在模式生物系統中表現異源性膜蛋白次單元體:微粒體甲烷單氧化酵素的B 次單元體

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姓名

張廷安(Ting-An Chang) 查詢紙本館藏

畢業系所

化學學系

論文名稱

在模式生物系統中表現異源性膜蛋白次單元體:微粒體甲烷單氧化酵素的B 次單元體
(Heterologous Expression of Membrane Protein Subunit in Host Organisms: The Subunit B of pMMO)

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摘要(中)

從嗜甲烷菌Methylcoccus capsulatus (Bath)中分離的微粒體甲烷單氧化酵素B次元體,在過去的研究中證實了它與還原態銅離子具有高度的親合力，這個現象是因為蛋白質C端水溶性次區塊所蘊含的九到十二個還原態銅離子所造成，這些銅離子在甲烷催化過程中，扮演著將電子傳進催化中心以活化甲烷的角色。然而，在最近報導的X光結晶結構中,B次單元體與大量還原態銅離子的結合情形並未得到證實；可能是因為這些銅離子容易在高氧濃度下或者嚴苛的純化過程中流失。為了要再度確認微粒體甲烷單氧化酵素B次單元體與銅離子的高度親合力，我們使用分子生物的技術在大腸桿菌TB1菌株的細胞膜上表現了此蛋白質，並且利用西方式點墨法與蛋白質電泳上，證實了我們的轉殖是成功的；隨後我們將此基因轉殖進入另外一株高銅耐受性的大腸桿菌BCRC®50305，並且在含有高銅離子濃度下的營養液中，培養此株大腸桿菌，並表現微粒體甲烷單氧化酵素B次元體。我們發現在高銅離子濃度下表現此蛋白質時，大腸桿菌的細胞膜會大量增生，這種行為非常類似嗜甲烷菌在培養時銅離子濃度逐漸增高時的行為。並且兩種細菌在此時，也都會在膜上堆積大量還原態的銅離子。這些現象也分別被電子顯微鏡與X光吸收光譜所證實。此外，表現重組微粒體甲烷單氧化酵素B次單元體時，大量增生的膜系統，並未能形成細胞膜的結構，而是以缺乏組織的方式堆積在細胞內部，形成一個類似胞器的新的細胞區間，並且在銅離子缺乏時導致細胞死亡。
此篇論文主要闡述銅離子與重組微粒體甲烷單氧化酵素B次元體間如何交互作用，導致細胞內產生各種不同的生理變化，與重組微粒體甲烷單氧化酵素B次元體，這一蛋白質在不同濃度銅離子下的不同摺疊狀態。

摘要(英)

The pmoB sub-domian of pMMO from Methylcoccus capsulatus (Bath) exhibits strong affinity towards reduced copper ions. It was considered that the C-terminal aqueous sub-domain of the protein could accommodate 9-12 reduced copper ions to serve as a reservoir of reducing equivalents for methane activation in the holo pMMO enzyme. However, the recent X-ray crystal structure did not show any features corresponding to these copper ions. One explanation was that these copper ions were lost when the preparation was grown under high oxygen tension or harsh purification condition used. To prove the high copper affinity of this sub-domain, we transferred the subunit B of pMMO into the membrane of E. coli TB1 by exploiting the techniques of molecular biology. The pMMO pmoB was introduced as a fusion protein with Maltose-bind protein. That recombinant process for the over-expression had successfully occurred in the membrane was evidenced by the western blotting with the detection of MBP fusion. The gel electrophoresis analysis also gave rise to the correct molecular weight pattern in the 1D SDS PAGE. Subsequently, we also transformed the designed vector with pmoB insertion into the E. coli strain BCRC®50305. BCRC®50305 belongs to one of the strains originating from the copper tolerant species W3110. The bacteria could be grown in the growth media up to 3.1 mM CuSO4 concentration. We subjected the strains with the pmoB gene to grow in LB buffer containing 1 mM Cu(II) ions. Under high copper ion stress, the cellular membranes of E. coli behaved like the ones in Methylococcus capsulatus (Bath) with abundant membrane accumulation and high content of reduced copper contents. These results were confirmed by electron microscopy and X-ray absorbance spectroscopy, where we discovered that the recombinant pmoB subunit had introduced into a much less structured membrane system within the cell.

