臺灣博碩士論文加值系統

綠色螢光蛋白(GFP)最早是從維多利亞多管發光水母身上所發現的，在數十年的研究努力下， GFP成功地發展出相當多樣性的螢光蛋白，且在生物醫學研究上，扮演著重要的角色。在眾多種類的螢光蛋白中，有一小部分的螢光蛋白，可以用光進行螢光放光特性的調控，稱之為光控螢光蛋白。例如，光致轉化螢光蛋白，在紫光的照射下會從原來的綠色螢光放光，轉變成為紅色螢光放光，此反應稱為光致轉化反應。光致轉化現象的關鍵在於發色團受到特定的光照後，會有化學反應的發生，使得發色團結構產生變化，進而影響螢光放光特性。
mEos2 FP是一個光致轉化螢光蛋白，原先為綠色螢光放光，經過405 nm的光照後，會轉變成紅色螢光放光。在先前的實驗中發現，mEos2的突變種S142E，有特殊的光學調控機制。mEos2(S142E) 在405 nm 光照下所形成的紅螢光型態，可以進一步被450 nm 的光所影響而改變分子的螢光放光，此光效應可能為光還原反應所導致。我們使用了高解析液相層析串聯質譜儀與電灑式游離質譜儀，實驗結果顯示，其發色團結構與原本的綠螢光型態不同但是與紅螢光型態一致。
於不同氧化還原環境下，光轉化實驗結果發現，mEos2(S142E) 紅螢光型態，在氧化環境下，大幅降低450 nm的光照效應；在還原環境下，加速了450 nm的光照效應。更進一步地觀測發色團結構的變化，從質譜結果來看，兩個型態(450 nm光照前/後)的發色團分子量並無差異。因此推斷 450 nm的光照效應，並未產生發色團結構變化，而是產生了螢光放光產率下降的物種。其一可能為受到450 nm的光照之下，螢光放光圖譜與原先綠螢光態相似，此現象的產生可能是450 nm的光，使得紅螢光態分子轉變成不放光型態，或是有光裂解產生，使得觀測到的螢光放光圖譜，為原先綠螢光態分子所放出的螢光。其二為在450 nm的光照之下，發生激發態電子轉移excited-state electron transfer，使得螢光產率大幅下降，導致測量到的螢光放光光譜為原先就存在的綠色螢光形態。未來實驗可以利用時間解析光譜分析，應可提供激發態動力學的訊息，進而驗證是否有激發態電子轉移反應發生。

Green fluorescent protein (GFP) was first discovered from the Aequorea victoria and it has developed more than fifty years. As a powerful tool for biomedical sciences research, GFP exhibits many applications to biotechnology. Among GFP variants, there are some special variants which can be manipulated by light such as photoconvertible fluorescent proteins (PCFPs). PCFPs can be photoconverted when the violet light induced. The mEos2 is one of PCFPs that originally emits green fluorescence and could be converted to red-fluorescence emission under the 405 nm irradiation.
We have found from previous studies that the mutant mEos2(S142E) behaves a special optical response under the 450 nm irradiation. Additional absorption and emission bands were detected after 405-nm photoconversion and were further confirmed as a neutral red-form of mEos2 S142E. This neutral red-form of mEos2 S142E could be further converted by 450-nm irradiation and a green emission emerged. Such a new appearing green emission could resulting from either photoconverting to fluorescent-less state or reversibly conversion to the original green form. To further confirm the structure of this new state, we incorporated HPLC/Mass spectrometer into experiments. The results showed that the chromophore’s structure is not similar the original green form but remains the red form structure.
Under different oxidation conditions, our experimental results show that the 450-nm conversion rate become slower when the mEos2(S142E) red form is under the oxidizing condition but accelerated when it is in the reducing environments. Therefore, a photo-reduction process might occur upon 450-nm irradiation. However, the mass spectrometry results show that the chromophore of the two different types (before/after 450 nm conversion) is identical. Hence, the 450 nm irradiation does not change the structure of chromophore directly but forms a flurescence-less form.
Since this form is highly affected by oxidation environment, 450-nm irradiation might trigger photoreduction reaction in the excited state of red-neutral form. Further experiments by time-resolved spectroscopy will provide more details to confirm this.

