Structure of Proteins in Cell Walls by REDOR NMR

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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.