Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97753
DC FieldValueLanguage
dc.contributor胡念仁zh_TW
dc.contributorNien-Jen Huen_US
dc.contributor.author李振權zh_TW
dc.contributor.authorChen-Chung Leeen_US
dc.contributor.other生物化學研究所zh_TW
dc.date2018zh_TW
dc.date.accessioned2019-03-22T06:04:19Z-
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Cabral, J.o.M., Biochemical studies of a membrane protein: The KtrAB ion transporter. 2014. 53. Smith, F.J., et al., Structural basis of allosteric interactions among Ca2+-binding sites in a K+ channel RCK domain. Nat Commun, 2013. 4: p. 2621. 54. Pliotas, C., et al., Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System. Biochemistry, 2017. 56(32): p. 4219-4234. 55. Hanelt, I., et al., Membrane region M2C2 in subunit KtrB of the K+ uptake system KtrAB from Vibrio alginolyticus forms a flexible gate controlling K+ flux: an electron paramagnetic resonance study. J Biol Chem, 2010. 285(36): p. 28210-9. 56. Giraldez, T. and B.S. Rothberg, Understanding the conformational motions of RCK gating rings. J Gen Physiol, 2017. 149(4): p. 431-441. 57. Roosild, T.P., et al., A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch. Cell, 2002. 109(6): p. 781-91. 58. 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dc.identifier.urihttp://hdl.handle.net/11455/97753-
dc.description.abstractKtrAB複合蛋白在枯草芽孢桿菌是個鉀離子通蛋白,能夠調控鉀攝取離子進入細胞進而調節滲透壓。KtrAB 複合蛋白是由細胞膜上鉀離子通道蛋白KtrB以及在胞質的調控蛋白KtrA所組成。KtrA 屬於Regulator of Conductance of K+ (RCK) 家族之一,其結構為八聚體環。有研究指出當KtrA的RCK N-terminal (RCK_N) domain結合ATP或是ADP後,KtrA會因此發生改變構型,進而調控KtrB離子通道的開關(gating)。先前研究發現新型二級訊號分子c-di-AMP,能夠結合在KtrA 的RCK C-terminal (RCK_C) domain。然而c-di-AMP對於KtrAB複合蛋白的作用機制尚未明確,因此在本研究我們試著釐清c-di-AMP及其他核苷酸對於KtrA的構型及寡聚體的影響。從我們解出的晶體結構發現KtrA RCK_N domain結合ATP似乎阻礙RCK_C domain結合c-di-AMP,因此我們認為先前純化的KtrA沒有經過大量透析以確保沒有內源性的ATP或是ADP結合,導致晶體結構無法看到c-di-AMP。我們透過等溫滴定量熱法 (ITC)和差異性掃描螢光法 (DSF)探討核苷酸與KtrA的結合,並證明KtrA排除ATP殘留後能與c-di-AMP及其他核苷酸結合。從DSF結果中,我們意外發現KtrA結合ATP後造成Tm值下降,但透過動態光散射(DLS)結果看到KtrA仍然上八聚體。過去文獻曾報導c-di-AMP結合KtrA會抑制KtrB的功能,因此我們因此懷疑是否c-di-AMP會造成KtrA八聚體構型改變或瓦解,因此我們利用native gel以及負染電子顯微鏡觀察有無添加c-di-AMP對於KtrA之寡聚體程度。結果發現KtrA結合ci-di-AMP並未造成八聚體瓦解,但因為解析度的限制目前無法得知KtrA八聚體環細微構型變化。未來我們將嘗試使用apo KtrA與c-di-AMP共結晶,並透過X射線晶體學和冷凍電子顯微鏡(cryoEM)得到KtrAB複合蛋白與c-di-AMP結合的高解析度結構。zh_TW
dc.description.abstractIn Bacillus subtilis, KtrAB complex is a potassium channel which can mediate the osmotic stress by controlling uptake of potassium ions (K+) into cells. KtrAB complex is composed of the potassium channel KtrB, and the cytoplasmic regulator KtrA. KtrA belongs to a Regulator of Conductance of K+ (RCK) family and forms an octameric ring. Structural and functional studies have shown that KtrA adopts different conformations while binding ATP and ADP at the RCK N-terminal (RCK_N) domain, by which mediating the gating of KtrB. Recent studies suggested that c-di-AMP, a new second messenger, can bind to KtrA RCK C-terminal (RCK_C) domain dimer