Structural Studies Of E Coli Integral Rhodanese Ygap Using Combinational Methods Of Nuclear Magnetic Resonance And Electron Paramagnetic Resonance

In this dissertation, we applied solution nuclear magnetic resonance (NMR) and site directed spin labeling electron paramagnetic resonance (SDSL-EPR) techique to study the structure and dynamic properities of YgaP from Escherichia coli. There are four chapters in this dissertation.Chapter1is a brief review of the rhodanese superfamily proteins. First, we introduced their catalytic mechanisms and the classification according to the characteristics of their different structures. The functions of rhodanese superfamily proteins, such as detoxification, synthesis of biological macromolecules, cell cycle regulation and so on, are also described here.Chapter2is a brief introduction to electron paramagnetic resonance(EPR) and its applications in biological studies. Here, we briefly described the conventional spin labels and site-directed spin labeling(SDSL) technique. Using SDSL-EPR, the dynamics and structural information of protein can be obtained by introducing the spin label into specfic sites on the protein These information includes motion, solvent accessibility and distance information. A few examples of SDSL-EPR in protein studies are presented here to show the unique advantages of this technique. In addition, we also introduced the applications of EPR in biological free radicals and cell membranes research.Chapter3brief described the three-dimensional structure of the rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. Compared with GlpE and PspE from Escherichia coli, they showed a lot of similarities in the structure. Then we studied the interaction between YgaP and its substrate by Na2S2O3titration experiment. The substrate binding pocket and fast conformational exchange between the ligand-free and persulfide-bound state of YgaP rhodanese domain were determined by chemical shift perturbation. The dissociation constant was calculated by the chemical shift changes of Ala66and Asn72.Chapter4brief described the advantage of resolving membrane protein structure information by SDSL-EPR. By cysteine scanning method, we intrudced the spin labels to50residues of YgaP transmembrane region one by one. By using electron paramagnetic resonance technology, we studied the dynamics and accessibilities of these spin-labeled residues. YgaP is found to form a dimer in DPC micelles, with two a-helical transmembrane regions according to the periodicity of3.6of dynamics and accessibility. The borders of two transmembrane helices were determined based on the information obtained with EPR. The interface between the two transmembrane helices were also illustrated here. Combining with the distance information, we built a topological model of the transmembrane region of YgaP. Furthermore, according to the solution NMR structure of the rhodanese domain of YgaP and the distance constraints between the rhodanese domain and the YgaP transmembrane region, the structure model of full-length YgaP was also built.