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波双共振（REDOR）NMR实验，以原位确定万古霉素和万古霉素类似物在活跃分裂的金黄色葡萄球菌细胞中的作用模式。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6频传输线NMR探头。三种新类型的REDOR实验将提供对全细胞（悬浮液和聚集在生物膜中）细胞壁中的标记的位点特异性检测，而不会受到细胞质标记或天然丰度背景的干扰。还将对成熟肽聚糖的厚外层已被去除的全细胞进行REDOR实验。这些原生质体将在回复正常细菌的不同阶段进行检查，因此具有不同量的附着的新生肽聚糖。结合在细胞质膜外表面附近的万古霉素具有治疗活性。除万古霉素外，REDOR实验中还将使用其他肽类抗生素，包括合成和天然爪蟾抗菌肽、乳链菌肽和默沙菌素。将在玻璃板上的全细胞、原生质体、回复原生质体、多层囊泡和机械排列的双层中检查结合。该项目的总体目标是使用REDOR来定义抗菌作用模式，从而帮助药物发现过程，旨在对抗预期的S。金黄色葡萄球菌对目前已知的所有抗生素都有抗药性。

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.