預激活綜合體的形成對於脲酶的成熟是必需的。所以作為預激活綜合體一部份，UreF/UreG/UreG綜合體的形成也是脲酶成熟的關鍵之一。從幽門螺桿菌UreF/UreH的晶體結構顯示出是一個由異源二聚體形成的二聚體，這UreF/UreH二聚體和幽門螺桿菌的脲酶都有擁有個獨特的二重對稱性。而UreF/UreH二聚體的長度和幽門螺桿菌脲酶獨特的二次軸上那兩個催化中心的距離很接近。這讓我們聯想到UreF/UreH二聚體的二聚化是否與脲酶的活性有關。所以跟據UreF/UreH的晶體結構，計計了三個証實可以破壞UreF二聚化的突變體(F33D/Q37A, R179A/Y183D and F33D/Q37A/R179A/Y183D)。而這些突變體與UreH的結合體都保留了和脲酶結舍的能力卻失去了和UreG結合的能力，所以都不可以結合成完整的預激活綜合體來熟化脲酶。為了UreF/UreH二聚面的虛擬篩選，AutoDock Vina和Dock6.5,這兩個篩選程式用了DUD去做了一些基準。而基於一個百分比的富集值和首個已知配體的百分比值， Dock6.5比AutoDock Vina優勝，所以會用Dock6.5來篩選可以綁定UreF的二聚分介面的分子。最後，分析Dock6.5前1排名的分子，這些分子可以跟據它們和UreF殘基的接觸分類。 / The formation of the pre-activation complex is essential for the urease maturation. Being part of the pre-activation complex, the formation of theUreF/UreG/UreH complex is crucial for the formation of the complete preactivation complex. The crystal structures of Helicobacter pylor iUreF/UreH had been determined showing a dimer of heterodimer formation. The structure of UreF/UreH complex and H. pylori urease shared a unique two-fold symmetry. Moreover, the length of the UreF/UreH complex is similar to the distance of the two catalytic centres on the two-fold symmetry axis. This brought to the question: whether the dimerization of the UreF in the UreF/UreH complex has an effect on the H. pylori urease activity. According to the UreF/UreH crystal structure, three UreF mutants (F33D/Q37A, R179A/Y183D and F33D/Q37A/R179A/ Y183D) were designed and all were able to break the dimerization of UreF. These mutants were not able to interact with UreG, hence the complete pre-activation complex could not be formed and the maturation of urease was inhibited. Working towards to the virtual screening of the UreF/UreH complex dimerization surface, two docking programs, AutoDock Vina and Dock 6.5 were benchmarked using the DUD set. Dock 6.5 out performed AutoDock Vina by comparing the EF1 (Enrichment Factor of the top1% ranked ligands) and the percentage ranking of the first true hit. Using Dock 6.5, UreF residues that make the most contacts with the ligands had been identified using the top 1% of the ranked ligands. / Detailed summary in vernacular field only. / Yuen, Man Hon Nicholas. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 72-74). / Abstracts also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iii / Table of Content --- p.iv / Figures List --- p.vi / Tables List --- p.vi / Chapter Chapter 1 --- introduction --- p.1 / Chapter Introduction --- p.1 / Chapter 1.1 --- What is urease? --- p.1 / Chapter 1.2 --- Role of urease in H. pylori --- p.3 / Chapter 1.3 --- Structure of urease --- p.4 / Chapter 1.4 --- The active site of urease --- p.6 / Chapter 1.5 --- Accessory proteins are needed for urease maturation --- p.8 / Chapter 1.6 --- Crystal structure of H. pylori UreF/UreH complex --- p.12 / Chapter 1.7 --- Objective --- p.14 / Chapter Chapter 2: --- Material and Methods --- p.15 / Chapter 2.1 --- General Techniques --- p.15 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.15 / Chapter 2.1.2 --- Agarose gel electrophoresis of DNA --- p.16 / Chapter 2.1.3 --- Polymerase Chain reaction, PCR --- p.17 / Chapter 2.1.3.1 --- Basic protocol --- p.17 / Chapter 2.1.3.2 --- Generation of HisGST-UreF mutants --- p.18 / Chapter 2.1.