Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36285
標題: 全新非機械性蛋白質奈米力學暨增強其機械力穩定度之驗證
Characterization and Nanomechanics of Unique Non-Mechanical Proteins with Rational Enhancing of Mechanical Stability
作者: 王建中
Wang, Chien-Chung
關鍵字: Atomic Force Microscopy
原子力顯微鏡
Single Molecule Force Spectroscopy
Protein Mechanics
Staphylococcal Nuclease
Cytochrome b562
單一分子力譜
蛋白質機械力學
金黃色葡萄球菌核酸水解
出版社: 生物科技學研究所
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摘要: 本研究主要著重在非機械性蛋白質 ( non-mechanical proteins ) 的奈米機械力學分析與驗證,以拓展工具箱因應人造彈性蛋白質的設計需求,其可自組裝成以蛋白質為基礎的生物性材料且具多元功能性( multi-functionality )的應用。更細節地說明,本研究的主要目的係為(一)系統性量測非機械性蛋白質之機械力穩定度方法學之建立,以基因工程設計內建標誌蛋白質的卡匣式系統,因應原子力顯微鏡(Atomic Force Microscopy, AFM) 力譜實驗分析中單一分子記錄的鑑別。(二)利用以AFM為主的單一分子力譜驗證非機械性蛋白質-金黃色葡萄球菌核酸水解酶之機械力穩定度,並利用配體結合的方式增加其機械力穩定度。(三)利用AFM為主的單一分子力譜驗證非機械性蛋白質-細胞色素b562之機械力穩定度,並利用鐵基質中心還原增加其機械力穩定度。 建立多功能具標誌蛋白質的卡匣式嵌合多聚蛋白質系統:原子力顯微鏡基礎單一分子力學譜最大的挑戰在於單一分子力譜事件的辨識,以及如何減少探針與基板之間的非專一性交互作用遮蔽訊號 。本研究我們建立一個具標誌蛋白質基因的卡匣式嵌合多聚系統,使目標蛋白質可輕易地在此系統替換指定模組位置,同時具有目前最出色的機械性蛋白質I27作為linker與標誌蛋白質, 減少非專一性的雜訊干擾和正確辨識單一分子的力譜事件,有系統地量測目標蛋白質的機械力穩定度。 金黃色葡萄球菌核酸水解脢之奈米機械力學之驗證:金黃色葡萄球菌核酸水解脢 (Staphylococcal Nuclease, SNase) 受鈣離子調控催化水解去氧核醣核酸(DNA)和核醣核酸(RNA)。我們使用以原子力顯微鏡為基礎的單一分子力學譜來驗證SNase 單獨的機械力穩定度以及其與SNase 抑制劑, deoxythymidine 3’- 5’- bisphosphate (pdTp)結合的複合物之機械力穩定度。我們發現當酵素對機械力去摺疊有反應時所需的力量約26 pN。而在抑制劑結合之後,以機械力去摺疊該酵素 –抑制劑複合物所需的力量增加到約 ~50 pN。抑制劑所誘發的酵素機械力穩定度的增加,與SNase 酵素抑制劑複合體在熱力學穩定度的實驗結果表現是符合一致的。SNase 的機械力穩定度的驗證,以期未來可拓展滿足人造彈性蛋白質設計需求的工具箱。此外,由於SNase 對於抑制劑的結合有強烈的機械性反應,同時亦是研究蛋白質摺疊系統中的模型蛋白質,提供未來研究酵素機械力學與催化之間的關係的獨特平台。 大腸桿菌細胞色素b562 之奈米機械力學之驗證:血鐵基質的氧化還原蛋白質提供獨一無二的機會以測量蛋白質於不同氧化態是否具有機械力穩定度之差異 。 我們首次利用原子力顯微鏡基礎單一分子力譜直接量測細胞色素b562在不同氧化態條件下的機械力穩定度差異,證實由還原劑誘導的氧化態改變使細胞色素b562的機械力穩定度產生改變,由去摺疊蛋白質機械力約27pN (-TCEP) 增加至約45pN(+ TCEP),統計上明顯增加約70%。 此研究除了首次證實血鐵基質的四螺旋綁束 ( four-helix bundles ) 結構型態具有顯著性機械力穩定度,細胞色素b562獨特的電致機械耦合性質(electromechanical coupling) 所展現整體長度收縮伸張之機械行為更為此研究之焦點。 本論文所研究驗證的非機械性蛋白質,可增添新的可用模組以擴增現有的蛋白質工具箱,滿足人造彈性蛋白質的設計需求或製造新穎的蛋白質基礎建構單元,進而自組裝成為蛋白質基礎的生物性奈米材料或水合膠,未來有潛力應用於組織工程 、藥物投遞、生物性材料以及奈米科學等領域。
The study focus on the characterization and nanomechanics of non-mechanical proteins in order to expand the toolbox of elastomeric proteins, which can be used for designing or being modules of protein-based building blocks that are incorporated into multifunctional nano-structured assembles. In detail, this research aims to: (1) the methodology established of measuring mechanical stability of non-mechanical proteins. We genetically construct the chimera polyprotein system with reference proteins, which are necessary to identify the single molecule force - extension recordings probed by AFM. (2) We use AFM-based single molecule force spectroscopy to directly measure mechanical unfolding forces of SNase alone and in the complex with (pdTp)-Ca+2 and demonstrated that the insertion of the nucleotide inhibitor significantly enhances the mechanical stability of the enzyme. (3) We use AFM-based single molecule force spectroscopy to directly measure mechanical unfolding forces of‭ ‬cytochrome b562 ‬ in the different oxidation states, and demonstrated that the reduction of iron center can enhance the mechanical stability of cytochrome b562. The methodology established of measuring mechanical stability of non-mechanical proteins. The current challenge on AFM based single - molecule