關鍵字(中)

★ 銅離子(I)結合蛋白
★ 微粒體甲烷單氧化酵素
★ 銅離子運輸
★ 膜蛋白表現

關鍵字(英)

★ pMMO
★ membrane-protein expression
★ copper trafficking
★ Cu(I)-binding protein

論文目次

Abstract (Chinese)…………………………………………………………………….I
Abstract (English) ……………………………………………………………………II
Table of contents……………………………………………………………………..IV
List of Figures……………………………………………………………………….VII
List of Tables…………………………………………………………………………IX
Abbreviations…………………………………………………………………………X
Chapter 1 Introduction………………………………………………………………...1
1.0 Preface…………………………………………………………………………......2
1.1 Methanotrophs……………………………………………………………………..3
1.2 pMMO and the role of the copper ions in pMMO…………………………….......7
1.3 pmoB and its role in providing a buffer of reducing equivalents in the catalytic cycle………………………………………………………………………………….16
1.4 Copper trafficking and metal chaperones in membrane - protein folding……….20
1.5 The procaryotic cell membrane and difficulties with expressing membrane proteins in the membrane. ……………………………………………………….22
1.6 Goals of this dissertation…………………………………………………………25
Chapter 2 Materials and Methods……………………………………………………26
2.1 Chemical reagents………………………………………………………………..27
2.1.1 Culturing of Methylococcus capsulatus(Bath).………………………………...27
2.1.2 Molecular cloning.……………………………………………………………..28
2.1.3 Purification, identification and measurement of properties of the membranes..29
2.2 Instrumentation…………………………………………………………………..29
2.3 Culturing of Methylococcus capsulatus(Bath)…………………………………..30
2.4 Construction of the pMALdesp pmpB plasmid and transform to the E.coli strain TB1……………………………………………………………………………….31
2.5 Transformation of plasmids into the E.coli strain BCRC® 50305……………….34
2.5.1 The wild type E.coli strain—BCRC® 50305…………………………………..34
2.5.2 Construction of the competent cell of BCRC® 50305…………………………34
2.5.3 Transformation of the pMALdesp pmoB plasmid into competent cells of BCRC®50305…………………………………………………………………34
2.6 Overexpression of pmoB in E.coli membranes ………………………………….35
2.7 Growth of the genetic recombinant in E. coli strain TB1 and BCRC® 50305 at the different copper concentrations…………………………………………………..35
2.8 Isolation and purification of membranes from E.coli under different conditions..35
2.9 Western blotting………………………………………………………………….36
2.10 Measurement of properties of the membranes …………………………………36
2.10.1 Measurement of the copper concentration of the membranes by BCA assays.36
2.10.2 Measurement of the weight of membranes in the cells……………………….37
2.11 X-ray absorption spectroscopy………………………………………………….37
2.12 Electron microscopy…………………………………………………………….37
Chapter 3 Results ……………………………………………………………………39
3.1 The nature of pMMO-enriched membranes from Methylococcus capsulatus (Bath)…………………………………………………………………………………40
3.2 Molecular cloning and gene transformation……………………………………...41
3.2.1 Construction of pMAL pmoB plasmid ………………………………………...41
3.2.2 Transfer of the pMAL pmoB plasmid into BCRC® 50305……………………43
3.3 Expression of pmoB in the E.coli membranes: Different folding states of the recombinant pmoB protein……………………………………………………….46
3.4 Properties of the membranes which are prepared under different conditions……53
3.5 The oxidation state of the copper ions in the recombinant pmoB protein……….55
3.6 Electron microscopy……………………………………………………………..57
Chapter 4 Conclusions……………………………………………………………….70
References……………………………………………………………………………74
Appendices…………………………………………………………………………...78
Appendix 1 pMAL-p2x plasmid map………………………………………………..78
Appendix 2 Appendix 2 MBP-pmoB sequence……………………………………...79

