Solid-state NMR observation of chitin in whole cells by indirect 15N detection with NC, NCC, CNC and CNCC polarization transfers

Abstract

Chitin is the most important nitrogen-containing polysaccharide found on Earth. This polysaccharide is a polymer of an N-acetylglucosamine and it is a crucial structural component of fungal cell walls and crustaceans. Magic-angle spinning solid-state NMR is emerging as a powerful analytical approach to study polysaccharides in the context of intact cell walls and whole cells. The presence of an acetamido group in chitin is attractive for 15N solid-state NMR. Here we investigate the use of various multi-step polarization transfer experiments incorporating indirect 15N detection at moderate spinning frequency, adapted from pulse sequences commonly employed for residue resonance assignment in biosolid proteins. The 13C,15N chitin spin topology slightly differs from amino acids, and we discussed the use of frequency-selective 15N-13C cross-polarization transfers followed by broadband or frequency-selective homonuclear 13C–13C transfers to detect chitin resonances. Demonstrated here for chitin found in the cell wall of the fungus Aspergillus fumigatus, the use of indirect 15N detection through multi-step polarization transfers could be advantageous to investigate more complex nitrogen-containing polysaccharides found in whole cells and peptidoglycan samples.