Overexpression Of Membrane Proteins And Their Structure And Function Studied By Magic Angle Spinning Solid-state Nuclear Magentic Resonance

In recent years, magic-angle spinning solid-state NMR is increasingly considered the most promising technologies in the study of structure and function of membrane proteins. Structure and function of membrane proteins are closely related with the membrane-mimetic environment. Detergent micelles are the most commonly used in X-ray crystallography and solution state NMR spectroscopy. However, membrane protein structure resolved in detergents environment may diffier from its real structure in native membrane. The structural characterization of membrane proteins in membrane environments are necessary to validate the structures determined in detergent environments. Membrane proteins could be studied under phospholipid bilayer which is more native-like membrane environment using ssNMR. Based on the advantage of ssNMR, it is considered as a powerful tool for studying native structure of membrane proteins.Membrane proteins are the bridge of material exchange and energy transfer between internal and external of the cell. They perform a variety of functions such as transporters, channels and enzymes. A lot of the membrane proteins also associated with human disease. To date, more than50%drugs is designed against membrane proteins. Therefore, it is very important to study the structure and function of membrane protein. However, due to extremely hydrophobic characteristics of membrane proteins, it’s difficult to prepare membrane protein samples. We need to screen a large amounts of conditions in overexpression, purification and reconstitution process without effective guidance. In this research, we worked out a set of membrane protein sample preparation process for magic angle spinning NMR spectroscopy. And then, we prepared a high-resolution proteoliposome of DAGK.(1) Overexpression and purification of membrane proteins. We optimized expression and purification conditions of four viroporin EV712B, HCV p7(1b), HCV p7(5a) and HIV-1Vpu. As a result, we designed a set of condition screening process of the expression of membrane protein with His6-tag, making the production of four viroporin to more than10mg/L M9culture and even up to30-40mg/L M9culture. We found that the expression was related to the position of His6-tag and provide useful reference for membrane protein expression vector designing. Moreover, through optimizing the influence factors of purification including the amount of resin, bounding time and wash level, we could obtain high purity (>95%pure) target membrane protein using one-step affinity chromatography.(2) MBP-Tag cleavage by TEV protease. We chose fusion membrane protein MBP-TEV.CS-MrpF as substrate to research the constraints of tag cleavage by TEV protease in detergent micelle. Firstly, we found that TEV cleavage site might make the structure of target membrane protein changed and then inhibit the expression. So, it should be designed carefully to introduce the protease cleavage site in the fusion membrane protein in order to avoid changing the structure of target protein. Secondly, we screened three detergents commonly used in membrane protein study that not inhibition TEV activity. Finally, we confirmed the steric occlusions of detergent-protein complex mainly inhibited the cleavage efficiency, and then eliminated the steric effect by extending the linker between TEV cleavage site and target membrane protein for fully exposion of TEV cleavage site.(3) Heterogeneous hydration dependence of the dynamics of DAGK in DMPC/DMPG lipid bilayers. We prepared high resolution proteoliposome of DAGK in DMPC/DMPG lipid bilayers. Our experiments revealed different hydration dependence of the dynamics of the highly mobile (J-residue) and immobile(D-residue) regions of membrane proteins. Based on the characterization of hydration dependence of dynamics and water accessibility of the protein, the general mechanism of hydration dependence of the different dynamical regions of the protein is revealed. In addition, we found that hydration dependence of the dynamics of D-residues is related to lipid bilayers phase. So we also study the hydration dependence of lipids phase transition.(4) Secondary and topology structure of DAGK in E.coli membranes. In this study,3D magic angle spinning (MAS) NMR was employed to characterize structure of E.coli DAGK reconstituted into its native E.coli membranes. Secondary structure and topology of DAGK revealed by solid-state NMR are different from those by solution state NMR and X-ray crystallography. Structure and function of membrane protein may different in membrane-mimetic environment. We examined the activity of DAGK proteoliposome in E.coli total in lipids, which consistent with the results reported in the previous literature. So, membrane protein structures characterized in native membrane environments are important to validate the structures determined in other membrane-mimetic environments. This study provided a good example for demonstrating the influence of solubilization environments to the structure of membrane proteins.