Structural and Functional Study on DNA binding of MazE2 from Mycobacterium tuberculosis
Mycobacterium tuberculosis에서 유래한 MazE2의 DNA binding에 대한 구조적, 기능적 연구

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dc.description학위논문 (석사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. 이봉진.-
dc.description.abstractMycobacterium tuberculosis was first discovered by Robert Koch in 1882 and is a human infectious strain that causes lung disease through the respiratory tract. M. tuberculosis differs from ordinary bacteria in that it has thick membranes on the cell surface and does not stain Gram stain. However, when stained with Ziehl-Neelsen, it is difficult to decolorize by acid, alcohol and boiling, and this property is called acid-fast. Gram staining is negative, but they do not have external membranes and are classified as acid-fast gram-positive bacteria. Treatment of patients infected with M. tuberculosis is mainly medication. Isoniazid, rifampin, and pyrazinamide are used, and streptomycin is also used.
Mycobacterium tuberculosis has the largest number of Toxin-Antitoxin system pairs among bacteria. The Toxin-Antitoxin system is classified into six types according to antitoxin properties. The target protein Rv0660c of this study is MazE, an antitoxin of MazEF system, which is one of Type2 Toxin-Antitoxin system.
The Toxin-Antitoxin system plays a role in inhibiting the growth of bacteria or leading to death by external stimuli-activated toxins. Antitoxin normally binds to toxin and inhibits the activity of toxin. but it breaks down in extreme situations. Thus, dissociated toxin induces growth inhibition and death. This also induces latency and lowers susceptibility to antibiotics.
We overexpress the N1-44 construct of Rv0660c to characterize the tertiary structure of this Rv0660c protein. His-tagged Rv0660c protein was purified using Immobilized Metal Affinity Chromatography (IMAC), and the His-tag was removed through thrombin cutting to further increase the protein purity. As a result, the crystal could be made and the structure could be obtained with a high resolution of 1.69 Å.
The DNA binding experiments of Rv0660c were carried out by NMR and EMSA. Rv0660c was labeled with 13C and 15N isotopes. Heteronuclear multidimensional NMR spectra were measured and backbone assignments were made through HNCO, HNCA, HNCACO, HNCOCA, HNCOCACB and HNCACB spectra. The TALOS program was used to predict the secondary structure and identify the parts which interact DNA through DNA titration. We confirmed the binding of Rv0660c with DNA in vitro from Electrophoretic Mobility Shift Assay (EMSA).
dc.description.tableofcontentsI. Introduction 1
1.1 Structure Based Drug Design (SBDD) 1
1.2 Characteristics of Mycobacterium tuberculosis 2
1.3 Epidemiology of Mycobacterium tuberculosis 2
1.4 Toxin–Antitoxin System 3
1.5 Characteristics of Rv0660c 4
1.6 Purpose of the study 5

Ⅱ. Materials and Methods 6
2.1 Materials 6
2.1.1 Reagents 6
2.1.2 Apparatus 7
2.2. Methods 7
2.2.1 Cloning of target protein 8
2.2.2 Protein over-expression and purification 9
2.2.3 Crystallization 10
2.2.4. X-ray data collection and structure determination 11
2.3 Structural and Functional studies by NMR spectroscopy 11
2.3.1 NMR data collection 12
2.3.2 Backbone assignment . 12
2.3.3 Secondary structure prediction based on TALOS 13
2.3.4 DNA synthesis and preparation 13
2.4 Electrophoretic Mobility Shift Assay (EMSA) 14

Ⅲ. Result 15
3.1 Protein preparation 15
3.1.1 Cloning, overexpression and purification 15
3.1.2 Crystallization 18
3.2 Crystal structure of Rv0660c1-44 19
3.3 NMR studies of 15N or15N-13C labeled Rv0660c1-44 20
3.3.1 2D 1H-15N HSQC 20
3.3.2 Sequential backbone assignment 21
3.3.3 Predicted secondary structure of Rv0660c1-44 22
3.4 Functional study 24
3.4.1 Protein-DNA binding 24
3.4.2 Comprehensive result of protein-DNA binding 27
3.4.3 Electrophoretic Mobility Shift Assay (EMSA) 29

Ⅳ. Disussion 30

Ⅴ. Referenece 31

국문초록 35
dc.format.extent1190298 bytes-
dc.publisher서울대학교 대학원-
dc.subjectSBDD(Structure Based Drug Design)-
dc.subjectToxin-Antitoxin system-
dc.subjectx-ray crystallography-
dc.subjectNMR(Nuclear Magnetic Resonance)-
dc.subjectEMSA(Electrophoretic Mobility Shift Assay)-
dc.titleStructural and Functional Study on DNA binding of MazE2 from Mycobacterium tuberculosis-
dc.title.alternativeMycobacterium tuberculosis에서 유래한 MazE2의 DNA binding에 대한 구조적, 기능적 연구-
dc.contributor.AlternativeAuthorPark SeongHyun-
dc.contributor.affiliation약학대학 약학과-
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Master's Degree_약학과)
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