Structure Characterization And Functional Study Of Membrane Protein By Electron Paramagnetic Resonance Based Hybrid Method

Recently, Site Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) has been widely used to study the structure and function of biological macro-moleculars. It can be applied to obtain the dynamics, solvent accessibility and distances information of proteins. Here, SDSL-EPR was used to determine the three-dimensional structure of transmembrane protein YgaP and the membrane topology of IFITM3 protein in detergent micelles. This dissertation is composed of four chapters.In chapter 1, a brief introduction of the Electron Paramagnetic Resonance theory and types of interactions in EPR were presented. Besides, a brief introduction of EPR spectrometer was included here.Chapter 2 presents the application of SDSL-EPR in biological macro-moleculars. First, we briefly described different biological spin labels and spin labeling methods. Second, the basic theory of SDSL-EPR and the dynamic, solvent accessibility and distance information derived from EPR analysis were presented here. Moreover, several examples of the application of EPR in transmembrane protein and nuclear acid were introduced.In chapter 3, combinational CW-EPR, pulsed EPR and rigid-body computational methods were applied to determine three-dimensional fold of the YgaP. Specifically, the systematic SDSL-EPR analysis was applied for the transmembrane domain of YgaP. The EPR dynamic and accessibility data were used to derive the secondary structure of the YgaP-TMD. Distance restraints were obtained using combined CW-EPR and DEER methods. The secondary structural information and distance restraints were applied for rigid-body structural computations of the dimeric YgaP-TMD. Besides, long distance restraints between the cytosolic rhodanese domain and YgaP-TMD were collected using DEER method. Then, in conjunction with the solution NMR structure of the cytosolic rhodanese domain of YgaP determined by our group previously, the three dimensional fold of full-length YgaP was finally determined using rigid body computational method. Besides, The dynamic, accessibility and distance measurements applied for YgaP in the presence of the rhodanese enzymatic product SCN" illustrated that the protein has some structural transitions upon SCN" binding, which provide insight into the thiocyanate exportation mechanism of YgaP in the E. coli membraneIn chapter 4, the combined approaches of electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) were applied to analyze the structure and membrane topology of the IFITM3 protein in DPC detergent micelles. The CW-EPR spectra analysis and accessibility data unequivocally demonstrated a single long transmembrane helix in the C-terminus of 1FITM3 and an intramembrane segment containing two short a-helices in the N-terminal hydrophobic region, supporting the proposed type Ⅱ transmembrane protein topology. Further secondary structure and backbone relaxation analysis using solution NMR method also verified this membrane topology. The resulting membrane topology of IFITM3 supports the mechanism of an enhanced restricted membrane hemi-fusion.