Abstract
Carbohydrate polymers in their cellular context display highly polymorphic structures and dynamics essential to their diverse functions, yet they are challenging to analyze biochemically. Proton-detection solid-state NMR spectroscopy offers high isotopic abundance and sensitivity, enabling rapid and high-resolution structural characterization of biomolecules. Here, an array of 2D/3D (1)H-detection solid-state NMR techniques are tailored to investigate polysaccharides in fully protonated or partially deuterated cells of three prevalent pathogenic fungi: Rhizopus delemar, Aspergillus fumigatus, and Candida albicans, representing filamentous species and yeast forms. Selective detection of acetylated carbohydrates reveals fifteen forms of N-acetylglucosamine units in R. delemar chitin, which coexists with chitosan as separate domains or polymers and associates with proteins only at limited sites. This is supported by distinct order parameters and effective correlation times of their motions, analyzed through relaxation measurements and model-free analysis. Five forms of α-1,3-glucan with distinct structural origins and dynamics were identified in A. fumigatus, important for this buffering polysaccharide to perform diverse roles of supporting wall mechanics and regenerating soft matrix under antifungal stress. Eight α-1,2-mannan sidechain variants in C. albicans were resolved, highlighting the crucial role of mannan sidechains in maintaining interactions with other cell wall polymers to preserve structural integrity. These methodologies provide novel insights into the functional structures of key fungal polysaccharides and create new opportunities for exploring carbohydrate biosynthesis and modifications across diverse organisms.