臺灣博碩士論文加值系統

細菌在浮游態與固著態之生活狀態間的轉換，受到細胞內二級信使 (3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) 濃度的嚴密調控，而 PilZ domain首先被發現能與c-di-GMP結合，以傳遞訊息並參與調控細胞的生理狀態。根據序列分析，PilZ domain 的 N 端具有由 5 個高度保留性殘基形成的 RxxxR 及D/NxSxxG 兩組序列，此特徵序列被證實是與c-di-GMP結合所需，然而有些 PilZ domain的保留性氨基酸序列有退化的現象。Xanthomonads 的四個 PilZ domain 蛋白中，有兩個完全缺乏或不具完整的保留性氨基酸序列。本研究以晶體 X-ray 繞射技術解析二個 Xc17 菌株的 PilZ domain 蛋白，Xc1028 及 Xc6012，的三度空間結構。Xc1028 是第四型纖毛系統的 PilZ 蛋白，完全缺乏 c-di-GMP 結合序列，Xc6012 是致病蛋白，其c-di-GMP 結合序列不完整，等溫滴定實驗結果顯示，Xc1028 與 Xc6012皆不直接與 c-di-GMP 結合。與具有完整的 c-di-GMP 結合特徵序列的典型 PilZ 蛋白 PA4608、PP4397、VCA0042 的結構比較，Xc1028 及 Xc6012 與這三個蛋白的 PilZ domain 整體結構相似，但由於 N 端二級結構的差異，造成 Xc1028 及 Xc6012 無法單獨與 c-di-GMP 結合。Xc6012 比已知的 PilZ domain 多二條

The transition between planktonic and sessile lifestyle for bacteria is highly regulated. Recently, the bacteria-specific second messenger (3’–5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) was found to be critically involved in the switch from such transition, and PilZ domain be one of the key receptors for this important second messenger. Currently, many PilZ domain-containing proteins have been identified, and several tertiary structures of PilZ domains capable of binding c-di-GMP with strong affinity have been determined. Sequences of such PilZ domains feature a c-di-GMP signature. Here we have used X-ray crystallography to determine structures of two pathogenic proteins Xc1028 and Xc6012 form X. campestris strain 17. Both of them are PilZ-domain proteins however do not possess a complete c-di-GMP signature. Comparing to the typical PilZ domains, Xc1028 and Xc6012 adopt a similar tertiary structure, however, significant structural differences are detected at the N-terminus of these PilZ-domain containing proteins that exhibit substantial difference in the c-di-GMP binding activity. Xc6012 tetramer comprises a tetramerization domain stabilized by a 12-helices bundle containing a parallel four-stranded coiled-coil located in the center, and four PilZ domains pointing outward. We further generated a series of Xc6012 variants and measured the unfolding temperatures and oligomeric states in order to investigate the nature of this novel tetramer.

