Nuclear Magnetic Resonance Methods Development And Applications For Membrane Protein Structure Determination

All cells and organelles are contained within a hydrophobic lipid bilayer membrane to get a stable environment for vital function. A wide variety of biological processes for communications between cells and surrounding environment are controlled by the integral membrane proteins. However, due to partially hydrophobic surfaces and lack of stability, membrane proteins continue to be among the most challenging targets in structural biology. Here, we will apply some new methods for membrane protein purification and NMR based structure biology study.A brief review of function and current structure studies of membrane proteins is introduced in Chapter1. Then a brief review of the basic theory and protein study methods of Nuclear Magnetic Resonance (NMR) are introduced in Chapter2.In Chapter3, we apply the combined use of solution NMR and EPR (Electron Paramagnetic Resonance) methods for the structural analysis of a Mycobacterium tuberculosis "outer membrane protein". Rv0899functions as a pore-forming protein and the deletion of this gene impairs the uptake of some water-soluble substances, such as serine, glucose, and glycerol. It has also been shown to play a part in low-pH environment adaption, which may play a part in pathogenic mycobacteria overcoming the host’s defense mechanisms. Here, biochemical and structural data indicated that Rv0899is a monomeric membrane-anchoring protein with two separate domains, rather than an oligomeric pore. Using NMR chemical shift perturbation and isothermal calorimetric titration assays, we show that Rv0899was able to interact with Zn2+ions, which may indicate a role in the process of Zn2+acquisition.In Chapter4, an efficient dipolar-based band-selective homonuclear CO-CA cross-polarization transfer method was applied in solid state NMR spectrum of protonated proteins. Due to increased relaxation rate, NMR signal is more broad and undetectable for detergent bound membrane protein in solution buffer. So we turned our attention to Solid state NMR which used for the immobilized sample study. The most efficient recoupling is achieved when the sum of effective radio-frequency fields on CO and CA resonances equals two times the spinning rate. More than30%of the CO magnetization can be transferred to CA using BSH-CP, and the transfer efficiency is increased by up to70%compared to the most frequently applied spin diffusion (PDSD) procedure. This BSH recoupling method has been adapted for a complete set of sensitivity-enhanced protein sequential resonance assignment experiment. In Chapter5, A novel and reliable gas chromatography method was developed to separate and quantify detergents frequently used in membrane protein samples. In membrane protein biochemical and structural studies, detergents are always used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. Coupling of GC-MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.In Chapter6, a brief review of the current and the future structure investigation of membrane protein is introduce. As of Sep.2013less than1.5%of protein structures determined were membrane proteins despite being20-30%of the total proteome and significant functional importance. Detergents used in protein purification procedures leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy could be an emerging method for membrane-protein structural biology that can overcome these technical problems. Meanwhile, the functional diversity of membrane proteins is determined not only by themselves, but also by their interactions with membranes. People should pay more attention on how to analysis membrane protein structure and function in lipids or its native membrane in the future.