Structure of Proteins in Cell Walls by REDOR NMR

描述（由申请人提供）：我们提出了新的固态旋转回声双共振（REDOR）核磁共振实验，以原位确定万古霉素和万古霉素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被标记为13C， 15N， 2H和19F的组合。标签的检测将使用定制的，高效的6频传输在线核磁共振探针。三种新型的REDOR实验将提供全细胞（悬浮细胞和聚集在生物膜中的细胞）细胞壁标记的位点特异性检测，而不受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，其厚的外层成熟肽聚糖已被去除。这些原生质体将在恢复到正常细菌的不同阶段进行检查，因此与不同数量的新生肽聚糖相连。靠近细胞质膜表面的万古霉素具有治疗活性。除万古霉素外，其他肽类抗生素包括人工合成和天然的抗菌素、nisin和mersacidin也将用于REDOR实验。结合将在整个细胞、原生质体、还原原生质体、多层囊泡和玻璃板上机械排列的双层中进行检查。该项目的总体目标是使用REDOR来确定抗菌作用模式，从而帮助药物发现过程，旨在对抗预期的金黄色葡萄球菌致命菌株，这些菌株对目前已知的所有抗生素都具有耐药性。

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.