以質譜技術探討非共價鍵結蛋白質聚合物之結構

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：23

、訪客IP：3.133.86.172

姓名

黃興鴻(Xing-Hong Huang) 查詢紙本館藏

畢業系所

化學學系

論文名稱

以質譜技術探討非共價鍵結蛋白質聚合物之結構
(Study of Noncovalent Protein Assembly by Mass Spectrometry)

相關論文

★ 以液相層析質譜儀檢測水樣與生物檢體中全氟界面活性劑之濃度	★ 利用液相層析串聯質譜技術檢測水環境中藥物殘留物之方法開發與應用
★ 以蛋白質體學探討在大腸桿菌中甲醇利用代謝途徑	★ Data-independent acquisition mass spectrometry analysis for identification of cerebrospinal fluid biomarker of reversible cerebral vasoconstriction syndrome
★ 直鏈式烷基苯基二甲基銨鹽類陽離子型界面活性劑在水環境中微量檢測方法的研究	★ 芳香族磺酸鹽類有機污染物在水環境中的分析與研究
★ 以固相萃取及氣相層析質譜儀對水環境中壬基苯酚類持久性有機污染物之分析與研究	★ 以固相萃取法及氣相層析質譜儀對水環境中動情激素類有機污染物之分析與研究
★ 利用熱裂解直接高溫衍生化法快速分析直鏈式烷基三甲基銨鹽之方法建立與探討	★ 利用感應偶合電漿質譜儀檢測半導體製程用化學品中微量金屬不純物之分析研究
★ 應用毛細管電泳間接偵測方法分離四級銨鹽界面活性劑	★ 利用毛細管電泳結合線上濃縮方法分離奈磺酸鹽之機制探討
★ 快速分析水環境中醫療藥品殘留物之研究與探討	★ 以毛細管電泳法與電灑游離質譜法探討內包錯合物之研究
★ 以氣相及液相層析質譜儀分析具荷爾蒙效應物質之方法開發	★ 以離子配對高效液相層析儀檢測螢光增白劑在不同基質中之研究

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

摘要
在這實驗當中，我們希望藉由電噴灑游離質譜法，來研究非共價鍵結聚集的蛋白質四級結構。在實驗當中，我們分析了兩種蛋白質以非共價鍵結聚集的四級結構，其中包含了單體分子量約為38 kDa的唾液酸合成酶以及單體分子量約為10.3 kDa的牛痘病毒鞘膜蛋白質。我們藉由改變質譜中的加速電壓以及壓力，來找出最適當的分析條件，在我們的結果當中證明這些儀器條件決定了蛋白質的四級結構是不是能維持到最後，而被我們所觀察到。當溶液的pH值被改變時，我們可以從質譜圖上，觀察到蛋白質四級結構的改變，這除了有助於判斷質譜中所觀察到的結構是否和溶液中一致，更能夠提供蛋白質在具活性條件下的四級結構資訊。另一方面，非共價鍵結聚合物的解離路徑可以藉由質譜中特有的碰撞誘導解離技術來加以推論。我們更可藉由碰撞誘導解離曲線，來推算非共價鍵結聚合物在無外力施加狀況下解離所需要的 Gibbs free energy。
　　實驗當中的唾液酸合成酶來自兩個不同的菌種，包括大腸桿菌(EcNeuB)以及鏈球菌(SaNeuB)，在pH值7.5下，藉由電噴灑游離質譜法，我們觀察到四聚體的唾液酸合成酶分子量高達160 kDa。從改變溶液pH值的結果，顯示了唾液酸合成酶四聚體只存在於pH 6.5~8.5中，也說明了唾液酸合成酶的結構對溶液酸鹼值的改變相當敏感。　
　　另外一方面，之前的實驗認為牛痘病毒鞘膜蛋白質A27L-aa，與牛痘病毒侵入細胞的機制有相當大的關聯。在我們的實驗結果證實了A27L-aa 是一個六聚體的四級結構，另外我們藉由觀察改變氨基酸後造成的結構改變，證實Leucine51在A27L-aa本身形成四級結構的作用上，扮演了最重要的角色。我們根據碰撞誘導解離實驗中，不只證實了A27L-aa 的六聚體以及突變體(L47,51,54A)的四聚體都會直接解離成單體。最後，我們根據碰撞誘導解離曲線得出，A27L-aa的六聚體Gibbs free energy 是4.89 kcal/mol而突變體的四聚體Gibbs free energy 是3.98 kcal/mol，證明突變體(L47,51,54A)的四聚體比 A27L-aa的六聚體更容易解離。
　　我們發現具高分子量的非共價鍵結聚合物在質譜當中，需要有更高的加速電壓以及氣體壓力來幫助樣品能夠被我們所偵測。但是，對於較小分子量的聚合物，過高的加速電壓會造成四級結構被破壞。除此之外，包括pH實驗以及碰撞誘導解離的實驗結果，都能提供蛋白質非共價鍵結聚合物的結構資訊，由此我們證實電噴灑游離質譜法確實能在分析蛋白質非共價鍵結構上提供全面性的資訊，讓我們可更加了解蛋白質結構。