參考文獻

1. Higgins, I. J.; Best, D. J.; Hammond, R. C., New findings in methane-utilizing
bacteria highlight their importance in the biosphere and their commercial potential.
Nature 1980, 286, (5773), 561-564.
2. Finkelstein, J., Structural biology: methanol maker. Nature 2005, 434, (7030), 151.
3. Murrell, J.C.; Gilbert, B.; McDonald, I.R.; Molecular biology and regulation of methane monoxygenase. Arch Microbiol 2000 173:325-332
4. Hanson, R. S.; Hanson, T. E., Methanotrophic bacteria. Microbiol Rev 1996, 60,
(2), 439-71.
5. Semrau, J. D.; Zolandz, D.; Lindstrom, M. E.; Chan, S. I., The role of copper in the
pMMO of Methylococcus capsulatus Bath: A structural vs. catalytic function.
Journal of Inorganic Biochemistry 1995, 58, (4), 235-244.
6. Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P.; auml, Crystal
structure of a bacterial non-haem iron hydroxylase that catalyses the biological
oxidation of methane. Nature 1993, 366, (6455), 537-543.
7. Basu, P.; Katterle, B.; Andersson, K. K.; Dalton, H., The membrane-associated
form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a
copper/iron protein. Biochem J 2003, 369, (Pt 2), 417-27.
8. Lieberman, R. L.; Shrestha, D. B.; Doan, P. E.; Hoffman, B. M.; Stemmler, T. L.;
Rosenzweig, A. C., Purified particulate methane monooxygenase from
Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a
copper-containing cluster. Proc Natl Acad Sci U S A 2003, 100, (7), 3820-5.
9. Lieberman, R. L.; Rosenzweig, A. C., Crystal structure of a membrane-bound
metalloenzyme that catalyses the biological oxidation of methane. Nature 2005,
434, (7030), 177-82.
10. Takeguchi, M.; Miyakawa, K.; Okura, I., The role of copper in particulate methane
monooxygenase from Methylosinus trichosporium OB3b. Journal of Molecular
Catalysis A: Chemical 1999, 137, (1-3), 161-168.
11. Semrau, J. D.; Chistoserdov, A.; Lebron, J.; Costello, A.; Davagnino, J.; Kenna, E.;
Holmes, A. J.; Finch, R.; Murrell, J. C.; Lidstrom, M. E., Particulate methane
monooxygenase genes in methanotrophs. J Bacteriol. 1995, 177, (11), 3071-3079.
12. Hakemian, A.S.; Rosenzweig, A.C., The biochemistry of methane oxidation.
Annu Rev Biochem. 2007, 76, 223-241
13. Lienerman, R.L.; Rosenzweig, A.C., Biological methane oxidation: reduction, biochemistry, and active site structure of particulate methane monooxygenase.
Crit. Rev. Biochem Mol Biol. 2004, 39(3) 147-164.
14. Nguyen, H. H.; Elliott, S. J.; Yip, J. H.; Chan, S. I., The particulate methane
monooxygenase from methylococcus capsulatus (Bath) is a novel
copper-containing three-subunit enzyme. Isolation and characterization. J Biol
Chem 1998, 273, (14), 7957-66.
15. Yu, S. S.; Chen, K. H.; Tseng, M. Y.; Wang, Y. S.; Tseng, C. F.; Chen, Y. J.;Huang, D. S.; Chan, S. I., Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (bath) with a hollow-fiber membrane bioreactor. J Bacteriol 2003, 185, (20), 5915-24.
16. Chan, S. I.; Chen, K. H.; Yu, S. S.; Chen, C. L.; Kuo, S. S., Toward delineating the
structure and function of the particulate methane monooxygenase from
methanotrophic bacteria. Biochemistry 2004, 43, (15), 4421-30.
17. Vinchurkar, M. S.; Chen, K. H.; Yu, S. S.; Kuo, S. J.; Chiu, H. C.; Chien, S. H.;
Chan, S. I., Polarized ATR-FTIR spectroscopy of the membrane-embedded
