In-cell Protein ¹⁹F Nuclear Magnetic Resonance Spectroscopv And Its Applications

In-cell protein nuclear magnetic resonance (NMR) spectroscopy is capable of providing information about protein conformation and function in living cells at atomic resolution. However, high concentrations of macromolecules and inhomogenous environment in cells make the application of in-cell NMR challenging. The aim of this dissertation is to develop 19F NMR methods for the investigation of protein conformation, dynamics and weak interactions in living cells.Systematically assessing protein labeling methods and strategies for In-cell NMR spectroscopy. Several proteins were labeled by 15N,13C,2H and 19F, respectively, and corresponding spectra in cells and in buffer were acquired. After comparison, we found that 15N labeling with deuteration improves the in-cell 15N-1H HSQC spectra. In contrast to E. coli, strong background signals in the methyl region are present in Xenopus leavis oocytes.19F NMR spectroscopy shows few background signals, and it can be used to study large proteins in cells. In terms of detectabilty and background signals, we found that 19F labeling is promising for protein NMR in both prokaryotic and eukaryotic cells.Investigating signal broadening mechanism of protein NMR spectroscopy in cells. Portein resonances are broadened in cells due to intracellular viscosity, weak interactions and magnetic inhomogeneity. Here, we employed 19F NMR methods to quantify these factors contributing to the line-width of NMR signals. We found that the signal broadening mechanism is different for different cell types. Intracellular viscosity and interactions are the main reasons for the low sensitivity of NMR experiments in E. coli, but for Xenopus oocytes, the main causes are weak interactions and inhomogeneous broadening.In addition, size dependence of protein rotational motion is due to weak interactions.19F NMR applications in studying conformational transition of calmodulin in cells. Ca2+-mediated calmodulin (CaM) conformational transition is key to a variety of signal transduction pathways. It has been postulated that signaling pathway activation is regulated by differentiable binding affinity of CaM with various targets at different Ca2+ level. But whether the conformational transition, binding affinity and the structure in living cells are similar as those in vitro studies needs to be confirmed. Here, we directly observed CaM conformational transition in intact Xenopus laevis oocytes. The Ca2+-free and -bound form of CaM in oocytes were quantified at different Ca2+levels using 19F NMR. We found that at the same low Ca2+level, Ca2+-bound CaM population was greatly increased upon binding with myosin light-chain kinase (MLCK) peptide, indicating that MLCK can be activated at lower Ca2+level for signal transduction.19F pseudocontact shift (PCS) in cells was also exploited for the first time and can be used to obtain long-range structural constrains of proteins in cells. The 19F method demonstrated here with CaM can be applied to other cellular signal transduction systems in living cells.The biophysical effect of a-synuclein (SYN) and its phosphorylation in cells, and protein enrichment method in living HeLa cells were also tried and preliminary results were obtained.In summary, we developed 19F NMR methods for studying both globular and disordered proteins in cells, and quantified the contributions of intracellular viscosity, weak protein interactions and magnetic inhomogeneity to NMR line-width in different cell types. We found that the size dependence of protein rotational motion in cells comes from weak interactions. We directly observed the conformational transition of CaM in cells by 19F NMR spectroscopy, and speculated that the structure of Ca-CaM-MLCK complex in cells and in buffer are similar according to the 19F PCS experiments. Besides, our preliminary results from in-cell NMR spectroscopy indicate that SYN and its phosphorylation form have different biophysical character and function in cells.