Development of in-cell NMR methodologies

The characterization of molecules within a biologically relevant environment distinguishes nuclear magnetic resonance (NMR) spectroscopy from other molecular-based biophysical techniques, such as X-ray crystallography and cryo-electron microscopy. Due to its exceptional stability and reduced ability to interact in a specific manner with other cellular components, the GB1 protein represents the quintessential probe to investigate the physiochemical effects imposed by the crowded environment on the structure and dynamics of proteins, without simultaneously compromising the ability to obtain in-cell NMR spectra due to binding events. The general aim of this thesis was to investigate the possible interactions of the GB1 protein with the Escherichia coli lysate and intracellular milieu with the purpose of inferring the physiochemical effects imposed by these two crowded environments on the structure and dynamics of proteins. Thus, the experimental parameters critical for performing in-cell NMR experiments, including bacterial growth and protein overexpression within E. coli cells, were initially optimized. Subsequently, by monitoring proton and nitrogen chemical shifts of backbone amides and lysines side chains, as well as carbon and proton chemical shifts of side chains containing carbonyl groups, the preferential behaviour of GB1 was analysed in lysate and within cells, considering the pure protein in water as the reference state. Furthermore, interactions with the local environment were further examined by determining the overall translational motion of the protein through diffusion-ordered NMR spectroscopy (DOSY). The results obtained suggest that the intracellular environment is much more viscous than its artificially crowded counterpart and that GB1 exhibits a distinct behaviour in E. coli than in lysate. Specifically, residues at or near the more flexible and solvent-exposed loop regions of the protein display an increased preference for interaction with cellular components within cells compared to lysate. Finally, a comparison of diffusion coefficients obtained with DOSY and fluorescence correlation spectroscopy (FCS), the standard analytical technique for studying protein diffusion, was made