利用核磁共振技術探討人類鈣離子結合之S100B在S100B-SIP189-219複合物內的水溶液結構以及其與SIP189-219蛋白之間的交互作用

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研究生：	謝育程 Xie, Yu-Cheng
論文名稱：	利用核磁共振技術探討人類鈣離子結合之S100B在S100B-SIP189-219複合物內的水溶液結構以及其與SIP189-219蛋白之間的交互作用 NMR Solution Structure of Calcium-bound Human S100B in Complexed with SIP189-219 and its Interation with SIP189-219
指導教授：	余靖
口試委員:	陳金榜莊偉哲
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	101
中文關鍵詞：	核磁共振、解離常數、螢光、化學位移
相關次數：	點閱：3 下載：0
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人類S100B隸屬於S100蛋白質家族，在人體中具有多樣的生理活性。其具有EF-hand之結構，能與鈣離子結合而改變構形並與多種蛋白質作用。在細胞內SIP與Siah-1、Skp-1、Ebi ...等蛋白協同參與並調節β-catenin的含量，間接影響Tcf/LEF轉錄因子的活性。有報導指出S100蛋白會與SIP中的SGS domain結合而抑制β-catenin的水解，導致癌細胞的增生。
在本文我們希望計算出在複合物中S100B 之水溶液結構，並研究其與SIP之間的交互作用。我們利用一系列的三維核磁共振實驗完成其骨架和支鏈的化學位移判定，再利用ARIA/CNS軟體解出複合物中S100B之水溶液結構。
接著使用HSQC滴定的實驗觀測在交互作用中受到最多化學位移擾動的氨基酸，搭配我們計算出的複合物中S100B結構和已發表存在S100A6複合物中的SIP189-219結構，並以此作為運行HADDOCK的資訊。結果顯示S100B- SIP189-219複合物的結構比S100A6- SIP189-219複合物的結構較為外擴。此外，我們也用恆溫滴定微卡計與螢光技術求得其解離常數，以證實S100B與SIP189-219有交互作用存在。

S100B, a member of S100 family, contains various biological activities in the human body. There are two EF-hand motifs, which may interact with calcium ions, in the protein. Upon calcium binding, S100B undergoes conformational changes to expose a hydrophobic surface as a binding site for various target proteins. For example, the target proteins such as Siah-1, Skp-1 and Ebi associate with Ca2+-S100B and subsequently adjust the expression level of β-catenin and the transcription activity of Tcf/LEF. Several studies indicate that S100 proteins may interact with the SGS domain of SIP to inhibit β-catenin degradation and enhance the proliferation of certain cancer cells.
In this thesis, we determine the solution structure of Ca2+-S100B in complex and study its interaction with SIP using ITC and fluorescence spectroscopy. By using a series of 3D NMR experiments, the backbone and side chain resonaces were alomost completely assigned.
HSQC titration experiments indicate the residues on one protein with significant chemical shift perturbation after associating with its binding partner. The chemical shift perturbation data on both S100B and SIP proteins serve as the input constraints for HADDOCK calculation. The atomic coordinates of S100B in complex calculated in the study were docked with the published structure of SIP189-219 bound to S100A6. The result reveals that the complex structure of S100B-SIP189-219 is more expanded then that of S100B-SIP189-219.