摘要(英)

Abstract
In this study, we attempted to use electrospray ionization mass spectrometry (ESI-MS) to investigate the noncovalent assembly of protein quaternary structure. Two proteins with different sizes, sialic acid synthases (monomer ~ 38 kDa) from E. coli and Streptococcus agalactiae, and the vaccinia virus envelope protein A27L (monomer ~ 10.3 kDa) were studied as models for noncovalent assembly. Optimization of various critical parameters in interface was studied in detail to preserve the integrity of noncovalent assemblies. Furthermore, variations in solution pH were found to induce dramatic changes on the ESI mass spectra, which can be used as a readout for characterization of noncovalent protein assemblies. Moreover, collision-induced dissociation technique was applied to probe dissociation pathway and stability of noncovalent complexes. In addition, to characterize different degree of association, the Gibbs free energy of these oligomers was determined accordingly
E. coli sialic acid synthase (EcNeuB) and Streptococcus agalactiae sialic acid synthase (SaneuB) have high sequence homology. Tetrameric form was observed in ESI-MS at as the highest order quaternary structure for both enzymes at pH 7.5. Tetrameric form only exist at physiological range (pH 6.5 ~ pH 8.5), demonstrating that quaternary structure of sialic acid synthases is pH-sensitive. The dependence of structure on solution pH also provides a high level of confidence for “structural-specific” evidence.
Previous studies have revealed that recombinant vaccinia virus protein, A27L-aa, may facilitate vaccinia virus entry into host cell. The chemical shift index studies also strongly indicated that the three hydrophobic leucine residues (L47, L51, and L54) may play an important role in self-assembly of A27L-aa. In this investigation, we demonstrated that the quaternary structure of A27L-aa is a hexameric form. Besides, the dramatic change of quaternary structure of A27L-aa from hexamer to tetramer on mass spectrum by mutation of leucine 51 to alanine 51 demonstrated that leucine 51 play crucial role in contributing hydrophobic interaction of A27L-aa self-assembly. Furthermore, CID experiment showed that both tetramer and hexamers of A27L-aa and its mutants will directly dissociated to monomers without any intermediates. Gibbs free energy of hexamer and tetramer can be calculated from CID curves for hexamer and tetramer respectively. The Gibbs free energy of A27L-aa is 4.89kcal/mol and triple mutation is 3.98kcal/mol. The result demonstrate that the subunit-subunit interaction of L47,51,54A is weaker than A27L-aa.
ESI-MS may have great potentials for study noncovalent protein assembly. Moreover, the more detail structural information can also be provided by using the ESI-MS.

關鍵字(中)

★ 非共價鍵結聚合物
★ 質譜技術

關鍵字(英)

