臺灣博碩士論文加值系統

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。

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.

Table of contents
摘 要1
ABSTRACT2
TABLE OF CONTENTS2
LIST OF FIGURES4
LIST OF TABLES8
CHAPTER 1 INTRODUCTION1
1.1 GENERAL INTRODUCTION TO PROTEIN STRUCTURE1
1.2 MASS SPECTROMETRY AS A POTENTIAL TOOL FOR PROTEIN STRUCTURE INVESTIGATION2
1.3 CHARACTERISTICS OF ESI-Q-TOF MASS SPECTROMETER5
1.3.1 General Introductions to Q-star pulsar i5
1.3.3 Introduction of collisional-induced dissociation6
1.4 INTRODUCTION OF SIALIC ACID SYNTHASE8
1.5 INTRODUCTION OF VACCINIA VIRUS ENVELOPE PROTEIN, A27L9
1.6 GOAL OF CURRENT STUDY10
CHAPTER 2 EXPERIMENTAL METHODS FOR STUDY PROTEIN STRUCTURE11
2.1 DNA MANIPULATION AND PROTEIN PURIFICATION OF SIALIC ACID SYNTHASE11
2.2 VACCINIA VIRUS ENVELOPE PROTEIN, A27L12
2.3 ELECTROPHORESIS12
2.3.1 SDS-PAGE12
2.3.2 Native PAGE13
2.4 SAMPLE PREPARATION OF ESI-MS14
2.4.1 High performance liquid chromatography (HPLC)14
2.4.2 Porous R114
2.4.3 Zip Tip15
2.4.4 Microcon15
2.5 THE EXPERIMENT OF INFLUENCE OF VARIANT PH15
2.6 THE PARAMETERS SETTING OF Q-STAR PULSAR I16
2.7 COLLISION-INDUCED DISSOCIATION EXPERIMENT FOR PROBE THE CHANGE OF PROTEIN CONFORMATION16
CHAPTER 3 RESULTS & DISCUSSION17
PART I. STRUCTURAL CHARACTERIZATION OF ESCHERICHIA COLI NEUB & STREPTOCOCCUS AGALACTIAE NEUB17
I.1 ELECTROPHORESIS18
I.1.1 SDS-PAGE18
I.1.2 Native PAGE19
I.2 DETERMINATION OF THE QUATERNARY STRUCTURE OF ECNEUB & SANEUB BY NANOFLOW ESI-MS19
I.2.1 Influence of instrument parameters on the detection of noncovalent NeuB complex20
I.2.2 Influence of solution pH on the quaternary structure of sialic acid synthases22
EcNeuB23
SaNeuB23
I.3 COMPARISON OF QUATERNARY STRUCTURE BETWEEN ECNEUB AND SANEUB24
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 MS26
II.1 DETERMINATION OF THE QUATERNARY STRUCTURE OF TRUNCATED A27L MUTANT, A27L-AA26
II.1.1 Determination of molecular weight of A27L-aa multimeric form under variant protein concentration27
II.1.2 Influence of instrument parameters on the detection of vaccinia virus envelope protein A27L-aa28
II.1.3 Influence of solution pH on the detection of A27L-aa29
II.2 VARIATION ON QUATERNARY STRUCTURE BY MUTATION ON VACCINIA VIRUS ENVELOPE PROTEIN30
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 mutation32
II-3 SUBUNIT-SUBUNIT INTERACTION PROBED BY COLLISIONAL-INDUCED DISSOCIATION (CID) TECHNIQUE33
II.3.1 Collisional-induced dissociation curves33
II.3.2 Gibbs free energy35
CHAPTER 4 CONCLUSION36
REFERENCES38

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.