Structural And Functional Studies On Class IA Phosphatidylinositol-3’-Kinase And Relative Protein Complexes

Proteins are functional biomacromolecules of great importance in vivo, and participate in virtually every process of organisms. The protein-protein complexes are composed of a great many unqiue proteins, the structural study of which is the crucial part of clarifying the life processes such as signaling, cell cycle, gene expression and so on. Therefore, this study has double meanings in both fields of biology and pharmacology.Phosphatidylinositol-3’-kinases, P13Ks, form a diverse family of lipid kinases that phosphorylate phosphatidylinositol at the D3-position. Over the past decade, research on it has demonstrated that this family of enzymes contains important regulators of cellular signaling.On the background of analytic chemistry and environmental toxicology, we studied the protein’s structure by the metonds of hydroxyl radical-mediated protein footprinting, modern biotechnology, high performance liquid chromatography and mass spectrometry. This paper has five chapers, as follows:Chapter One:We described the composition and function of P13Ks and the research status of the structure study. Besides, we introduced hydroxyl radical-mediated protein footprinting with mass spectrometry, which provides a novel avenue toward examining the structure and dynamic processes of proteins and large biological 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 studied the structure of nSH2-iSH2, the connecting domain in the regulation unit of p85. The iSH2domain of p85is sufficient to bind p110a but does not affect its activity. Inhibition of p110a requires the presence of the nSH2domain linked to the iSH2domain. So, the nSH2-iSH2domain fragment is sufficient to mediate inhibition and phosphopeptide disinhibition of p110a. We determined the binding site of the two domains:(351-360) DTADGTFLVR,(392-426) YGFSDPLTF SSVVELINHYR and (426-436) LLYPVSK.Chapter Three:We studied the structure of p85dimer. 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 p85dimer in the SH3and BCR domains:(21-36) EEDIDLHLGDILTVNK,(37-68) GSLVALGFSDGQEARPEEIGWLNGYNETTGER,(106-136) TEADVEQQ ALTLPDLAEQFAPPDIAPPLLIK and (137-143) LVEAIEK.Chapter Four:We explored the relationship between structure and function of proteins by spectroscopy methods. Under the simulated physiological condition, we studied the effect of one kind of Pops (dicofol) on two kind of functional enzymes-trypsin and a-CT and the mechanism of the reactions. What’s more, we found out the conformational changes of proteins influence the enzymes’activity, which meant the function of the enzymes would be effected by the structure change. This is an efficient way to study the structure-activity relationship.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, and the study of structure-activity relationship, which contributes to the development of proteomics and could provide some reference gist for diagnosis, precaution, and therapy for some relevant diseases.