Deciphering Complex Biomolecular Interactions By In Situ Solid-State NMR Spectroscopy

Abstract

Understanding the systematic details of biological macromolecular systems requires information at the atomic level. Structural biology techniques such as X-ray crystallography, cryo-electron microscopy (cryo-EM), and nuclear magnetic resonance (NMR) - both solution and solid-state- have significantly advanced our knowledge of the structures and functions of biomolecular complexes, helping us understand diseases and develop new drugs to tackle them. X-ray crystallography provides atomic details of proteins and nucleic acids mostly in their static condition. On the other hand, cryo-electron tomography (cryo-ET) allows for the study of large macromolecular complexes in native environments, including membrane proteins, at the atomic level. However, both techniques face limitations when it comes to studying flexible systems. Solution-state NMR provides structural and dynamic information of flexible biomolecular systems, limited to ~50 kDa molecular weight using conventional approaches. In contrast, solid-state NMR (ssNMR) can probe large macromolecular complexes within their native environment without any size limitations. The work in this thesis described the structural and dynamic properties of three biomolecular complex systems, such as the β-barrel assembly machinery (BAM), the cell wall of Aspergillus fumigatus, and the cell wall of Fomes fomentarius, by employing CP–based and INEPT-based ssNMR spectroscopy. The BAM complex comprises five proteins, BamA to BamE, that play a vital role in inserting and folding outer membrane proteins into the E.coli outer membrane. Our primary focus was on the structural conformation of BamC and BamE in their native bacterial membrane to better understand their roles within the complex. Additionally, we investigated the cell wall composition of two different fungal cell walls – A.fumigatus from a therapeutic perspective and F.fomentarius from bio-sustainable perspectives. This thesis employs CP-based and INEPT-based solid-state NMR spectroscopy to investigate the structure and dynamics of three biomolecular systems: the β-barrel assembly machinery (BAM) complex in Escherichia coli, the cell wall of Aspergillus fumigatus, and Fomes fomentarius. For the BAM complex, we examined BamC and BamE in their native bacterial membranes. BamC showed environment-dependent dynamics: in the BAM complex, its N-terminus was rigid (via interaction with BamD), while folded domains showed intermediate motion; in native membrane, the N-terminus became flexible, with folded domains rigid through lipid interactions. The inter-domain linker remained flexible in both environments. BamE was rigid in both contexts but shifted from protein–protein interactions (with BamA and BamD) within BAM to protein–lipid interactions in native membranes. Both proteins localized to the inner leaflet of the outer membrane. In A. fumigatus, the rigid cell wall domain consisted primarily of α-glucan, β-glucan, and chitin with α-helical amino acids, while mobile domains contained random-coil amino acids. The antimicrobial peptide L-Cathelicidin (L-CATH2) initially targets mobile components, later targeting rigid domains, increasing water accessibility without inducing wall remodeling. Crystalline polysaccharides at the cell wall–water interface were identified via ssNMR. For F. fomentarius, ssNMR revealed that the crust layer’s rigid domain contained α-glucan, β-glucan, and chitin, while context and H-tube layers contained β-glucan and chitin. All layers’ mobile domains were lipid-rich with minimal protein, confirmed by ATR-FTIR.