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標題: | 螺旋酶A次單元C末端區域之特定位置脯胺酸增減對蛋白結構與功能的影響 Structural and Functional Effects of Site-Specific Introduction and Removal of Proline on the C-terminal Domain of Gyrase A subunit |
作者: | Ting-Hsuan Kuo 郭庭瑄 |
指導教授: | 詹迺立(Nei-Li Chan) |
關鍵字: | 螺旋酶,脯胺酸, gyrase,proline, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 組成蛋白質的二十種胺基酸中,脯胺酸具有獨特的環狀結構,因此在形成胜肽鍵時其主鏈上的 phi 二面角會被限制在約-75度。且脯胺酸的醯胺環在形成胜肽鍵後,其氮原子無法作為氫鍵的提供者。因此,脯胺酸除了在 α-螺旋胺基端的第一圈和 β-摺板邊緣不參與氫鍵的位置外,較不常於此兩種二級結構中出現。因為這些特性,增加或移除脯胺酸的突變可能會造成結構與功能的重大改變。本實驗室先前的研究發現,Escherichia coli螺旋酶A次單元 (EcGyrA) 之 C-末端區域(CTD)的脯胺酸636 (P636) 和此酵素能夠於DNA中引入負超螺旋的活性有關。EcGyrA-CTD結構的組成單元為 β-摺板構成之「葉片」,再由六個葉片以環狀排列而形成β-風車狀結構。此環狀結構的外圍分佈著許多鹼性胺基酸,形成彎曲且帶正電的表面,可讓帶負電的DNA纏繞其上。P636位在葉片1和2間,可能使Gyr-CTD結構產生明顯的彎折,造成DNA結合面扭曲,協助其以右手旋的方式進行纏繞。先前研究發現,若將脯胺酸突變為丙胺酸 (P363A) 會降低GyrA-CTD的方向性纏繞活性以及螺旋酶引入負超螺旋的能力。
鑒於 P636對DNA結合面結構扭曲之重要性,因此我們推測若於特定位置插入脯胺酸可能會導致螺旋酶活性的改變。透過葉片 2和 3 之結構對排 (alignment) ,我們發現有一白胺酸殘基(L687)位於結構中對等於P636的位置。我們將此白胺酸殘基突變為脯胺酸,希望觀察此突變是否會影響GyrA-CTD結構以及螺旋酶活性。本研究的具體目標在於進行EcGyrA-CTD P636A和L687P突變蛋白的結構與功能性分析。兩種突變可在大腸桿菌C43(DE3) strain中大量表現,並可利用Co2+-親合性管柱和分子篩管柱純化得到高純度的蛋白樣品。 EcGyrA-CTD P636A和L687P突變的晶體可在含有 0.2 M sodium chloride、0.1 M Bis-Tris pH 6.5、22-31% PEG-3350、100 mM MES pH 5.8、和 30-40% v/v MPD的結晶溶液中生成。P636A和 L687P突變晶體的X-射線繞射解析度分別為1.80 和1.97 A、空間群P212121。我們成功以分子取代法建立了P636A與L687P之晶體結構,並觀察到P636A結構之變化,但並未如預期般大幅影響GyrA-CTD葉片1的彎折。在突變位置周圍可發現些許的結構改變,推測此改變可能已足以解釋其活性的相對變化,但其影響活性之具體機制可能仍需進一步的研究。 Proline has unique characteristics that are different from other amino acids. Because of its cyclic structure, the backbone φ dihedral angle of proline is restricted to approximately -75°. Besides, the peptide bond N-terminal to the proline lacks the capacity to act as a hydrogen bond donor. As a result, proline is often avoided in the α-helices and β-sheets, except for the first turn of an α-helix or the edge strands of a β-sheet. Due to these properties, mutations involved introduction and removal of proline may lead to drastic structural and functional changes in protein. Our lab has previously identified a proline residue (P636) within the C-terminal domain (CTD) of the Escherichia coli gyrase A (EcGyrA) subunit that contributes directly to gyrase’s unique (-) supercoiling activity. The EcGyrA-CTD adopts a so-called β-pinwheel fold with six circularly arranged β-sheet “blades”. The outer rim of EcGyrA-CTD harbors many basic residues, forming a curved and positively charged surface capable of wrapping a piece of DNA around itself. The presence of P636 in between blades 1 and 2 appears to produce a significant kink in EcGyrA-CTD, which twists the proposed DNA binding surface to allow handed DNA-wrapping by this domain. The proline to alanine mutation (P636A) was found to decrease both the directional wrapping activity of EcGyrA-CTD and the (-) supercoiling activity of gyrase. Given that P636 is crucial for the twisting of the proposed DNA binding surface, and that this proline-induced structural twist appears to link directly to gyrase function, we wonder if gyrase activity can be further modulated by additional site-specific introduction of proline. Through structural alignment, we identified a leucine (L687) residue located at an equivalent position as P636. We hypothesized that the specific introduction of proline to this position may further alter the structure and function of EcGyrA-CTD. The specific aim of this research is to perform structural and functional analysis on P636A and L687P mutant forms of EcGyrA-CTD. Both P636A and L687P mutants were expressed in E. coli C43 (DE3) strain, purified to homogeneity by Co2+-affinity and size-exclusion chromatography. Crystals of EcGyrA-CTD P636 mutant and L687P mutant can be obtained using a crystallization buffer containing 0.2 M sodium chloride, 0.1 M Bis-Tris pH 6.5, 22-31% PEG-3350 and 100 mM MES pH 5.8 and 30-40% v/v MPD. Datasets of both P636A and L687P mutants were collected to a resolution of 1.80 and 1.97 A, respectively, with space group P212121. The crystal structures of P636A and L687P were determined by molecular replacement, which reveal significant mutation-induced structural alternations. However, the predicted structural effect on the kink between blade 1 and 2 was not observed. The subtle displacement of surrounding residues may explain the different activities between mutants, but the mechanisms leading to the unique directional DNA-wrapping activity of EcGyrA-CTD still require further investigation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45753 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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