Proton-Detected Solid-State NMR for Deciphering Structural Polymorphism and Dynamic Heterogeneity of Cellular...

Area of Science:

• Biochemistry and Biophysics
• Structural Biology
• Microbiology

Background:

• Carbohydrate polymers (polysaccharides) have vital cellular roles but are difficult to analyze biochemically due to polymorphic structures and dynamics.
• Proton-detection solid-state NMR spectroscopy provides high sensitivity and resolution for biomolecular structural characterization.

Purpose of the Study:

• To apply advanced 2D/3D proton-detection solid-state NMR techniques to investigate fungal polysaccharides in their native cellular context.
• To characterize the structural heterogeneity and dynamics of key polysaccharides in pathogenic fungi: Rhizopus delemar, Aspergillus fumigatus, and Candida albicans.

Main Methods:

• Utilized 2D/3D 1H-detection solid-state NMR spectroscopy on fully protonated or partially deuterated fungal cells.
• Performed selective detection of acetylated carbohydrates.
• Analyzed molecular dynamics using relaxation measurements and model-free analysis to determine order parameters and effective correlation times.

Main Results:

• Identified 15 forms of N-acetylglucosamine units in R. delemar chitin, coexisting with chitosan and associating with proteins at limited sites.
• Resolved five distinct forms of alpha-1,3-glucan in A. fumigatus, crucial for cell wall mechanics and matrix regeneration under stress.
• Characterized eight alpha-1,2-mannan side chain variants in C. albicans, essential for cell wall integrity through interactions with other polymers.

Conclusions:

• Developed and applied novel NMR methodologies for high-resolution analysis of fungal polysaccharides.
• Provided unprecedented insights into the functional structures and dynamic behaviors of chitin, alpha-1,3-glucan, and alpha-1,2-mannan in pathogenic fungi.
• Opened new avenues for studying carbohydrate biosynthesis, modifications, and their roles in fungal biology and pathogenesis.