一、緒論......................................................................................................................................1
1.1維多利亞多管發光水母(Aequorea Victoria)螢光蛋白的重要發現與發展.........................1
1.2珊瑚生物(Anthozoa)體中的螢光蛋白的重要發現與發展.....................................................6
1.3光控螢光蛋白(optical highlighter fluorescent proteins) ......................................................7
1.3.1光控螢光蛋白簡介.................................................................................................................7
1.3.2光激活化螢光蛋白.................................................................................................................8
1.3.3光致轉換螢光蛋白.................................................................................................................8
1.3.4光致轉化螢光蛋白.................................................................................................................9
1.4 mEos2 FP光致轉化螢光蛋白................................................................................................10
1.4.1 mEos2 FP 的演變歷程.........................................................................................................10
1.4.2 mEos2發色團的反應機制..................................................................................................13
1.5 實驗目的.................................................................................................................................16
1.5.1 mEos2(S142E)突變種的設計概念與由來.........................................................................17
1.5.2 mEos2(S142E)的特性.........................................................................................................18
1.5.3 mEos2(S142E) 450 nm光轉換...........................................................................................20
1.5.4 405 nm與450 nm光轉化的反應機制.................................................................................21

二、 材料與方法......................................................................................................................22
2.1 mEos2(S142E)點突變設計與質體DNA放大與萃取............................................................22
2.2 蛋白質的表達.........................................................................................................................22
2.3 蛋白質的純化.........................................................................................................................23
2.4 光譜實驗.................................................................................................................................24
2.4.1 UV-VIS吸收光譜原理........................................................................................................24
2.4.2 螢光放光光譜原理...............................................................................................................25
2.4.3 mEos2(S142E)突變種的405 nm與450 nm的光轉化實驗..............................................26
2.5 mEos2(S142E)於還原劑(DTT)的環境下光轉化實驗..........................................................27
2.6 mEos2(S142E)於氧化劑(K3[Fe(CN)6])的環境下光轉化實驗.............................................28
2.7 蛋白質質譜分析實驗.............................................................................................................29
2.7.1 蛋白質大分子量質譜分析...................................................................................................29
2.7.2蛋白質水解與高效層析電灑式二次離子質譜儀...............................................................30
2.7.3 目標Peptide的純化與質譜量測實驗.................................................................................32
2.7.4 膠體中蛋白質消化水解實驗(In-solution digestion with trypsin) .....................................34
2.7.5 水溶液中蛋白質消化水解實驗(In-solution digestion with trypsin) .................................35
2.7.6 科技部貴重儀器中心質譜儀實驗記錄...............................................................................35

三、實驗結果...........................................................................................................................36
3.1 405 nm及450 nm光轉化蛋白質電泳實驗............................................................................36
3.2 405 nm 450 nm 光轉化實驗光譜測定..................................................................................39
3.3 氧化劑環境450 nm光轉化實驗光譜測定...........................................................................41
3.4 添加還原劑DTT與450 nm光轉化實驗光譜測定.............................................................43
3.5 蛋白質質譜分析.....................................................................................................................45
3.5.1實驗目的...............................................................................................................................45
3.5.2 水解螢光蛋白序列與發色團分子量預測...........................................................................46
3.5.3 胺基酸序列HYGNR的精準分子量分析..........................................................................48
3.5.4 胺基酸序列HYGNR質譜分析結果..................................................................................50
3.5.4.1 分析方法與數據分析........................................................................................................50
3.5.4.2 HPLC 液相層析與一次離子與二次離子質譜結果........................................................51
四、討論與結論.......................................................................................................................55
4.1 實驗結果的探討.....................................................................................................................55
4.2 450 nm 的光照效應................................................................................................................56
4.2.1 450 nm的光還原反應..........................................................................................................56
4.2.2 mEos2螢光蛋白的光致轉換(photoswitching)現象...........................................................60
4.2.3 mEos2(S142E)突變種photoswitching現象的實驗測試....................................................62
4.3 結論.........................................................................................................................................63
五、文獻Reference................................................................................................................64
六、附錄.....................................................................................................................................68