謝誌 ........................................................................................................................................... II
Abstract .................................................................................................................................... III
摘要 .......................................................................................................................................... IV
圖目錄 ...................................................................................................................................... IV
縮寫表 ...................................................................................................................................... VI
第一章 前言 ........................................................................................................................... 1
1-1 S100B 之性質與結構 ................................................................................................. 1
1-2 CacyBP/SIP之性質與結構 ......................................................................................... 3
1-3 S100蛋白與SIP的交互作用 ..................................................................................... 5
1-4 實驗動機 ...................................................................................................................... 6
1-5 生物核磁共振技術介紹 .............................................................................................. 7
1-6 蛋白質分子的NMR光譜循序判定(sequential assignment) ..................................... 8
1-6.1 蛋白質分子的骨架循序判定 ............................................................................... 8
1-6.2 蛋白質分子的支鏈循序判定 ............................................................................. 10
1-7 限制條件的找尋 ........................................................................................................ 11
1-7.1 距離限制條件(NOE distance constraints) .......................................................... 11
1-7.2 雙面角限制條件 ................................................................................................. 11
1-7.3 氫鍵限制條件 ..................................................................................................... 12
1-7.4 結構計算 ............................................................................................................. 12
1.8 HADDOCK(high ambiguity driven docking)介紹 .................................................... 13
第二章 材料與方法 ............................................................................................................... 16
2-1 蛋白質的表現與純化 .................................................................................................. 16
2-1.1 人類S100B蛋白質之取得 .................................................................................. 16
2-1.2人類S100B蛋白質之表現 ................................................................................... 16
2-1.3 人類S100B蛋白質之純化 .................................................................................. 18
II
2-1.4 SIP(殘基189-219)蛋白之取得與表現 .............................................................. 21
2-1.5人類SIP189-219蛋白質之表現 ................................................................................ 21
2-1.6 SIP189-219蛋白之純化 .......................................................................................... 23
2-1.7蛋白質濃度的測定 ................................................................................................ 26
2-2 蛋白質之基本性質鑑定 .............................................................................................. 27
2-2.1蛋白質質量鑑定 .................................................................................................... 27
2-3 蛋白質之間的交互作用 .............................................................................................. 29
2-3.1 螢光放射光譜(Fluorescence spectrum) ............................................................... 30
2-3.2恆溫滴定微卡計(isothermal titration calorimetry, ITC) ....................................... 31
2-3.3 核磁共振實驗 ....................................................................................................... 33
第三章 結果與討論 ............................................................................................................... 38
3-1 S100B與SIP189-219蛋白質在大腸桿菌之表現 ........................................................ 38
3-1.1 異丙基硫化半乳糖(IPTG)對大腸桿菌之誘導作用 ........................................... 38
3-1.2 S100B與SIP 189-219蛋白質的大量表現 ............................................................ 39
3-2. S100B蛋白之純化 ...................................................................................................... 41
3-2.1 陰離子交換層析管柱(monoQ)純化 .................................................................... 41
3-2.2 疏水交互作用層析管柱(HIC) 純化 ................................................................... 42
3-3 SIP 189-219融合蛋白之純化 ........................................................................................... 43
3-3.1鈷離子親和性管柱純化 ........................................................................................ 43
3-3.2 SIP189-219蛋白與Trx tag之分離 ........................................................................ 44
3-5 S100B及與SIP 189-219蛋白質之分子量鑑定 ........................................................... 45
3-6 S100B與SIP 189-219蛋白質之二級結構鑑定 ........................................................... 47
3-7 在複合物中(S100B與SIP 189-219複合物)S100B之NMR實驗 ............................... 48
3-7.1在複合物中S100B蛋白之1H-15N HSQC ........................................................... 49
3-7.2在複合物中S100B蛋白之HNCA與HN(CO)CA ............................................. 50
3-7.3在複合物中S100B蛋白之CACB(CO)NH ......................................................... 51
III
3-7.4在複合物中S100B蛋白之HNCO ....................................................................... 52
3-7.5在複合物中S100B蛋白之CC(CO)NH ............................................................... 53
3-7.6在複合物中S100B蛋白之HBHA(CO)NH ......................................................... 54
3-7.7在複合物中S100B蛋白之TOCSY和 COSY ................................................... 55
3-7.8在複合物中S100B蛋白之15N-edited 和13C-edited NOESY ....................... 55
3-8 在複合物中S100B之二級結構預測 ......................................................................... 56
3-8.1 TALOS ................................................................................................................ 56
3-8.2 CSI(Chemical shift index) ..................................................................................... 58
3-9 在複合物中S100B蛋白質結構計算 ......................................................................... 59
3-10 在複合物中S100B結構計算與已發表的未結合S100B ....................................... 63
3-11 在複合物中SIP189-219 NMR實驗 .............................................................................. 63
3-12 S100B與SIP189-219交互作用之探討 ...................................................................... 65
3-12.1 S100B(M9)與SIP189-219 的HSQC滴定實驗 ................................................... 65
3-12.2 SIP189-219 (M9)與S100B的HSQC滴定實驗 .................................................. 66
3-12.3 S100B的結合位置 ............................................................................................ 68
3-12.4 HADDOCK(High Ambiguity Driven Docking)................................................. 69
3-12.5 ITC .................................................................................................................... 71
3-12.6 螢光 ................................................................................................................... 72
第四章 結論 ........................................................................................................................... 76
參考文獻 ................................................................................................................................. 77
附錄一：在複合物中S100B的HNCA和HN(CO)CA的strips ........................................ 80
附錄二：在複合物中SIP189-219的HNCA和HN(CO)CA的strips ..................................... 83
附錄三：在複合物中S100B完整化學位移表 .................................................................... 85
                                

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