Structural insights into native intact Mycobacterium abscessus by conventional and ultrahigh-field solid-state NMR at 1.2 GHz.

We present an ultrahigh-field magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize intact nontuberculous mycobacteria (NTM). Hydrated and dried whole-cell Mycobacterium abscessus samples were investigated by combining conventional high-field ssNMR at 750 MHz with ultrahigh-field ssNMR at 1.2 GHz and ultrafast MAS at 100 kHz. To improve sensitivity and enable multidimensional experiments, 13C/15N isotope labeling was performed after growth in synthetic cystic fibrosis medium (SCFM). We utilized 1D13C and 2D 1H-13C and 13C-13C ssNMR experiments to characterize the chemical composition, dynamics, and structural organization of the M. abscessus cell envelope. The 13C isotope-labeling efficiency was found to be non-uniform across different molecular classes, with high incorporation into polysaccharides and lower incorporation into lipid and peptide-associated signals. INEPT- and CP-based experiments selectively probed flexible and rigid fractions of the samples, revealing substantial differences in linewidth, dynamics, and sensitivity between hydrated and dried preparations. Conventional 750 MHz experiments provided high-resolution multidimensional spectra and enabled identification of distinct chemical environments associated with peptidoglycan, arabinogalactan, mycolic acids, lipids, and peptide-associated components. Ultrahigh-field ssNMR at 1.2 GHz combined with ultrafast MAS and 1H detection improved resolution and sensitivity per mg of sample. The largest improvement of 100 kHz and 1.2 GHz system is observed for the CP spectra of the dry sample, where significantly better FWHM of 1H: 250 Hz (∼0.2 ppm) and 13C: 300 Hz (∼1 ppm) is observed compared to 10 kHz and 750 MHz system with 1H: 300 Hz (∼0.4 ppm) and 13C: 600 Hz (∼3.2 ppm). This allowed the detection of new aromatic and possible nucleic-acid-associated signals. Together, these results demonstrate that ultrahigh-field and ultrafast-MAS ssNMR enables detailed characterization of intact NTM cell envelopes under near-native conditions and provides a framework for future molecular investigations of antimicrobial interactions.