Sensitivity Enhanced Solid-State NMR of Soluble Proteins in Cells

In the recent years, there have been no unique protein structure topologies deposited in the PDB, which may reflect the finiteness of unique protein structures that exist in nature. Moreover, traditional structural studies have neglected the cellular context of the biomolecules, as there were no techniques that enabled atomistic studies in a highly heterogeneous cellular environment. Developments in biomolecular NMR and cryo-EM techniques have fueled the conception and the growth of cellular structural biology as a field. While cryo-EM focusses on very large molecular machines and protein assemblies, NMR has tackled small and heterogenous proteins. The two techniques thus complement each other. An analogous complementarity also exists within NMR, where cellular studies using solution-state NMR has been limited to small and soluble proteins while solid-state NMR has enabled studies on rather large insoluble proteins. The motivation behind pursuing the research presented in this thesis is to design and implement solid-state NMR approaches which would enable the study of soluble and promiscuous proteins within the cellular milieu. Despite their soluble nature, promiscuous proteins have remained out of the reach for solution-state NMR as they interact with their binding partners leading to reduced tumbling in crowded cellular environments. We have developed and tested an innovative DNP-ssNMR approach which is compatible with both mammalian and bacterial cells.