臺灣博碩士論文加值系統

訊息傳導因子(Signal Recognition Particle)是在生物體中將蛋白質運送到膜上的一普遍的系統。 在所有的訊息傳導因子系統中，葉綠素內的訊息傳導因子是最為獨特的，因為在葉綠素訊息傳導因子的系統中並沒有RNA的參與且可以以共轉譯轉譯及後轉譯轉移的方式運送蛋白質。在葉綠素的訊息傳導因子中包含兩個蛋白質，cpSRP54及cpSRP43。cpSRP54在很多生物體內都有同源物(homologue)，主要負責共轉譯轉移的機制；cpSRP43是一個新穎訊息傳導因子，主要負責後轉譯轉移的機制。cpSRP43的存在與否會決定cpSRP54的生理活性。cpSRP43由下列單元所組成： Chromo1, Ank1-4, Chromo2 和 Chromo3。最近的研究顯示Chromo2和運送蛋白質形成複合物後載到目標位置有關，因為此一區域能和cpSRP54鍵結。 Chromo2 在運送蛋白質到葉綠素或類囊體膜中扮演重要的角色。在本篇論文中主要要利用多維核磁共振的技術解出Chromo2在水溶液中的結構。利用2D NOESY、2D TOCSY、15N-HSQC NOESY、 15N-HSQC TOCSY、 HNCA和 HN(CO)CA來判定蛋白質中每一個胺基酸質子的化學位移；利用反選擇性標定蛋白來確定胺基酸質子的判定。在結構計算的部分我們利用HNHA來得到雙面角的資訊並加入TALOS和CSI來預測Chromo2的二級結構。主要用ARIA此一軟體來計算結構。除了判定所有胺基酸的質子化學位移並計算出初步結構之外，也利用圓二色光譜儀和螢光光譜儀來鑑定Chromo2的物理性質。

The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. cpSRP is present within two pools in the chloroplast: a co-translationally active SRP54 homologue (cpSRP54) and a post translationally active cpSRP, which is a complex of cpSRP54 and the novel SRP component, cpSRP43. The presence or absence of cpSRP43 seems to determine the targeting activity of cpSRP54. cpSRP43 has the following modules: Chromo1, Ank1-4, Chromo2 and Chromo3. The recent studies show that the Chromo2 is required for both targeting complex formation and integration because this module interacts with cpSRP54. Hence, Chromo2 plays a crucial role in the formation of “transit complex” contains cpSRP54, cpSRP43 and Lhcb1 which is responsible for targeting the proteins into the chloroplast and into the thylakoid membrane. In this context, we tempted to solve the solution structure of Chromo2 domain using multi-dimensional NMR spectroscopy. We carried out variety of three dimensional NMR experiments such as, 15N-HSQC NOESY, 15N-HSQC TOCSY, HNCA, HN(CO)CA and HNHA. Also, we preformed 2D NOESY& TOCSY. Assignments of all protons, 15N and 13Ca have been done using 2D NOESY& TOCSY, HSQC 15N-HSQC NOESY, 15N-HSQC TOCSY, HNCA and HNCA datasets. TALOS and CSI are performed to predict dihedral angles and secondary structure of Chromo2. The structure is being calculated by dynamical simulated annealing protocol using ARIA (which is a structure calculation software generous provided by Michael Nigles). Also, we characterized Chromo2 domain using biophysical techniques such as UV-CD, Fluorescence, and FPLC. The results will be discussed in greater detail.

摘要··························· 1
第一章 前言 ······················· 2
1.1 光合作用和葉綠體(Chloroplast)的基本構造········ · 2
1.2 葉綠體訊息傳導因子(Chloroplast signal recognition particle)在類囊體膜中的功能···················· · 3
1.2.1葉綠體訊息傳導因子協助蛋白質傳輸的機制······ ·················· 4
1.3 核磁共振光譜技術··················· 10
1.3.1 蛋白質分子的NMR光譜判定··············13
1.3.2 限制條件的找尋·················· 20
1.3.2.1 距離限制條件(NOE distance constraints) ········ 22
1.3.2.2 雙面角限制條件(Dihedral angle constraints) ······· 22
1.3.3 計算蛋白質分子的三維結構·············· 25
第二章 實驗材料與方法 ················· 30
2.1蛋白質之表現······················30
2.1.1重組質體之性狀轉殖(Transformation)與大腸桿菌菌落之培養························ 30
2.1.2 蛋白質小量及大量表現(Small Scale Expression and Large Scale Expression) ····················· 33
2.1.3 蛋白質的標定(Labeling of protein) ··········· 35
2.1.4 蛋白質的反選擇標定(Selective Reverse Labeling) ····· 38
2.1.5 利用高壓均質機獲取大腸桿菌中的Chromo2蛋白質···· 40
2.2 Chromo2之純化及濃縮················· 42
2.2.1 GST層析管柱···················· 42
2.2.2以凝血脢Thrombin來切斷GST fusion Chromo2·······44
2.2.3 Chromo2流析液之緩衝液交換及濃縮··········· 44
2.2.4 GST fusion Chromo2 再純化··············· 45
2.3 Chromo2之基本性質鑑定············· ··· 46
2.3.1 蛋白質電泳技術(SDS-PAGE) ············· 46
2.3.2 高效能液相層析法················· 49
2.3.3蛋白質的定量Bradford method (protein assay) ······· 50
2.4 Chromo2的物理性質測試················ 51
2.4.1 CD光譜儀(圓二色光譜儀) ··············· 51
2.4.2 螢光光譜儀···················· 53
2.5 核磁共振實驗···················· 54
儀器與藥品······················· 57
第三章 結果與討論 ··················· 63
3.1 大腸桿菌中Chromo2蛋白質之表現與純化········· 63
3.1.1 由大腸桿菌中取得蛋白質··············· 64
3.1.2 蛋白質的純化與分離················ 65
3.1.3 蛋白質的定量Bradford method (protein assay) ······ 68
3.2 蛋白質的構形鑑定·················· 69
3.2.1 高效能液相層析法················· 69
3.2.3 電灑質譜技術：ESI-Mass··············· 70
3.2.4 DNA定序····················· 71
3.3 Chromo2之穩定度探討················· 72
3.3.1 不同的酸鹼度···················· 72
3.3.2 不同變性劑濃度下·················· 76
3.4 核磁共振實驗····················· 80
第四章 結論 ······················ 91
Reference························· 92
附錄圖表······················ 95-118

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