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.1.5 --- SDS-polyacryamide gel electrophoresis, SDS-PAGE --- p.19 / Chapter 2.1.6 --- Staining of polyacrylamide gel --- p.20 / Chapter 2.2 --- Expression and Purification of Recombinant Proteins --- p.21 / Chapter 2.2.1 --- General bacterial culturing, harvesting and lysis procedures --- p.21 / Chapter 2.2.2 --- Purification of wild type HisGST-UreF and mutants with UreH --- p.22 / Chapter 2.2.3 --- Purification of Urease (UreAC) --- p.23 / Chapter 2.2.4 --- Purification of His-SUMO-UreG --- p.24 / Chapter 2.3 --- Static light scattering, SLS --- p.25 / Chapter 2.4 --- In vitor Urease Activity --- p.26 / Chapter 2.5 --- In vitor Urease Activity --- p.27 / Chapter 2.6 --- Virtual Screening --- p.28 / Chapter 2.6.1 --- Docking with Dock 6.5 --- p.28 / Chapter 2.6.2 --- Docking with AutoDock Vina --- p.29 / Chapter 2.6.3 --- Enrichment factor calculation --- p.29 / Chapter 2.7 --- Reagents and Buffers --- p.30 / Chapter 2.7.1 --- Buffers for competent cells preparation --- p.30 / Chapter 2.7.2 --- Nucleic acid electrophoresis buffers --- p.30 / Chapter 2.7.3 --- Media fr bacterial culture --- p.30 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.31 / Chapter 2.7.5 --- Reagents and Buffers for in vitro Urease Activity Assay --- p.32 / Chapter 2.7.6 --- Reagents and Buffers for in vitro Urease Activity Assay --- p.32 / Chapter Chapter 3 --- Dimerization of UreF is Essential for Urease Maturation --- p.33 / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Results --- p.34 / Chapter 3.2.1 --- Mutant design --- p.34 / Chapter 3.2.2 --- When expressed alone, the UreF mutants were found in the inclusion Body --- p.36 / Chapter 3.2.3 --- Co-expressing UreFmutants with UreH would solublize UreF mutants and the interactions between UreF mutants and UreH were retained --- p.36 / Chapter 3.2.4 --- UreF oligomerizationstate determination by size exclusion chromatography / static light scattering (SEC/LS) --- p.39 / Chapter 3.2.5 --- UreF dimerization is necessary for the interaction between the UreF/UreH complex and UreG --- p.41 / Chapter 3.2.6 --- UreF dimerization is not involved in the interaction between the UreF/UreH complex and Urease(UreA/UreC) --- p.43 / Chapter 3.2.7 --- UreF dimerization is essential for in vitro Urase Maturation --- p.45 / Chapter 3.2.8 --- UreF dimerization is essential for in vivo Urase Maturation --- p.47 / Chapter Chapter 4 --- Benchmarking Virtual Screening Performance of AUTODOCK VINA and DOCK 6.5 - Towards Virtual Screening of Inhibitors for Uref/UreH Complex Dimerization --- p.53 / Chapter 4.1 --- Introduction --- p.53 / Chapter 4.2 --- Benchmarking AutoDock Vina and Dock 6.5 --- p.54 / Chapter 4.2.1 --- Description of the Directory of Useful Decoys (DUD) set --- p.54 / Chapter 4.2.2 --- Benchmarking AutoDock Vina and Dock 6.5 shoewing Dock 6.5 has a better overall EF1 --- p.57 / Chapter 4.2.3 --- Dock 6.5 has a higher first hit percentile --- p.60 / Chapter 4.2.4 --- Analysis of the binding site for the top 1% ranked ligand for UreF Dimerization surface --- p.63 / Chapter 4.3 --- Discussion --- p.68 / Chapter Chapter 5 --- Conclusion --- p.71 / References --- p.72
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328754 |
| Date | January 2012 |
| Contributors | Yuen, Man Hon Nicholas., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (vi, 74 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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