Structure And Dynamic Study Of IFITM3 Using Solution Nuclear Magnetic Resonance And Electron Paramagnetic Resonance

In this thesis, we will discuss the study of structure and dynamic information of IFITM3 (Interferon-Induced Transmembrane Protein 3) with solution Nuclear Magnetic Resonance (solution-NMR) and Electronic Paramagnetic Resonance (EPR). There are five chapters in this thesis.Chapter 1 is a brief review of membrane proteins and its significant role in life. We illustrate the common structure, component and the interaction that keep the structure stable. Then we discuss the methods to the study of membrane proteins, which include X-ray crystallography, NMR and cryo Electron microscopy. In these methods, solution NMR can study structure and dynamic of proteins and protein complex in a relative wide time scale (from ps to hours). However its applications are restrict in small proteins.Chapter 2 is about the theory and methods in the study of membrane protein structure and function. We discuss the basic theory of NMR, including spin property of nuclear, Zeeman Effect, transition between different energy levels, relaxation and chemical shift. Then we introduce the method of solution NMR research, including the composition of NMR spectroscopy, spectra acquisition, data analysis, pulse technology, decoupling, and water suppressing. We also introduce technology ID 1H spectrum,2D and 3D multi-resonance spectrum, relaxation experiment, chemical shift assignment and structure calculation.Chapter 3 is the background information about IFITM3.We introduce virus and its relation with disease. Then, based on researches of IFITM proteins before, we review the roles of IFITM proteins, most of which are the restriction to many type of virus in immune process. We also briefly introduce the structure information (three topological models) of IFITM proteins.Chapter 4 focuses on the solution NMR research of IFITM3. Before carring out NMR experiment, we do some Homological analysis of IFITM3 and predict its second structure and transmembrane property. IFITM3 gene is cloned into an expression vector and expressed in E.coli. Expression condition, purification method and usage of detergents are optimized through a series of experiments. In this chapter, solution NMR experiments and data analysis of IFITM3 are discussed in detail, including sample preparation, optimization of NMR conditions,2D and 3D spectrum acquisition, data analysis, assignment of chemical shifts and structure calculation. Then we show and discuss the results of these data. From these data, we obtain a low-resolution 3D structure of IFITM3, which contains a long N terminal coil, two alpha-helices and a loop between them.Chapter 5 is about the study of IFITM3 using EPR. The background information of EPR and its application on protein is discussed briefly. Site-Directed Spin Labeling EPR (SDSL-EPR) experiment technology on proteins is introduced. The experimental protocol includes mutation introduction, protein purification, EPR sample preparation, spectrum acquisition and data analysis. From the result of EPR expreitments of IFITM3, we confirm a topological model of IFITM3:An extracellular C terminus and a relative long cytoplasmic N terminus; N helix passes through lipid bilayer while C helix dock on its surface.