force spectroscopy has been to identify single-molecule AFM force-extension recordings and reduce the background signal masked by the non-specifically interactions between tip and substrate. The proteins usually were immobilized on substrate and picked up by the tip non-specifically. Therefore, it recommends that a longer protein in size is necessary in AFM pulling experiments. Otherwise, the build-in mechanically reference proteins can be very helpful to identify the single molecule records due to its well-defined mechanical properties. Thus, we combined genetic and molecular approaches to fill this lacuna. In the study, we genetically engineered a chimera polyprotein system, in which interest are flanked by I27 domains of titin. I27 domains serve here as molecular handles allowing to apply stretching forces to the N and C termini of protein and also serve as a mechanical reference allowing to identify single-molecule AFM force-extension recordings. The characterization and nanomechanics of Staphylococcal nuclease. Staphylococcal nuclease (SNase) catalyzes the hydrolysis of DNA and RNA in a calcium-dependent fashion. We used AFM-based single-molecule force spectroscopy to investigate the mechanical stability of SNase alone and in its complex with an SNase inhibitor, deoxythymidine 30,50-bisphosphate. We found that the enzyme unfolds in an all-or-none fashion at ~26 pN. Upon binding to the inhibitor, the mechanical unfolding forces of the enzyme-inhibitor complex increase to ~50 pN. This inhibitor-induced increase in the mechanical stability of the enzyme is consistent with the increased thermodynamical stability of the complex over that of SNase. Because of its strong mechanical response to inhibitor binding, SNase, a model protein folding system, offers a unique opportunity for studying the relationship between enzyme mechanics and catalysis. The characterization and nanomechanics of E.coli cytochrome b562. Heme redox proteins offer a unique opportunity to examine the effect of redox reactions on the mechanical stability of heme proteins, which can be probed by SMFS. Here, we used SMFS to directly measure the effect of heme and its oxidation state on the mechanical properties of cytochrome b562 (cyt b562). Our‭ ‬results show that the reduced cytochrome b562 has indeed a higher mechanical stability as compared to the oxidized cytochrome. The average unfolding force ﹤Funfolding ﹥of cytochrome b562 in the presence of TCEP increased to 45.5 ± 1.9 pN (mean ± SEM), as compared to 27.0 ± 1.4 pN (mean ± SEM) measured without TCEP. We conclude that heme reduction in cytochrome b562 triggered by TCEP increased its mechanical stability by almost 70%. Our present single-molecule level study offers a different perspective on the effect of redox reactions on heme proteins in that that it finds redox-related changes in the mechanical stability of a heme protein. Otherwise, the proposed redox-related change in the molecular flexibility and the folded length of cytochrome b562 could be the basis of protein-based “piezoelectric” nanomaterials and actuators.
URI: http://hdl.handle.net/11455/36285
其他識別: U0005-1608201120033800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1608201120033800
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