近十年間，離子液體在生物催化和蛋白質化學方面的廣泛應用引起了鑒定蛋白質在離子液體中特性的研究興趣。本研究以古菌Pyrococcus horikoshii的酰基磷酸酶acylphosphatase (PhAcP) 和50% (v/v) 離子液體1-乙基-3-甲基咪唑四氟硼酸鹽 ([EMIM][BF₄]) 作為研究模型，首次利用多維核磁共振譜對蛋白質在離子液體中的結構和穩定性作高解析度的分析。我們首先通過蛋白質主鏈共振歸屬，得出PhAcP每個被歸屬的胺基酸在50% (v/v) [EMIM][BF₄] 中¹³C[superscript α]、¹³C[superscript β]、¹³CO、¹⁵N、H[superscript N]和H[superscript α]原子的化學位移。¹³C的化學位移相對無序纏捲狀態的¹³C化學位移的偏差分析 (¹³C secondary shift)，以及二級結構之間的nuclear Overhauser effect (NOE) 連接顯示PhAcP在50% (v/v) [EMIM][BF₄] 中的二級結構與PhAcP的自然結構大致相同，其三級結構亦無顯著變化。此外，我們以二維的¹H-¹⁵N HSQC實驗觀察在318K、328K和338K這三個溫度下的硫氰酸胍 (GdnSCN) 誘導蛋白質變性，發現同一溫度下無論50% (v/v) [EMIM][BF₄]是否存在，PhAcP的變性曲線都互相重疊，得到的 [GdnSCN]₁[subscript /]₂值也相同，由此可推斷50% (v/v) [EMIM][BF₄] 對PhAcP的穩定性沒有影響。 / The extensive application of ionic liquid in biocatalysis and protein chemistry in the past decade arouses interest in the characterization of protein behavior in ionic liquid. This study demonstrates the use of multi-dimensional nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and conformational stability of protein in ionic liquid at a high resolution for the first time, with Pyrococcus horikoshii acylphosphatase (PhAcP) and 50% (v/v) 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF₄]) as a study model. The backbone amide resonances of PhAcP in 50% (v/v) [EMIM][BF₄] were assigned in order to obtain the chemical shifts of ¹³C[superscript α], ¹³C[superscript β], ¹³CO, ¹⁵N, HN and H[superscript α] of each assigned residue. The estimation of secondary structure by the ¹³C secondary shift analysis and the nuclear Overhauser effect (NOE) connectivities observed within secondary structures together suggest that PhAcP has secondary structures arranged in native-like topology and there is no major alteration in the tertiary structure in 50% (v/v) [EMIM][BF₄]. Guanidine thiocyanate (GdnSCN)-induced denaturation was performed at 318K, 328K and 338K and monitored by 2D ¹H-¹⁵N HSQC experiments to study the conformational stability of PhAcP in 50% (v/v) [EMIM][BF₄]. The overlapping denaturation curves and consistent [GdnSCN]₁[subscript /]₂ values obtained at each temperature indicate no observable trend of stability alteration. / Detailed summary in vernacular field only. / Lee, Tsz Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 57-63). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgements --- p.iii / Contents --- p.iv / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.ix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction to ionic liquid --- p.1 / Chapter 1.1.1 --- Ionic liquid as reaction medium, co-solvent and additive in biocatalysis and protein chemistry --- p.1 / Chapter 1.1.2 --- The impact of ionic liquid on protein structure and stability is poorly understood --- p.3 / Chapter 1.2 --- PhAcP in [EMIM][BF₄] as a model to study the structure and stability of protein in ionic liquid by NMR spectroscopy --- p.6 / Chapter 1.2.1 --- The application of [EMIM][BF₄] with protein --- p.6 / Chapter 1.2.2 --- The background of PhAcP --- p.9 / Chapter 1.2.3 --- Overview of the study --- p.10 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Expression and purification of PhAcP --- p.12 / Chapter 2.1.1 --- Expression of PhAcP in Escherichia coli system --- p.12 / Chapter 2.1.2 --- Purification of PhAcP --- p.14 / Chapter 2.2 --- Solubility determination --- p.15 / Chapter 2.3 --- NMR experiments --- p.17 / Chapter 2.3.1 --- General procedures and sample preparation --- p.17 / Chapter 2.3.2 --- ¹H-¹⁵N HSQC spectra in various concentrations of [EMIM][BF₄] --- p.18 / Chapter 2.3.3 --- Structural characterization --- p.18 / Chapter 2.3.4 --- Stability characterization --- p.19 / Chapter Chapter 3 --- Results --- p.21 / Chapter 3.1 --- Can the solubility of PhAcP in [EMIM][BF₄] reach the millimolar range required for NMR study? --- p.21 / Chapter 3.2 --- Determination of the [EMIM][BF₄] concentration for a feasible NMR study --- p.23 / Chapter 3.3 --- Backbone resonance assignment of PhAcP in 50% (v/v) [EMIM][BF₄] --- p.26 / Chapter 3.4 --- Structural characterization of PhAcP in 50% (v/v) [EMIM][BF₄] --- p.29 / Chapter 3.4.1 --- Secondary structure estimation by ¹³C secondary shifts --- p.29 / Chapter 3.4.2 --- NOE connectivities within secondary structures --- p.35 / Chapter 3.5 --- Characterization of the conformational stability of PhAcP in 50% (v/v) [EMIM][BF₄] by guanidine thiocyanate-induced denaturation --- p.40 / Chapter Chapter 4 --- Discussion --- p.46 / Chapter 4.1 --- The structure of PhAcP in 50% (v/v) [EMIM][BF₄] resembles the native conformation --- p.46 / Chapter 4.2 --- The conformational stability of PhAcP has no observable change in 50% (v/v) [EMIM][BF₄] --- p.47 / Chapter 4.3 --- Insight into the application of enzyme in ionic liquid --- p.48 / Chapter 4.4 --- Limitation of the study --- p.49 / Chapter 4.5 --- Insight into future studies --- p.50 / Chapter Chapter 5 --- Conclusions --- p.51 / Appendix --- p.53 / References --- p.57
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328091 |
| Date | January 2013 |
| Contributors | Lee, Tsz Ying., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (ix, 63 leaves) : ill. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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