A New Insight into Fungal Cell Wall Architecture by Functional Genomics and Solid-State NMR along with Recent Advancements in Dynamic Nuclear Polarization for Analyzing Biomolecules

This dissertation summarizes the findings related to the way by which supramolecular architecture of fungal cell wall changes with genetic mutation, dispensing genes responsible for biosynthesis of cell wall polysaccharides. This is necessary because without perfect picture of how supramolecular assembly changes with genetic mutation it is hard to assess new anti-fungal targets. Alongside this we have highlighted how recent advancement into Dynamic Nuclear Polarization (DNP) methods improved characterization of biomolecules both in case of labeled and unlabeled samples. First study utilized Solid-state NMR (SSNMR) which is a non-destructive technique hence enabled us for the first time to deduce how nanoscale packing, hydration, and mobility of inner cell wall polysaccharides: α-1,3-glucan, β-glucan and chitin changes when either α-1,3-glucan or chitin is removed. Although galactomannan (GM) and galactosaminogalactan (GAG) lies in the exterior still GM deficiency brought about changes in the inner cell wall which is mysterious. GAG has an important role is masking β-1,3-glucan from recognition by human immune system hence its identification using SSNMR was very crucial. At the same time, we were surprised how polysaccharide deficiency brought about drastic changes in the protein region. Apart from this we have also treated the control strain with hot alkali and demonstrated that α-1,3-glucan cannot be removed completely which indicates its involvement in forming fibrils in the inner cell wall with β-glucan and chitin. The biggest surprise came in when repeated alkaline treatment could not remove valine. This is a clear indication that valine is involved in forming glycoprotein linkages. This model which depicts architectural change will improve our understanding of the way fungi responds to environmental stress. The recent advancements in magic angle spin dynamic nuclear polarization (MAS-DNP) enabled atomic-level characterization of biomolecules. Main factor contributing to this success has been preparation of DNP juice compatible with several biomolecules and hence allowing huge sensitivity enhancements. There are several interactions which were brought to light by MAS-DNP. For example, interactions between carbohydrates, proteins, and ligands. Hence in the future we can expect to see more utilization of MAS-DNP on biological research which will induce rapid development in its instrumentation