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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

基本信息

批准号：
6542277
负责人：
JACOB SCHAEFER
金额：
$ 27.16万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2006-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): We propose new solid-state rotational-echo double-resonance (REDOR) NMR experiments to determine in situ the mode of action of vancomycin and vancomycin analogues in actively dividing cells of Staphylococcus aureus. Both the vancomycins and the bacteria will be labeled with combinations of 13C, 15N, 2H, and 19F. Detection of the labels will use custom-built, high-efficiency 6-frequency transmission-line NMR probes. Three new types of REDOR experiments will provide site-specific detection of labels in cell walls of whole cells (both in suspension and aggregated in biofilms) with no interferences from cytoplasmic labels or from the natural-abundance background. REDOR experiments will also be performed on whole cells whose thick, outer layer of mature peptidoglycan has been removed. These protoplasts will be examined in various stages of reversion to normal bacteria, and so with varying amounts of attached nascent peptidoglycan. The vancomycin bound close to the exoface of the cytoplasmic membrane is therapeutically active. In addition to vancomycin, other peptide antibiotics including synthetic and natural magainins, nisin, and mersacidin will be used in REDOR experiments. Binding will be examined in whole cells, protoplasts, reverting protoplasts, multi-lamellar vesicles, and mechanically aligned bilayers on glass plates. The overall goal of the project is to use REDOR to define antibacterial modes of action thereby aiding the drug-discovery process aimed against anticipated lethal strains of S. aureus that are resistant to every presently known antibiotic.

描述(申请人提供)：我们提出了新的固态旋转回波双共振(REDOR)核磁共振实验，以原位确定万古霉素及其类似物在金黄色葡萄球菌活跃分裂细胞中的作用模式。万古霉素和细菌都将用13C、15N、2H和19F的组合进行标记。标签的检测将使用定制的、高效的6频传输线核磁共振探头。三种新类型的REDOR实验将提供对整个细胞细胞壁(悬浮和聚集在生物膜中)中的标记进行现场特定检测，而不会受到细胞质标记或自然丰度背景的干扰。REDOR实验也将在整个细胞上进行，这些细胞的成熟肽多聚糖的厚外层已经被去除。这些原生质体将在回复到正常细菌的不同阶段进行检查，并因此与不同数量的新生肽聚糖结合。结合在细胞膜表面附近的万古霉素具有治疗活性。除万古霉素外，还将在REDOR实验中使用其他多肽抗生素，包括合成和天然的麦芽菌素、乳酸链球菌素和麦芽菌素。结合将在整个细胞，原生质体，回复原生质体，多层小泡，以及玻璃板上机械排列的双层中进行检测。该项目的总体目标是使用REDOR来定义抗菌作用模式，从而帮助针对预期的致命金黄色葡萄球菌菌株的药物发现过程，这些菌株对目前已知的每一种抗生素都有抗药性。