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研究生:莊孟哲
研究生(外文):Juang, Meng-Je
論文名稱:人體突觸蛋白與肌醇六磷酸之複合物結構探討
論文名稱(外文):Study on Complex Structure between Human Synaptotagmin I C2B Domain and Inositol Hexakisphosphate
指導教授:余靖余靖引用關係
指導教授(外文):Yu, Chin
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:117
中文關鍵詞:核磁共振突觸結合蛋白六磷酸肌醇
外文關鍵詞:Nuclear Magnetic ResonanceSynaptotagmin IInositol hexakisphosphate
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突觸結合蛋白 I (Synaptotagmin I, Syt I)是神經細胞突觸囊泡上的一種膜蛋白,而C2A與C2B則是其具有重要功能的胞質片段。近年的研究顯示,C2A會與鈣離子作用,而C2B則會與多磷酸肌醇作用。在鈣離子引發的神經傳導物質釋放的過程中,C2A與鈣離子的結合會活化神經傳導物質從神經突觸前膜釋放,但當C2B與多磷酸肌醇結合則會抑制此過程。利用親和性層析方法先將C2AB片段與老鼠大腦萃取物混合後,再以含有六磷酸肌醇之沖湜液沖湜,發現另一複合蛋白的次體、μ2, the subunit of clathrin assembly protein, AP-2。此結果顯示,當六磷酸肌醇與C2B結合時,會改變Syt I–AP-2間的作用力,進而抑制神經傳導物質的釋放。我們利用多維核磁共振的技術來研究C2B與六磷酸肌醇的複合物結構。我們的研究結果顯示,當六磷酸肌醇與C2B結合後,會引發C2B蛋白之結構改變,進而使C2B喪失了與AP-2的結合力。此研究結果在藥物學的治療上針對神經失調疾病將有助於開發出更好的新藥。
Abstract
Synaptotagmin I (Syt I) is a synaptic vesicle membrane protein that contains two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains. The C2A domain binds Ca2+ and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). It has been reported that Ca2+ regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Syt I is also known to be present in neuronal growth cone vesicles. Affinity elution chromatography from the C2 domain of Syt I-immobilized Sepharose using IP6 as the eluent found that several proteins, including an adaptin, specific subunits of the clathrin assembly protein, AP-2 were eluted from the mouse brain. It suggests that inositol high polyphosphate-binding to the C2B domain of Syt I alter the state of protein–protein interaction including the Syt I–AP-2 interaction. Thus, the inositol high polyphosphate may result in the inhibition of events involved in the synaptic vesicle trafficking. In this study, we used NMR to solve the C2B-IP6 3D complex structure. Our data provide new evidence for the hypothesis that the conformational change of C2B binding to IP6 alter the interface of C2B- AP-2. This information will aid in the design of better pharmacological treatments for neurological disorders.
Table of Contents
Chapter 1. Introduction
1.1 Overview of the synaptic vesicle cycle. …………………… 5
1.2 The Function and Structure of Synaptotagmin I………7
1.3 The role of Syt I C2B domain …………………………… 11
1.4 Inositol hexakisphosphate (IP6) ……………………………… 14
1.5 The aim of this study …………………………………………… 16
Chapter 2. Molecular Cloning, Overexpression, and Purification of the Human Synaptotagmin I C2B Domain
2.1 Introduction ………………………………………………………… 17
2.2 Materials and Methods …………………………………………… 19
2.3 Results and Discussions ………………………………………… 23
Construction of the C2B domain. …………………………………… 23
Purification of the C2B domain. …………………………………… 25
Isothermal Titration Calorimetry (ITC). ……………………… 30
The C2B domain is stabilized by the binding of IP6. …… 32
NMR analysis of the C2B domain. …………………………………… 34
Chapter 3. Resonance assignments of the C2B-IP6 complex structure and the characterization of IP6 binding site on C2B domain
3.1 Introduction ………………………………………………………… 36
3.2 Materials and Methods …………………………………………… 39
3.3 Results and Discussions ………………………………………… 42
Triple-resonance experiments of the C2B-IP6 complex. …… 42
Heteronuclear experiments of the C2B-IP6 complex. ………… 45
Extent of assignments and data deposition of the C2B-IP6 complex. ……………………………………………………………………… 46
Chemical Shift Perturbation Experiments. ……………………… 48
Chemical Shift Index. …………………………………………………… 52
Chapter 4. Structure Calculation and HADDOCK of the C2B-IP6 complex structure
4.1 Introduction………………………………………………………………69
TALOS…………………………………………………………………………69
ARIA-CNS……………………………………………………………………71
HADDOCK……………………………………………………………………73
4.2 Materials and Methods………………………………………………75
Nuclear Overhauser Effects (NOEs)………………………………75
Hydrogen-Deuterium Experiments…………………………………76
Structure calculation………………………………………………77
Molecular Docking………………………………………………………78
4.3 Results and Discussion………………………………………………………………………80
Solution structure of the C2B-IP6 complex…………………80
HADDOCK structure of C2B-IP6 complex…………………………84
Chapter 5. Conclusion………………………………………………………87

References ……………………………………………………………………90
APPENDIX …………………………………………………………………………99
A. The sequential connectivity of the C2B domain in C2B-IP6 complex using HNCA and HN(CO)CA strips………………99
B. Chemical shift values of IP6………………………………108
C. The galley proof of this thesis…………………………109

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