interface. However, the functional effects of c-di-AMP on KtrAB complex remains unclear. In the project, we intend to clarify the conformational and/or oligomerizational impacts of c-di-AMP and nucleotides on KtrA. Previously we determined a crystal structure of KtrA indicating c-di-AMP-binding at RCK_C domain is hindered by ATP-binding at RCK_N domain. It is assumed that endogenous ATP was not removed by extensive dialysis. We utilized Isothermal titration calorimetry (ITC) and Differential Scanning Fluorimetry (DSF) to characterize the interactions between KtrA and nucleotides, indicating a significant binding of c-di-AMP after removal of ATP. Surprisingly, DSF results suggested that the melting temperature (Tm) of KtrA became lower while binding with ATP. However, dynamic light scattering (DLS) results revealed that KtrA remains octameric state in the presence of ATP. As shown in a previous study that KtrA binding to c-di-AMP would inhibit the channel activity of KtrB. Therefore, we speculated that whether c-di-AMP would change the octameric assembly or conformation. We performed native gel electrophoresis and negative stain electron microscope to study the oligomeric state of KtrA. The results demonstrated that KtrA remains its octameric state in the presence of c-di-AMP, whereas the subtle change in octameric assembly is still vague due to the resolution limit. We are currently preparing crystallization trials of ATP-free KtrA with c-di-AMP. The high-resolution structure of c-di-AMP-bound KtrAB complex will also be attempted using X-ray crystallography and cryoEM.en_US
dc.description.tableofcontents中文摘要 i Abstract ii 目錄 iii 圖表目錄 viii 第一章 前言 - 1 - 一、 鉀離子的對於細胞的功能及重要性 - 1 - 二、 鉀離子通道蛋白 - 1 - (一) KcsA鉀離子通道蛋白 - 2 - (二) RCK (Regulator of Conductance of K+) domain - 3 - 三、 KtrAB complex 鉀離子通道蛋白 - 4 - (一) KtrB -鉀離子通道蛋白 - 4 - (二) KtrA八聚體環(octamer ring)鉀離子調控蛋白 - 5 - (三) KtrAB complex gating的調控機制 - 6 - 四、 c-di-AMP新型二級訊號分子 - 7 - 五、 KtrA與c-di-AMP的近期研究 - 8 - 六、 本論文研究目的 - 9 - 第二章 材料與方法 - 10 - 一、 Tagless KtrA蛋白之大量表現及結合ATP之純化 - 10 - (一) Tagless KtrA蛋白大量(large scale)表現 - 10 - (二) 取得水溶性蛋白Tagless KtrA混合液 - 10 - (三) 第一道純化管柱–離子交換樹脂(GigaCap®Q-650M column) - 10 - (四) 第二道親和力純化管柱-ADP agarose - 11 - (五) 透析排除ATP - 11 - (六) 第三道純化管柱-膠體過濾(gel filtration) - 11 - (七) 蛋白質確認及取得 - 12 - (八) 蛋白質濃度測定 - 12 - 二、 Tagless KtrA蛋白之大量表現及排除ATP結合之純化 - 12 - (四) 第二道純化管柱-膠體過濾(gel filtration) - 12 - (五) 透析排除ATP - 13 - (六) 第三道純化管柱-膠體過濾(gel filtration) - 13 - (七) 蛋白質確認及取得 - 13 - (八) 蛋白質濃度測定 - 13 - 三、 KtrA-His6質體建構、蛋白大量表現及純化 - 13 - (一) KtrA-His6質體建構 - 13 - (二) KtrA-His6 蛋白大量(large scale)表現 - 15 - (三) 取得水溶性KtrA-His6蛋白混合液 - 15 - (四) 第一道純化管柱-親和性管柱(Ni-NTA) - 15 - (五) 第二道純化管柱-膠體過濾(gel filtration) - 16 - (六) 蛋白質確認及取得 - 16 - (七) 蛋白質濃度測定 - 16 - 四、 KtrA-His6 I78V質體建構、蛋白大量表現及純化 - 16 - (一) KtrA-His6 I78V質體建構 - 16 - (二) KtrA-His6 I78V蛋白大量(large scale)表現及純化 - 17 - 五、 KtrA-TEV-His6質體建構、蛋白小量(small scale)表現 - 17 - (一) KtrA-TEV-His6 質體建構 - 17 - (二) KtrA-TEV-His6 蛋白小量(small scale)表現 - 19 - 六、 His12-KtrA蛋白之大量(large scale)表現及純化 - 19 - (一) His12-KtrA蛋白大量-(large scale)表現 - 19 - (二) 取得水溶性蛋白His12-KtrA混合液 - 19 - (三) 第一道純化管柱–親和性管柱(Ni-NTA) - 19 - (四) 第二道純化管柱–His TrapTMHP column - 20 - (五) 第三道純化管柱–膠體過濾法(gel filtration) - 20 - (六) 蛋白質確認及取得 - 20 - (七) 蛋白質濃度測定 - 20 - 七、 KtrB膜蛋白之大量表現及純化 - 20 - (一) KtrB蛋白質過表現 - 20 - (二) 取得膜蛋白KtrB solubilized混合液 - 21 - (三) 第一道純化管柱–親和性管柱(Ni-NTA) - 21 - (四) 第二道純化管柱–His TrapTMHP column - 22 - (五) 第三道純化管柱–膠體過濾法(gel filtration) - 22 - (六) 蛋白質確認及取得 - 22 - (七) 蛋白質濃度測定 - 23 - 八、 KtrAB complex純化 - 23 - 九、 VcDncV純化及c-di-AMP合成 - 23 - (一) VcDncV蛋白過表現 - 23 - (二) 取得水溶性蛋白VcDncV混合液 - 24 - (三) 純化管柱–親和性管柱(Ni-NTA) - 24 - (四) 蛋白質確認及取得 - 24 - (五) c-di-AMP合成 - 24 - (六) c-di-AMP純化- Reverse phase chromatography - 25 - (七) c-di-AMP濃度計算 - 25 - 十、 結晶條件篩選測試 - 25 - (一) KtrA和KtrAB complex結晶前置準備 - 25 - (二) 點晶及晶體觀察 - 25 - 十一、Isothermal Titration Calorimetry (ITC) - 26 - 十二、Differential Scanning Fluorimetry (DSF) - 26 - 十三、Native gel - 27 - 十四、X-ray晶體繞射數據收集 - 27 - 十五、負染電子顯微鏡(Negative Stain Electron Microscope) - 27 - 十六、動態光散射儀(Dynamic Light Scattering, DLS) - 27 - 第三章 結果 - 28 - 一、 不同KtrA construct之蛋白水溶性與純化結果 - 28 - (一) KtrA-His6 Cloning - 28 - (二) KtrA-TEV-His6 Cloning - 29 - (三) KtrA-His6 I78V cloning - 29 - (四) His12-TEV-KtrA 純化 - 30 - (五) Tagless KtrA純化 - 31 - (六) His-tag對於KtrA之影響 - 31 - 二、 KtrA與c-di-AMP及ATP之親和力 - 32 - (一) VcDncV純化及c-di-AMP 生合成 - 32 - (二) His12-TEV-KtrA與c-di-AMP之親和力 - 32 - (三) tagless KtrA與c-di-AMP之親和力 - 33 - (四) KtrA結合ATP對於結合c-di-AMP之影響 - 33 - 三、 KtrA結合c-di-AMP及其他核苷酸之熱穩定性 - 34 - (一) His12-TEV-KtrA結合ATP及c-di-AMP之熱穩定性 - 34 - (二) KtrA結合c-di-AMP及其他核苷酸之熱穩定性 - 34 - (三) 結合ATP與c-di-AMP造成KtrA構型之影響 - 35 - 四、 KtrA結合不同核苷酸之寡聚體 - 35 - (一) KtrA結合ATP及c-di-AMP之構型 - 35 - (二) apo KtrA結和ATP、ADP、AMP及c-di-AMP之寡聚體 - 35 - (三) 動態光散射儀(Dynamic Light Scattering, DLS) - 36 - (四) ATP對於KtrA構型之影響 - 36 - 五、 全長KtrA與c-di-AMP共結晶 - 36 - (一) KtrA-His6能與c- di-AMP共結晶 - 37 - (二) apo KtrA與c-di-AMP 共結晶 - 37 - (三) Tagless KtrA apo-form晶體 - 37 - 六、 KtrA在水溶液之構型 - 37 - (一) 電子顯微鏡(Electron Microscope) - 37 - 七、 apo KtrA與KtrB形成KtrAB complex - 38 - (一) KtrB純化 - 38 - (二) KtrAB complex 純化 - 38 - 第四章 討論 - 40 - 一、 KtrA調控機制-ATP與c-di-AMP之異位調控 - 40 - 二、 KtrA之ATP殘留問題 - 41 - 三、 核苷酸對於KtrA熱穩定及寡具體之影響 - 41 - 四、 KtrA與c-di-AMP共結晶的瓶頸 - 42 - 五、 未來研究目標 - 43 - 參考文獻 - 44 -zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2018-08-01起公開。zh_TW
dc.subject鉀離子通道蛋白zh_TW
dc.subjectKtrABzh_TW
dc.subjectc-di-AMPzh_TW
dc.subjectKtrABen_US
dc.subjectRCK,en_US
dc.subjectc-di-AMPen_US
dc.title核苷酸對於KtrA八聚體環的寡聚體化及熱穩定之影響zh_TW
dc.titleThe effects of nucleotides on oligomerization and thermostability of KtrA octameric ringen_US
dc.typethesis and dissertationen_US
dc.date.paperformatopenaccess2018-08-01zh_TW
dc.date.openaccess2018-08-01-
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item.openairetypethesis and dissertation-
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