★ noncovalent complex
★ ESI-MS
★ ESI

論文目次

Table of contents
摘要 1
ABSTRACT 2
TABLE OF CONTENTS 2
LIST OF FIGURES 4
LIST OF TABLES 8
CHAPTER 1 INTRODUCTION 1
1.1 GENERAL INTRODUCTION TO PROTEIN STRUCTURE 1
1.2 MASS SPECTROMETRY AS A POTENTIAL TOOL FOR PROTEIN STRUCTURE INVESTIGATION 2
1.3 CHARACTERISTICS OF ESI-Q-TOF MASS SPECTROMETER 5
1.3.1 General Introductions to Q-star pulsar i 5
1.3.3 Introduction of collisional-induced dissociation 6
1.4 INTRODUCTION OF SIALIC ACID SYNTHASE 8
1.5 INTRODUCTION OF VACCINIA VIRUS ENVELOPE PROTEIN, A27L 9
1.6 GOAL OF CURRENT STUDY 10
CHAPTER 2 EXPERIMENTAL METHODS FOR STUDY PROTEIN STRUCTURE 11
2.1 DNA MANIPULATION AND PROTEIN PURIFICATION OF SIALIC ACID SYNTHASE 11
2.2 VACCINIA VIRUS ENVELOPE PROTEIN, A27L 12
2.3 ELECTROPHORESIS 12
2.3.1 SDS-PAGE 12
2.3.2 Native PAGE 13
2.4 SAMPLE PREPARATION OF ESI-MS 14
2.4.1 High performance liquid chromatography (HPLC) 14
2.4.2 Porous R1 14
2.4.3 Zip Tip 15
2.4.4 Microcon 15
2.5 THE EXPERIMENT OF INFLUENCE OF VARIANT PH 15
2.6 THE PARAMETERS SETTING OF Q-STAR PULSAR I 16
2.7 COLLISION-INDUCED DISSOCIATION EXPERIMENT FOR PROBE THE CHANGE OF PROTEIN CONFORMATION 16
CHAPTER 3 RESULTS & DISCUSSION 17
PART I. STRUCTURAL CHARACTERIZATION OF ESCHERICHIA COLI NEUB & STREPTOCOCCUS AGALACTIAE NEUB 17
I.1 ELECTROPHORESIS 18
I.1.1 SDS-PAGE 18
I.1.2 Native PAGE 19
I.2 DETERMINATION OF THE QUATERNARY STRUCTURE OF ECNEUB & SANEUB BY NANOFLOW ESI-MS 19
I.2.1 Influence of instrument parameters on the detection of noncovalent NeuB complex 20
I.2.2 Influence of solution pH on the quaternary structure of sialic acid synthases 22
EcNeuB 23
SaNeuB 23
I.3 COMPARISON OF QUATERNARY STRUCTURE BETWEEN ECNEUB AND SANEUB 24
PART II. STRUCTURAL CHARACTERIZATION OF VACCINIA VIRUS ENVELOPE PROTEIN, A27L-AA, L47A, L51A, L54A, AND TRIPLE MUTATION (L47, 51, 54A) BY NANOFLOW ESI-Q-TOF MS 26
II.1 DETERMINATION OF THE QUATERNARY STRUCTURE OF TRUNCATED A27L MUTANT, A27L-AA 26
II.1.1 Determination of molecular weight of A27L-aa multimeric form under variant protein concentration 27
II.1.2 Influence of instrument parameters on the detection of vaccinia virus envelope protein A27L-aa 28
II.1.3 Influence of solution pH on the detection of A27L-aa 29
II.2 VARIATION ON QUATERNARY STRUCTURE BY MUTATION ON VACCINIA VIRUS ENVELOPE PROTEIN 30
II.2.1 DETERMINATION OF THE QUATERNARY STRUCTURE OF THREE KINDS OF A27L-AA MUTANTS (L47A, L51A, AND L54A) 30
II.2.2 Influence of solution pH on triple mutation 32
II-3 SUBUNIT-SUBUNIT INTERACTION PROBED BY COLLISIONAL-INDUCED DISSOCIATION (CID) TECHNIQUE 33
II.3.1 Collisional-induced dissociation curves 33
II.3.2 Gibbs free energy 35
CHAPTER 4 CONCLUSION 36
REFERENCES 38

