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Structural and biochemical studies of protein-ligand interactions insights for drug development
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Figures
List of Abbreviations
List of Symbols
1 Helicobacter pylori 5’-methylthio/S-adenosylhomocysteine nucleosidase: background and significance
1.1 Role of bacterial MTAN
1.2 Helicobacter pylori MTAN as a drug target
1.3 Active site of HpMTAN
1.3.1 Purine binding subsite
1.3.2 Ribose binding subsite
1.3.3 5’-alkylthio binding subsite
1.4 Enzyme mechanism
1.5 Human MTAP vs. HpMTAN
1.6 Overall structure of HpMTAN
1.7 Ligand induced conformational changes of HpMTAN
2 Kinetic and structural insight into differential substrate binding to HpMTAN
2.1 Overview
2.2 Materials and methods
2.2.1 Mutagenesis
2.2.2 Protein expression and purification
2.2.3 MTAN steady-state kinetic assays
2.2.4 Tris inhibitory assay
2.2.5 Crystallization
2.3 Results and discussion
2.3.1 Kinetic characterization of wt-MTAN
2.3.2 Tris inhibitory assay
2.3.3 Crystallization studies
2.3.3.1 Interactions between homocysteine chain and 5’-alkylthio binding subsite
2.3.3.2 Comparison of HpMTAN-D198N-SAH with HpMTAND198-MTA
2.3.3.3 Comparison of HpMTAN-D198N-SAH with HpMTANSRH-ADE
2.4 Conclusion
3 Development of high-throughput binding assay to identifying HpMTAN inhibitors
3.1 Rational for assay development
3.2 Approach for the assay development
3.3 Counter-screen assay
3.4 Material and methods
3.4.1 Protein preparation and adenine removal from HpMTAN active site
3.4.2 Fluorescence labeling of SAH
3.4.3 HPLC purification of SAH-488
3.4.4 Characterization and concentration determination of SAH-488
3.4.5 SAH-488 binding assay
3.4.6 SAH displacement assay
3.4.7 Assay validation
3.4.8 Screening of compounds of NIH clinical collection
3.4.9 Counter-screen assay
3.5 Results and discussion
3.5.1 SAH-488 synthesis, purification and characterization
3.5.2 SAH-488 binding constant
3.5.3 SAH inhibition constant
3.5.4 Assay validation
3.5.5 Screening of compounds of NIH clinical collection
3.5.6 Hit validation and false positive hits
3.6 Conclusion and future work
4 Structural and biochemical analysis of I1: Telomere binding protein in vaccinia virus
4.1 Overview
4.2 Structure of vaccinia virus
4.3 Replication of vaccinia virus
4.4 Protein parameters, expression and purification of I1
4.4.1 Co-affinity purification of recombinant I1
4.4.2 SDS-PAGE gel analysis
4.4.3 Rationale for interest in truncated form I1
4.4.4 SEC purification of truncated I1
4.5 Crystallization
4.6 Truncated I1 and DNA binding assay
4.7 Issues with truncated I1 expressed using construct (p101-I1)
4.8 Construct design for expressing an engineered truncated I1
4.8.1 Mass spectrometry
4.8.2 Cloning, protein expression and purification of I1 truncated
4.9 Conclusion and future studies
5 Structure and functional analysis of Mycobacterial dihydroneopterin triphosphate pyrophosphatase: A nudix hydrolase involve in folate biosynthesis
5.1 Tuberculosis and Mycobacterium tuberculosis
5.2 Folate biosynthetic pathway
5.3 Protein expression and purification of M.smeg MutT3
5.4 MutT3 substrate specificity using ATP, GTP, CTP and UTP as a substrate
5.5 Kinetic parameters using ATP as a substrate
5.6 Crystallization attempts
5.7 Chemo-enzymatic synthesis of DHNTP
5.7.1 Cloning, protein expression and purification of E.coli FolE
5.7.2 Chemo-enzymatic synthesis of DHNTP
5.7.3 Purification and characterization of DHNTP
5.8 MutT3 steady-state kinetics using DHNTP as a substrate
5.9 Conclusion and future work
ReferencesBài viết liên quan
