Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells.
Solid-state NMR is a powerful and non-perturbative method to measure and define chemical composition and architecture in bacterial cell walls, even in the context of whole cells. Most NMR studies on whole cells have used selectively labeled samples. Here, we introduce an NMR sequence relay using frequency-selective REDOR (fsREDOR) and spin diffusion elements to probe a unique amine contribution in uniformly (13)C- and (15)N-labeled Staphylococcus aureus whole cells that we attribute to the d-alanine of teichoic acid. In addition to the primary peptidoglycan structural scaffold, cell walls can contain significant amounts of teichoic acid that contribute to cell-wall function. When incorporated into teichoic acid, d-alanine is present as an ester, connected via its carbonyl to a ribitol carbon, and thus has a free amine. Teichoic acid d-Ala is removed during cell-wall isolations and can only be detected in the context of whole cells. The sequence presented here begins with fsREDOR and a chemical shift evolution period for 2D data acquisition, followed by DARR spin diffusion and then an additional fsREDOR period. fsREDOR elements were used for (13)C observation to avoid complications from (13)C-(13)C couplings due to uniform labeling and for (15)N dephasing to achieve selectivity in the nitrogens serving as dephasers. The results show that the selected amine nitrogen of interest is near to teichoic acid ribitol carbons and also the methyl group carbon associated with alanine. In addition, its carbonyl is not significantly dephased by amide nitrogens, consistent with the expected microenvironment around teichoic acid.
Stanford University, Department of Chemistry, 380 Roth Way, Stanford, CA 94305, USA.
David M
DM
Rice
Stanford University, Department of Chemistry, 380 Roth Way, Stanford, CA 94305, USA.
Joseph A H
JA
Romaniuk
Stanford University, Department of Chemistry, 380 Roth Way, Stanford, CA 94305, USA. Electronic address: cegelski@stanford.edu.
Lynette
L
Cegelski
D000409:Alanine / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D002473:Cell Wall / Q000737:chemistry; D004058:Diffusion; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013211:Staphylococcus aureus / Q000166:cytology*; D013682:Teichoic Acids / Q000737:chemistry
D000588:Amines; D013682:Teichoic Acids; D000409:Alanine
Cell wall; Frequency-selective REDOR; REDOR; S. aureus; Solid-state NMR; Teichoic acid; Whole-cell NMR