Decoding the Structural Complexity of Fungal α‐ and β‐Glucan Matrix via Statistical Analysis of Solid‐State NMR Chemical Shifts

Fungal cell walls are carbohydrate‐rich matrices whose structural organization regulates morphogenesis, mechanical integrity, and antifungal susceptibility. Although covalent linkage patterns of α‐ and β‐glucans are well characterized, their conformational structures and higher‐order supramolecular architectures and species‐dependent variations remain incompletely defined. Here, we integrate high‐resolution solid‐state nuclear magnetic resonance (NMR) spectroscopy with statistical analysis of 13 C chemical shifts to elucidate the structural organization of α‐1,3‐ and β‐1,3‐glucans in living cells of pathogenic fungi, including species of Aspergillus , Candida , and Cryptococcus . Comparative heatmap analysis reveals that β‐1,3‐glucans from diverse species predominantly adopt a conserved triple‐helical conformation in most intact cell walls, despite local conformational heterogeneity and branching. In contrast, α‐1,3‐glucans primarily assemble into rigid, hydrogen‐bonded bundled‐sheet architectures, with minor populations existing as less aggregated forms under specific genetic or pharmacological perturbations. Principal component analysis of ring‐carbon 13 C chemical shifts further resolves differences in backbone connectivity and branching topology across glucan classes. Together, these findings establish chemical shifts as quantitative reporters of glucan supramolecular architecture and provide a scalable framework for comparative structural analysis of fungal cell walls across species and conditions.