Structural Insights Into Tsi1-Tse1 Complex And Studies On The Interaction Mechanism Of Hydrogen Peroxide And Two Proteins

Protein is one of the most important biological macromolecules of all livings. Thus, studies on protein structure have been an increasingly important field. The function of biological macromolecule mainly depends on the three dimensional structure, movement and interaction. The relationship between the primary and secondary structure of protein, and research about the mechanism of protein-protein interaction both play a key role in helping human to understand more about the life process. Protein-protein interaction and some other protein-macromolecule interaction, such as DNA, RNA, lipid and glucide, etc., control a range of biological processes that are closely correlated to individual development and cell communication, including replication, transcription, metastasis, translation, cell apoptosis and other signal pathways.In the first chapter of this thesis, the author introduced the background and methods about the studies on protein structure and protein-protein interaction. Based on the review about the related studies, we compared the pros and cons of the various methods and their targeted object as well as the basic principles of the protein oxidation labeling technology.In the second chapter, the biological components and functions of Type Ⅵ secretion system (T6SS) of gram-negative bacteria are introduced. Additionally, the structure and function of our targeted proteins, the Tse1 effect protein and the Tsi1-Tse1 complex are given as the background information. Afterwards, we elucidated the structure of the Tsi1-Tse1 complex in T6SS of Pseudomonas aeruginosa through the hydroxyl radical labeling coupled with mass spectrometry technology. The main experimental processes were as follows:(1) the solutions of Tse1,Tsi1 and Tsi1-Tse1 complex were exposed into the gamma ray at the same time. The certain dose was controlled by the accurate exposure time. The water molecules were decomposed into hydroxyl radicals as a result of the radiolysis, which can label the side chains of different amino acid residues of the two single proteins and the complex. (2) The oxidation extent of each peptide was determined quantitatively and qualitatively with the HPLC-MS analysis. For the same peptide, if its oxidation extent in the single protein was lower than that in the protein complex, then we suggested that it might be on the interaction interface of the two kinds of protein monomers. The experimental results showed that the oxidation behavior of the four peptides-(49-68) DNCSGFVQSVAAELGVPMPR, (109-123) TYGHVAWISGPLYR, (126-145) YPMCWCGSIAGAVGQSQGLK, (159-173) LNYYVYSLASCSLPR, is in accordance with the above hypothesis, because their oxidation extents in the Tsil-Tsel complex were much lower than that in the single Tsel protein. Therefore, we concluded that these four peptides were on the interface of Tsel and Tsil. Similarly, the oxidation extent of another four peptides in Tsil:(63-71) IEDGGIWSR, (83-104) LMHEFSGSSAELVSYDSATCK, (116-120) WAVDK and (144-153) SLAPFCQTAK was apparently higher than that in the Tsil-Tsel complex. We thus set them as the possible peptides involved in the interface of Tsil and Tsel.In the third part, the toxicity of hydrogen peroxide (H2O2) to trypsin was investigated by multiple spectroscopic techniques (including fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy, circular dichroism spectroscopy, etc.) and the molecular docking method at the molecular level. And the effects of H2O2 on the structure and function of trypsin were also studied. The intrinsic fluorescence of trypsin was proved to be quenched in a static process based on the results of fluorescence lifetime experiment. Hydrogen bonds interaction and van der Waals forces were the main force to generate the Trypsin-H2O2 complex on account of the negative ΔH0 and ΔS0. The binding of H2O2 changed the conformational structures and internal microenvironment of tryptophan illustrated by UV-vis absorption, fluorescence, synchronous fluorescence, three-dimensional (3D) fluorescence. With the addition of H2O2, the percent of a-helix in trypsin increased from 4.5%to 5.7%, while the content of p-sheet experienced a decrease from 45.4% to 42.8%. However, the binding site was far away from the active site of trypsin, so the trypsin activity was only slightly affected by H2O2, which was further explained and proved by molecular docking investigations.In the fourth part, we investigated the effects of hydrogen peroxide on the human serum albumin (HSA) with the similar classical spectroscopic methods and the molecular docking, which was a good contrast with the results of chapter three. On the whole, there are a lot of similarities between the two interactions, H2O2-Trypsin and H2O2-HSA. At the same time, we noticed that differences were also observed, as they are quite distinct from each other. The results showed that the quenching interaction between hydrogen peroxide and HSA were slightly stronger than that between H2O2 and trypsin, while the quenching mechanism should also be attributed to static quenching. And there was also only one binding site between H2O2 and HSA. However, the effects of hydrogen peroxide on the tyrosine and tryptophan were almost the same. In addition, the skeleton of HSA was loosened because of the addition of H2O2. In terms of the changes of secondary structure for the two proteins, with the addition of hydrogen peroxide, both trypsin and HSA experienced an increased a-helix and a decreased P-sheet.