodopsin; stable isotope; stable isotope double label; structural biology; transfection; vancomycin

We propose new solid-state rotational-echo double resonance (REDOR) and Double REDOR NMR experiments to characterize the structures of peptides, proteins, or DNA complexed or aggregated with cell walls or membranes. Many of these complexes will be examined in situ. All of the systems are heterogeneous, insoluble, and not suited to analysis by diffraction or solution-state NMR methods. Our goal is not the determination of total structure, but rather the analysis of restricted regions (interfaces, channels, binding sites) important to biological function. Our approach uses (i) REDOR methods for internuclear distances as great as 15 Angstrom units; (ii) specific stable-isotope labeling schemes for three and four different kinds of nuclei; and (iii) NMR probes tuned simultaneously to four, five, and even six radiofrequencies. In the last grant period, we examined (i) peptidoglycans in bacterial cell walls; (ii) antimicrobials in multilamellar vesicles; and (iii) antifungals in oriented phospholipid bilayers. As a part of this program, we have also developed analytical methods for interpreting REDOR experiments of multi-spin systems. Based on this work, we are now in position to extend our solid-state NMR approach to cell-wall and membrane problems of immediate biomedical interest. We will use REDOR to characterize the inhibiting complexes of vancomycin with staphylococcus aureus cell-wall precursors in situ to determine (i) the mode of action of vancomycin and vancomycin derivatives. Some anti- fungals are believed to work by aggregation leading to the formation of ion channels or pores. REDOR will detect directly aggregates of antifungals in multilamellar vesicles which will lead to our characterization of (ii) bilayer pore formation by magainins and amphotericin B. We intend to develop the methodology needed to extend REDOR to the broad class of ligand-activated G-protein coupled receptors and use this technology to determine (iii) the conformation of a G- protein fragment bound to rhodopsin. Finally, we are working with surface-active particles that complex DNA and have potential as transfection vehicles in gene therapy. Key design problems are to optimize (iv) DNA compaction and transfection by amphiphilic particles, which we believe can be solved using insights from REDOR. Because stable free radicals having magnetic properties that are ideal for dynamic nuclear polarization can be buried in the hydrophilic core of these particles, new types of electron-based REDOR experiments will be developed to increase the range of quantitative distance measurements in biological solids by an order of magnitude.

我们提出了新的固态旋转回波双共振（REDOR）， 双REDOR NMR实验来表征肽的结构， 与细胞壁或细胞膜复合或聚集的蛋白质或DNA。 许多这些复合物将在现场检查。 的所有系统 是不均匀的，不溶的，不适合通过衍射进行分析 或溶液状态NMR方法。 我们的目标不是确定 总体结构，而是对有限区域的分析 （界面、通道、结合位点）对生物功能很重要。 我们的方法使用（i）REDOR方法的核间距为大 （ii）特定的稳定同位素标记方案， 三种和四种不同类型的核;和（iii）NMR探针调谐 同时发射四个五个甚至六个射频 在 在上一个资助期，我们研究了（i）细菌细胞中肽聚糖 壁;（ii）多层囊泡中的抗微生物剂;和（iii） 定向磷脂双层中的抗真菌剂。 作为其中的一部分 程序，我们还开发了分析方法，解释 多自旋系统的REDOR实验。 基于这项工作，我们现在 能够将我们固态NMR方法扩展到细胞壁， 膜问题的直接生物医学利益。 我们将使用REDOR 为了表征万古霉素的抑制复合物， 金黄色葡萄球菌细胞壁前体原位测定（i） 万古霉素和万古霉素衍生物的作用方式。 一些反- 真菌被认为是通过聚集作用， 离子通道或孔。REDOR将直接检测 多层囊泡中的抗真菌剂，这将导致我们的 （ii）通过爪蟾抗菌肽形成双层孔的表征， 异甘草素B。 我们打算制定所需的方法， 配体激活的G蛋白偶联受体 并使用该技术来确定（iii）G- 与视紫红质结合的蛋白质片段。 最后，我们正在与 表面活性颗粒，复合DNA，并有潜力作为 基因治疗中的转染载体。 关键的设计问题是 优化（iv）通过两亲性颗粒的DNA压缩和转染， 我们相信可以通过REDOR的见解来解决。 因为 具有磁性的稳定自由基， 动态核极化可以被埋在亲水核中， 这些粒子，新类型的电子为基础的REDOR实验将 用于增加定量距离测量的范围 在生物固体中的数量级。