Methods for acquisition and assignment of multidimensional high-resolution magic angle spinning NMR of whole cell bacteria.
High-resolution magic-angle spinning (HR-MAS) NMR was developed in late 1990s, and it has evolved quickly for the study of a variety of biological matrixes. Recently, it has been used as an effective means to study the cell wall structures of intact bacteria. (1)H-(13)C heteronuclear single quantum coherence (HSQC) HR-MAS NMR can provide rapid analysis of the cell wall structure in live bacterial cells, thus allowing observation of drug effects, gene mutation, species differentiation, and environmental effects. However, this rapid analysis is dependent on having an established framework of HR-MAS NMR experiments and a detailed assignment of the whole-cell NMR spectra. This study examines parameters and describes strategies for the effective application of 2D and 3D HR-MAS NMR techniques to assign and study bacterial cell wall structures using Mycobacterium smegmatis as a model organism. Important parameters for successful whole-cell HR-MAS NMR studies, including pulse sequences, rotor synchronization, acquisition times, labeling strategies, temperature, number of cells, and cell viability, are described. A four-prong approach is presented for assignment of the complex whole-cell spectra, including the use of 3D HCCH-TOCSY and HCCH-COSY HR-MAS NMR.
Li
Wei
W
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. wli@utmem.edu
Lee
Robin E B
RE
Lee
Richard E
RE
Li
Jinghu
J
D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D020102:Mycobacterium smegmatis / Q000737:chemistry*; D012680:Sensitivity and Specificity