參考文獻

References
(1) Winston, R. L.; Fitzgerald, M. C. Mass Spectrom Rev 1997, 16, 165-179.
(2) Feild, M. J.; Nguyen, D. C.; Armstrong, F. B. Biochemistry 1989, 28, 5306-5310.
(3) Hensley, P. Structure 1996, 4, 367-373.
(4) Schuster, T. M.; Toedt, J. M. Curr Opin Struct Biol 1996, 6, 650-658.
(5) Svergun, D. I.; Barberato, C.; Koch, M. H.; Fetler, L.; Vachette, P. Proteins 1997, 27, 110-117.
(6) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71.
(7) Bakhtiar, R.; Nelson, R. W. Biochem Pharmacol 2000, 59, 891-905.
(8) Siuzdak, G.; Lewis, J. K. Biotechnol Bioeng 1998, 61, 127-134.
(9) Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A. J Mass Spectrom 2001, 36, 849-865.
(10) B. Ganem, Y. T. L., J. D. Henion J. Am. Chem. Soc. 1991, 113, 7818.
(11) Veenstra, T. D. Biochem Biophys Res Commun 1999, 257, 1-5.
(12) Kaltashov, I. A.; Eyles, S. J. Mass Spectrom Rev 2002, 21, 37-71.
(13) M. Wilm, M. M. Anal Chem 1996, 68, 1.
(14) A. A. Rostom, C. V. R. J. Am. Chem. Soc. 1999, 121, 4718.
(15) Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Proc Natl Acad Sci U S A 2000, 97, 5185-5190.
(16) M. A. Tito, K. T., K. Valegard, J. Hajdu, C. V. Robinson J. Am. Chem. Soc. 2000, 122, 3550.
(17) Verentchikov, A. N.; Ens, W.; Standing, K. G. Anal Chem 1994, 66, 126-133.
(18) Tang, X. J.; Brewer, C. F.; Saha, S.; Chernushevich, I.; Ens, W.; Standing, K. G. Rapid Commun Mass Spectrom 1994, 8, 750-754.
(19) McLafferty, F. W. Science 1981, 214, 280-287.
(20) Loo, J. A. Mass Spectrom Rev 1997, 16, 1-23.
(21) Sanglier, S.; Leize, E.; Dorsselaer, A.; Zal, F. J Am Soc Mass Spectrom 2003, 14, 419-429.
(22) Rostom, A. A.; Robinson, C. V. Curr Opin Struct Biol 1999, 9, 135-141.
(23) K. Levsen, a. H. S. Mass Spectrom. Rev. 1983, 2, 77.
(24) Jennings, K. R. Int. J. Mass Spectrom. Ion. Phys 1968, 1, 227.
(25) W. F. Haddon, a. F. W. M. J. Am. Chem. Soc. 1968, 90, 4745.
(26) R. G. Cooks, L. H., and J. H. Beynon Org. Mass spectrom. 1975, 10, 625.
(27) J. H. Baynon, R. M. C., and T. Ast Int. J. Mass Spectrom. Ion. Phys 1972, 7, 88.
(28) K. C. Kim, M. U., J. H. Baynon, and R. G. Cooks Int. J. Mass Spectrom. Ion. Phys 1974, 15, 23.
(29) R. W. Kondrat, a. R. G. c. Anal Chem 1978, 50, A81.
(30) Cooks, R. G. Wiley, New York.
(31) Varki, A. Science 1999, 283, 791-792.
(32) Roggentin, P.; Kleineidam, R. G.; Schauer, R. Biol Chem Hoppe Seyler 1995, 376, 569-575.
(33) Norman, K. E.; Katopodis, A. G.; Thoma, G.; Kolbinger, F.; Hicks, A. E.; Cotter, M. J.; Pockley, A. G.; Hellewell, P. G. Blood 2000, 96, 3585-3591.
(34) Taylor, G. Curr Opin Struct Biol 1996, 6, 830-837.
(35) Reeves, P. R.; Hobbs, M.; Valvano, M. A.; Skurnik, M.; Whitfield, C.; Coplin, D.; Kido, N.; Klena, J.; Maskell, D.; Raetz, C. R.; Rick, P. D. Trends Microbiol 1996, 4, 495-503.
(36) Bliss, J. M.; Silver, R. P. Mol Microbiol 1996, 21, 221-231.
(37) Annunziato, P. W.; Wright, L. F.; Vann, W. F.; Silver, R. P. J Bacteriol 1995, 177, 312-319.
(38) Haywood, A. M. J Virol 1994, 68, 1-5.
(39) Bentz, J. Viral Fusion Mechnism, CRC press, Boca Raton, FL 1993.
(40) Montgomery, R. I.; Warner, M. S.; Lum, B. J.; Spear, P. G. Cell 1996, 87, 427-436.
(41) B. Moss, i. D. M. K., P. M. Howley (Eds) 2001, 2849-2884.
(42) J. J. Esposito, F. F., in: D. M. knipe, 2001, 2885-2922.
(43) Chung, C. S.; Hsiao, J. C.; Chang, Y. S.; Chang, W. J Virol 1998, 72, 1577-1585.
(44) Vazquez, M. I.; Rivas, G.; Cregut, D.; Serrano, L.; Esteban, M. J Virol 1998, 72, 10126-10137.
(45) Lin, T. H.; Chia, C. M.; Hsiao, J. C.; Chang, W.; Ku, C. C.; Hung, S. C.; Tzou, D. L. J Biol Chem 2002, 277, 20949-20959.
(46) Rodbard, D.; Chrambach, A. Anal Biochem 1971, 40, 95-134.
(47) Alichanidis, E.; Wrathall, J. H.; Andrews, A. T. J Dairy Res 1986, 53, 259-269.
(48) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. J Mass Spectrom 1999, 34, 105-116.
(49) Smith, R. D. L.-W., K. J. Biol. Mass Spectrom. 1993, 22, 493-501.
(50) Kaltashov, I. A. C., I. V. J Am Soc Mass Spectrom 1998, 9, 569.
(51) R. D. Smith, X. C., Brenda L. Schwartz, Ruidan Chen, and Steven A. Hofstadler Biological and Biotechnological applications of ESI-MS 1996, 294-314.
(52) Light-Wahl, K. J. S., B. L.; Smith, R. D. J. Am. Chem. Soc. 1993, 116, 5271-5278.
(53) Jellen, E. E.; Chappell, A. M.; Ryzhov, V. Rapid Commun Mass Spectrom 2002, 16, 1799-1804.

指導教授

丁望賢、陳玉如
(Wang-Hsien Ding、Yu-Ju chen)

審核日期

2003-7-10

推文