Elucidating Functional Structure of Complex Carbohydrates in Intact Cells via Development of 1H-Detected Solid-State NMR Approaches

This project aims to develop new methodologies to investigate complex carbohydrates in their native cellular environments, advancing carbohydrate science and providing fundamental insights that may ultimately benefit human health and biotechnology. Carbohydrate biopolymers are essential for a wide range of biological processes yet remain among the most difficult to study due to their structural complexity. This pressing need for a versatile set of analytical tools will be addressed here through the development of innovative nuclear magnetic resonance (NMR) approaches to characterize carbohydrate biopolymers. These efforts align with the emerging era of ultra-high field NMR technologies and will provide molecular-level understanding of the role of carbohydrates in chemistry and biology. The project will offer rigorous training in biophysical and bioanalytical research and participants will engage in scientific collaborations, including in-depth training at the National High Magnetic Field Laboratory, preparing them for future careers. Outreach initiatives will offer valuable exposure to students and the community on topics such as carbohydrate structure, analytical chemistry, and spectroscopy. The research will also enrich the curriculum and enhance a graduate-level course by incorporating new concepts and methodologies. The research will address key fundamental biochemical questions related to the distinct structural and functional roles of complex carbohydrates in cellular environments. Specifically, the research will use fungal cells as a model system to investigate how variations in carbohydrate structure, which include intricate differences in branching, glycosidic linkages, conformations, and hydrogen-bonding networks, arise from biosynthetic pathways, and how structural modifications occur during interactions with chitin microfibrils and melanin. To achieve these objectives, the project will develop advanced proton-detected solid-state NMR experiments that enable faster data acquisition, require smaller sample amounts, and offer increased sensitivity to carbohydrate structural changes. These improvements will make the methods particularly effective for studying polymorphic molecules and whole-cell samples available in limited quantities. The research will also integrate these techniques with Dynamic Nuclear Polarization (DNP) to further enhance spectral sensitivity. Consequently, this project will establish an optimized platform for rapid and high-resolution structural characterization of cellular carbohydrates and contribute valuable proton solid-state NMR data to public carbohydrate databases. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.