目次
中文摘要 i
Abstract ii
表目次 vi
圖目次 vii
縮寫檢索表 ix
第一章、前言 1
一、細菌的二級信使bis-(3’-5’)-cyclic dimeric guanosine monophosphate 1
二、PilZ domain 1
三、Xanthomonads的PilZ domain 蛋白 3
第二章、材料與方法 4
一、菌種、質體與引子 4
二、染色體DNA (Chromosomal DNA)之抽取 4
三、聚合酶連鎖反應（PCR） 4
四、定點突變聚合酶連鎖反應（Site-directed point mutagenesis PCR） 4
五、瓊脂醣凝膠之製備及電泳實驗 (Agarose gel electrophoresis) 5
六、PCR產物之萃取 5
七、質體DNA之抽取 5
八、LIC 5
九、E. coli勝任細胞之製備 7
十、轉殖作用（Transformation） 7
十一、PCR check與DNA定序 7
十二、南方墨點法 7
(ㄧ) DNA電泳分離與轉漬 (blotting) 7
(二) 探針製備 (probing) 8
(三) 雜交 (hybridization) 與顯影 8
十三、蛋白質之大量表現 8
(一) Native蛋白質之大量表現 8
(二) Se-Met標定蛋白質之製備 8
十四、以Ni-CAM affinity純化蛋白質 9
十五、以凝膠過濾法純化目標蛋白 9
十六、以凝膠過濾法測定蛋白之分子量 9
十七、蛋白濃度之測定 10
十八、利用X-ray晶體繞射技術解析蛋白之結構 10
(一) 蛋白質結晶濃度測試 10
(二) 結晶條件篩選 10
(三) 抗凍試劑篩選 10
(四) 繞射數據收集 11
(五) 決定相位角 11
十九、結構模型的建立與精算 12
二十、利用沉降速度超高速離心(SV-AUC)分析蛋白的分子量及聚合度 12
二十一、利用等溫滴定熱焓實驗(ITC)分析蛋白質與配體的結合強度 14
二十二、利用差示掃描量熱法(DSC)分析蛋白的熱穩定度 14
二十三、胞外蛋白分泌檢測 14
(一) 纖維素酶 (cellulase) 分泌檢測 14
(二) 澱粉酶 (amylase) 分泌檢測 15
(三) 蛋白酶 (protease) 分泌檢測 15
二十四、致病性檢測 15
第三章、結果與討論 16
一、XcpilZ 基因之選殖、晶體結構解析及與配體結合強度的分析 16
(一) xcpilZ基因之分析 16
(二) XcPilZ蛋白表現載體之構築、蛋白質表現及純化 16
(三) XcPilZ蛋白質之結晶與繞射數據之收集 16
(四) XcPilZ結構模型的建立 16
(五) XcPilZ晶體結構的描述及與已知結構的PilZ domain的結構比較 17
(六) XcPilZ與c-di-GMP結合強度的等溫熱焓分析結果 18
(七) XcPilZ與XcFimXGGDEF-EAL結合強度的等溫熱焓分析結果 18
二、Xc6012 基因之選殖、xc6012 knockout突變株構築及表徵檢測 19
(一) xc6012基因之分析 19
(二) Xc6012蛋白表現載體及xc6012 knockout突變株XD62之構築 19
(三) 突變株XD62表徵(phenotype)之檢測 20
1. 泳動能力檢測 20
2. 胞外酵素活性檢測 20
3. 致病性檢測 20
三、蛋白質表達及純化及晶體結構解析 20
(一) 點突變Xc6012蛋白表現載體之構築、蛋白質大量表達及純化 20
(二) Xc6012蛋白質之結晶與繞射數據之收集 21
(三) Xc6012結構模型的建立及描述 21
1. 結構精算及結果 21
2. Xc6012單體三級結構的描述 22
3. Xc6012四級結構的描述 22
(四) Xc6012與已知結構的PilZ domain在序列及結構方面之比較 23
四、野生型及點突變Xc6012蛋白之性質分析 24
(一) 聚合度(polymerization)分析 24
1. 分子篩選層析法(SEC) 24
2. 沉降速度超高速離心 (SV-AUC) 25
(二) 以差示掃描量熱法進行熱穩定性 (thermo-stability)分析 26
(三) Xc6012蛋白質與c-di-GMP 結合強度的分析 27
第四章、結論 28
第五章、參考文獻 30
附錄一、實驗材料 92
附錄二、本實驗所使用之引子 95
表目次
表一、本論文所使用的菌種及質體 36
表二、Xanthomonas及Stenotrophomonas的 xc6012 同源基因與上下游基因產物 37
表三、Xc1028蛋白的晶體繞射數據及結構精算 38
表四、Xc6012蛋白之繞射數據及結構精算 39
表五、Xc6012單體及四聚體的可及表面積 (accessible surface area) 41
表六、位於Xc6012四聚體介面的胺基酸及被包埋在單體介面的面積 42
表七、Xc6012、3DSG、1YLN及2GJG的b-barrel及Loop6主鏈的熱擾動參數 47
表八、野生型及各點突變Xc6012蛋白連續體積分佈擬合之各項參數 50
表九、野生型及各點突變Xc6012蛋白DSC實驗之Tm值 51
圖目次
圖1 XcPilZ與同源蛋白的序列比對 52
圖2 pT1028 上 his6-xcpilZ 的鹼基序列定序結果 53
圖3 Se-Met XcPilZ蛋白的純化結果。 54
圖4 Se-Met XcPilZ的三度空間結構 55
圖5 XcPilZ與PA4608、VCA0042及PP4397在三級結構及二級結構拓之比較 56
圖6 XcPilZ與已知PilZ domain的三度空間結構之比對結果 57
圖7 XcPilZ、PA4608、VCA0042 及 PP4397之 b-barrel 與 C 端

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