STRUCTURE OF PROTEINS IN CELL WALLS BY REDOR NMR

我们提出了一种新的固态旋转回波双共振(REDOR)和 双红核磁共振实验来表征多肽的结构， 蛋白质或DNA与细胞壁或细胞膜复合或聚集。 这些复合体中的许多将进行现场检查。所有的系统 是多相的，不溶的，不适合用衍射法分析 或溶液状态核磁共振方法。我们的目标不是决心 总体结构，而不是对受限区域的分析 (界面、通道、结合部位)对生物功能很重要。 我们的方法使用(I)REDOR方法计算核间距离 作为15埃单位；(2)特定的稳定同位素标记计划 三种和四种不同的原子核；以及(Iii)调整的核磁共振探针 同时达到四次、五次甚至六次射频频率。在 上一次资助期间，我们检查了细菌细胞中的肽聚糖。 (Ii)多层囊泡中的抗菌剂；以及(Iii) 定向磷脂双层中的抗真菌药物。作为这件事的一部分 程序，我们还开发了口译的分析方法 多自旋系统的雷多尔实验。基于这项工作，我们现在 能够将我们的固态核磁共振方法扩展到细胞壁和 立即引起生物医学关注的膜问题。我们将使用Redor 万古霉素与万古霉素形成的抑制络合物的表征 金黄色葡萄球菌细胞壁前体的原位检测(I) 万古霉素及其衍生物的作用方式。一些反- 真菌被认为是通过聚集而起作用的，从而导致 离子通道或毛孔。Redor将直接检测 多层囊泡中的抗真菌药物将导致我们的 Mainins和Mainins对(II)双层孔道形成的表征 两性霉素B我们打算开发所需的方法学来推广 广泛的配体激活的G蛋白偶联受体 并利用这项技术来确定(Iii)G-的构象 与视紫红质结合的蛋白质片段。最后，我们正在与 表面活性颗粒使DNA复杂化，并具有潜在的 基因治疗中的转基因载体。关键的设计问题是 优化(Iv)两亲性颗粒DNA紧凑和转染法， 我们相信可以通过瑞德的洞察力解决这个问题。因为 稳定的自由基，具有理想的磁性 动态核极化可以被掩埋在亲水的核心中 这些粒子，新型的基于电子的红外线实验将是 开发的目的是增加定量距离测量的范围 在生物固体中增加一个数量级。

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.