Research On The Structure Of YdiV-YdiV&YdiV-FlhD By γ Radiation Coupled With Mass Spectrometry

Proteins and protein-protein complexes perform a vast array of functions within living organisms and participate in virtually every process of organisms, including the expression of genetic information. Proteins differ from one another primarily in their sequence of amino acids, which usually determines its activity. The structural study of proteins and their complexes can clarify the life processes in molecule level. Therefore, a new structural analysis method of protein complexes has double meanings in both fields of biology and pharmacology.YdiV, a member of the EAL protein family, acts as a negative regulator of cell motility of Escherichia coli. It represses bacterium motility by interacting with F1hD4C2complex and shutting down the downstream genes transcription. It has been reported that YdiV binds to the F1hD4C2complex through interaction with the FlhD subunit and the binding of YdiV does not lead to a separation of the FlhD and FlhC subunits of F1hD4C2. However, if the external signal is strong enough and maintains long enough, the intracellular YdiV concentration eventually reaches a threshold and YdiV begins to squeeze into the ring-like structure of F1hD4C2complex. Finally, DNA is displaced from the F1hD4C2. As a result, the subsequent expression of flagellar and motility is repressed.There are two molecules YdiV (Mol A and Mol B) in an asymmetric unit. The research on the structure of YdiV-YdiV homodimer and YdiV-FlhD complex are equally important.In our research, we studied the structure of protein and protein-protein complexes by the metonds of hydroxyl radical-mediated protein footprinting, high performance liquid chromatography and mass spectrometry. This study has five chapters, just as follows:Chapter one:We introduced the information of YdiV and its research status. Furthermore, we also proposed a novel avenue toward examining the structure of proteins and large complexes that are difficult to study due to size limitations in NMR or difficulties of crystallization. The solvent-accessible and reactive amino acid side chains underwent stable oxidative modification, oxidized protein samples were typically analyzed by proteolysis (peptides are generated) and mass spectrometry coupled to high-performance liquid chromatography. Specifically, quantitative LC-MS was used to determine the oxidation extents and rates through dose-response analysis while LC-MS/MS analysis was used to determine the site(s) of oxidation. The footprinting approach is quite powerful for revealing the structural details of protein interactions and protein-protein complex.Chapter two:We chose BSA (Bovine serum albumin) as the target protein. To optimize the experimental protocol, we use spectroscopic methods to study the structural changes. The studies have shown that the microenvironment of amino acid residues changes significantly with the increase of exposure dose. However, it cannot cause the changes of protein’s structure. In our research, we solved the problem of post-oxidation in solution after stopped radiation and improved the method of hydroxyl radical-mediated protein footprinting. It extend the range of y-ray application, which will also increase our international influence in the field of protein complex assessment and biological effect of environmental pollutants evaluation on molecular level.Chapter three:We studied the structure of YdiV dimer. The products after hydrolysis were monitored by mass spectrometry coupled with HPLC (C18). The footprinting products detected and separated by LC-MS were further structurally characterized by tandem mass spectrometry. After comparing the oxidative degree of the peptides before and after dimerization, we finally determined the binding site of the YdiV dimer:(182-188)SFEPFIR.Chapter four:We studied the structure of YdiV-FlhD complex, we utilize the γ-ray exposure to achieve the oxidation labeling of the solvent accessible side-chain of protein and protein-protein complex, and then combine enzyme digestion technology, LC-MS and molecular docking method to establish the reliable and novel strategy in the structural assessment of protein-protein complex. We determined the binding site of the two domains:(153-161)AVFDGLFTR,(167-176)SFIQQQITHR,(177-183)SFEPFIR,(238-2 45)MHTSELLK,(261-266)HIYDINLSYLLLAQR,(273-293)GINEEMATTLAALTL PQMVKI and (294-305)LAETNQLVCHFR.Chapter five:We concluded the research parts above and analysed the development direction and perspectives of this new method for structure study of proteins, especially protein-protein complexes.This study has enriched the structure study of protein-protein complexes, which contributes to the development of proteomics and could provide some reference gist for diagnosis, precaution, and therapy for some relevant diseases.