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NMR study on the structure and interaction of human HOXC9-homeodomain
Human HOXC9-homeodomain의 NMR을 이용한 구조 연구와 상호작용

DC Field Value Language
dc.contributor.advisor이봉진-
dc.contributor.author한정화-
dc.date.accessioned2017-07-19T11:10:42Z-
dc.date.available2017-07-19T11:10:42Z-
dc.date.issued2013-02-
dc.identifier.other000000010138-
dc.identifier.urihttps://hdl.handle.net/10371/133427-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 제약학과, 2013. 2. 이봉진.-
dc.description.abstractAbstract

NMR study on the structure and interaction
Of human HOXC9-homeodomain

HOX genes are a family of transcription factors, which share a common 61 amino acid DNA-binding domain, the homeobox. This contains a helix-turn-helix motif for DNA-protein interaction. HOX genes are important regulators of embryogenesis and envelopment along the anterior–posterior axis. They are involved in formation of body patterns, organogenesis, cell and organ differentiation, cell adhesion, migration and cell cycle control. Some HOX genes can act as oncogenes, playing a role in tumor development. HOXC9 is also Known that associated with angiogenesis and neuroblastoma.
The purpose of this study is to determine three dimensional structures of proteins from Homeodomain, and identify the function of the transcription factor by DNA-binding activity. To achieve the goal, Homeodomain of HOXC9 was selected and cloned through the known genome sequence. They were expressed using E.coli expression system and codon optimization process was performed to obtain large quantities of proteins. The expressed proteins were carried out solubility test to optimize the best soluble condition of the proteins. After purification, the secondary structures of the proteins were predicted via Circular Dichroism (CD). The final samples were predicted their three dimensional structure through NMR spectroscopy, and the part of the structure was analyzed. S
As the results, HOXC9 is consist of three α –helixes and it has monomer form. The secondary structure and 3D structure of HP0268 were predicted by TALOS+ and MODELLER programs respectively.
From this study, we established technical purification methods, optimized conditions for NMR experiments. We complete 95% of the backbone assignments, determinate secondary structure of HOXC9 and confirm HOXC9-DNA interaction by EMSA so we identify function as transcription factor of HOXC9. Additional experiment about binding of Smad4-MH1 with HOXC9 is on-going. This experiment provide the fundamental information for the drug development target to human HOXC9 protein and help to search the lead compound for structure based drug design like high throughput screening.
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dc.description.tableofcontentsContents
I. General Introduction
1.1. Characteristics of HOXC9 ……………………………………… 01
1.2. Diseases involving HOXC9
1.2.1. Angiogenic Processes …………………………………… 03
1.2.2. Prognostic marker in neuroblastoma……………………04
1.3. Interaction with SMAD4-MH1………………………………05
1.4. The need for structure base drug design …………………… 06
1.5. Purpose of this study ……………………………………………08
II. Materials and methods
2.1. Materials
2.1.1. Reagents……………………………………………………… 09
2.1.2. Apparatus …………………………………………………… 10
2.1.3. Chromatography Media (=column resin) …………… 11
2.2. Methods
2.2.1. Cloning of target proteins …………………………………12
2.2.2. Over-expression and purification……………………… 13
2.2.3. DNA synthesis and preparation………………………… 15
2.2.4. DNA binding test (EMSA)…………………………………16
2.2.5. Structural studies by CD spectroscopy ……………… 16
2.3. Structural studies by NMR spectroscopy
2.3.1. NMR spectroscopy …………………………………………18
2.3.2. Peak assignment…………………………………………… 18
2.3.3. Secondary structure and Modeling structure ……… 19

III. Results
3.1. Overexpression and purification ……………………………… 21
3.2. CD studies of HOXC9 …………………………………………… 24
3.2.1. The pH dependent stability of HOXC9…………………24
3.2.2. The salt dependent stability of HOXC9 ……………… 25
3.3. NMR studies of 15N -13C labeled proteins
3.3.1. Measurement of 1H-15N HSQC …………………………26
3.3.2. Sequential assignment………………………………………27
3.3.3. Secondary and 3D structure prediction…………………32
3.4. DNA binding test……………………………………………………35

IV. Discussion……………………………………………………………… 37
V. References………………………………………………………………40
국문초록………………………………………………………………………42
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dc.formatapplication/pdf-
dc.format.extent1421963 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectHOXC9-
dc.subjectNMR-
dc.subject.ddc615-
dc.titleNMR study on the structure and interaction of human HOXC9-homeodomain-
dc.title.alternativeHuman HOXC9-homeodomain의 NMR을 이용한 구조 연구와 상호작용-
dc.typeThesis-
dc.contributor.AlternativeAuthorJeong Hwa Han-
dc.description.degreeMaster-
dc.citation.pages54-
dc.contributor.affiliation약학대학 제약학과-
dc.date.awarded2013-02-
Appears in Collections:
College of Pharmacy (약학대학)Dept. of Manufacturing Pharmacy (제약학과)Theses (Master's Degree_제약학과)
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