domains of the particulate methane monooxygenase. Biochemistry 2004, 43, (42),
13283-92.
18. Yu, S.S.; Ji, C.Z.; Wu, Y.P.; Lee, T.L.; Lai, C.H.; Lin, S.C.; Yang, Z.L.; Wang, V.C.; Chen, K.H.; Chan, S.I., The C-terminal aqueous-exposed domain of the 45kDa subunit of the particulate methane monoxygenase in Methylcoccus capsulatus (Bath) is a Cu(I) sponge. Biochemistry 2007, 46, 13762-13774
19. Chan, S.I.; Wang, V.C.; Lai, J.C.; Yu, S.S.; Chen, P.P.; Chen, K.H.; Chen, C.L.; Chan, M.K., Redox potentiometry studies of particulate methane monoxygenase support for a trinuclear copper cluster active site. Angew. Chem. Int. Ed. 2007, 46, 1992-1994
20.Chen, P.P.; Yang, R.B.; Lee, J.C.; Chan, S.I., Facile O-atom insertion into C-C and C-H bonds by a trinuclear copper complex designed to harness a singlet oxene. Proc. Natl. Acad. Sci. U.S.A. 2007, 104(37):14570-14575
21. Chen, P.P.; Chan, S.I., Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase. J Inorg Biochem. 2006 100(4) 801-809
22. Yu, S.S.; Wu, L.Y.; Chen, K.H.; Luo, W.I.; Huang, D.S.; Chan, S.I., The stereospecific hydroxylation of [2,2-2H2]butane and chiral dideuteriobutanes by the particulate methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem. 2003 278(42) 40658-40669
23. Huang, D.S.; Wu, S.H.; Wang, Y.S.; Yu S.S.; Chan S.I., Determination of the carbon kinetic isotope effects on propane hydroxylation mediated by the methane monooxygenases from Methylococcus capsulatus (Bath) by using stable carbon isotopic analysis. Chembiochem. 2002 3(8) 760-765
24. Chen, C.L.; Chen, K.H.; Ke, S.C.; Yu, S.S.; Chan, S.I., Preparation and characterization of a (Cu, Zn)-pMMO from Methylcoccus capsulatus (Bath). Journal of Inorganic Biochemistry 98(2004) 2125-2130
25. Ng, K.Y.; Yu, S.S.; Chan, S.I., unpublished data, 2008
26. Rosen, B.P., Transport and detoxification systems for transition metals, heavy
metals and metalloids in eukaryotic and prokaryotic microbes. Comparative Biochemistry and Physiology Part A 133 (2002) 689–693
27. Resing, C.; Grass, G. Escherichia coli mechanisms of copper homeostasis in
a changing environment. FEMS Microbiology Reviews 27 (2003) 197-213
28. KrÓliczewski, J.; Szczepaniak, A., In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli. Biochimica et Biophysica Acta 1598(2002) 177-184
29 Smyth, D.R.; Mrozkiewicz, M.K.; McGrath, W.J.; Listwan, P.; Kobe, B., Crystal structures of fusion proteins with large-affinity tags. Protein Science 2003 July;12(7) 1313-22
30. Arechaga, I.; Miroux, B.; Karrasch, S.; Huijbregts, R.; Kruiift, B.D.; Runswick, M.J. ; Walker, J.E., Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F1F0 ATP synthase. FEBS letters 482(2000) 215-219
31. 王琢堅, Implications of the redox behavior of the copper clusters in the particulate
methane monooxygenase on the methane hydroxylation mechanism. 國立台灣大學化學研究所碩士論文, 2005.
32. 林素卿, Reconstitution of membrane proteins or subunits for structural and functional studies. 國立台灣大學化學研究所博士論文, 2007
33. 楊秉恭, Models for the trinuclear copper clusters of the particulate methane monoxygenase from nethanotrophic bacteria: synthesis, spectroscopic characterization of trinuclear copper complexes. 國立中央大學化學研究所碩士論文, 2006
34. 陳家全李家維楊瑞森, 生物電子顯微鏡學行政院國家科學委員會精密儀器發展中心編印, 1991

指導教授

蔡惠旭、陳長謙
(Hui-Hsu Gavin Tsai、Sunney I. Chan)

審核日期

2008-1-24

推文