[1] The Royal Swedish Academy of Sciences, Scientific Background on the Nobel Prize in Chemistry 2008 , The green fluorescent protein:discovery, expression and development
[2] Day, R.N. and M.W. Davidson, The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev, 2009. 38(10): p. 2887-921.
[3] Shimomura, O., F.H. Johnson, and Y. Saiga, Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol, 1962. 59: p. 223-39.
[4] Cody, C.W., et al., Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry, 1993. 32(5): p. 1212-8.
[5] aniel Th di , et al. Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy . J. Phys. Chem. Lett. 2017, 8, 4424−4430.
[6] Chalfie, M.; Kain, S. Green fluorescent protein: properties, applications, and protocols. 2nd. Wiley-Interscience; Hoboken, NY: 2006.
[7] Heim, R., D.C. Prasher, and R.Y. Tsien, Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A, 1994. 91(26): p. 12501-4.
[8] Tsien, R.Y., The green fluorescent protein. Annu Rev Biochem, 1998. 67: p. 509-44.
[9] Ormo, M., et al., Crystal structure of the Aequorea victoria green fluorescent protein. Science, 1996. 273(5280): p. 1392-5.
[10] Chattoraj, M., et al., Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci U S A, 1996. 93(16): p. 8362-7.
[11] Chalfie, M., et al., Green fluorescent protein as a marker for gene expression. Science, 1994. 263(5148): p. 802-805.
[12] Inouye, S. and F.I. Tsuji, Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett, 1994.
341(2-3): p. 277-80.
[13] Van Roessel, P. and A.H. Brand, Imaging into the future: visualizing gene expression and

protein interactions with fluorescent proteins. Nature cell biology, 2002. 4(1): p. E15.
[14] Xenopoulos, P., S. Nowotschin, and A.-K. Hadjantonakis, Live imaging fluorescent proteins in early mouse embryos, in Methods in enzymology. 2012, Elsevier. p. 361-389.
[15] Stewart Jr, C.N., Go with the glow: fluorescent proteins to light transgenic organisms. Trends in biotechnology, 2006. 24(4): p. 155-162.
[16] Baird, G.S., D.A. Zacharias, and R.Y. Tsien, Circular permutation and receptor insertion within green fluorescent proteins. Proceedings of the National Academy of Sciences, 1999. 96(20): p. 11241-11246.
[17] Brejc, K., et al., Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proceedings of the National Academy of Sciences, 1997. 94(6): p. 2306-2311.
[18] ] Elowitz, M. B., et al Photoactivation turns green fluorescent protein red. Curr. Biol. 7, 809–812 (1997).
[19] Virgile Adama, et al. Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. PNAS November 25, 2008 vol. 105 no. 47 18343–18348
[20] Matz, M.V., K.A. Lukyanov, and S.A. Lukyanov, Family of the green fluorescent protein: journey to the end of the rainbow. Bioessays, 2002. 24(10): p. 953-959.
[21] Labas, Y.A., et al., Diversity and evolution of the green fluorescent protein family. Proceedings of the National Academy of Sciences, 2002. 99(7): p. 4256-4261.
[22] Matz, M.V., et al., Fluorescent proteins from nonbioluminescent Anthozoa species. Nature biotechnology, 1999. 17(10): p. 969.
[23] Perron, A., et al., Red-shifted voltage-sensitive fluorescent proteins. Chemistry & biology, 2009. 16(12): p. 1268-1277.
[24] Shaner, N.C., G.H. Patterson, and M.W. Davidson, Advances in fluorescent protein technology. Journal of cell science, 2007. 120(24): p. 4247-4260.
[25] Patterson, G.H. and J. Lippincott-Schwartz, A photoactivatable GFP for selective photolabeling of proteins and cells. Science, 2002. 297(5588): p. 1873-1877.
[26] Andresen, M., et al., Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(37): p. 13070-13074.

[27] Ranajay Saha1*, et al., Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins SCIENTIFIC REPORTS | 3 : 1580 | DOI: 10.1038/srep01580
[28] Ann L. McEvoy.. et al., mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities. Published: December 11, 2012, doi:10.1371/journal.pone.0051314
[29] Ando, R., et al., An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proceedings of the National Academy of Sciences, 2002. 99(20): p. 12651-12656.
[30] 許甄听, 螢光蛋白mEos2與其突變種之光致轉化研究(Unravel the key residues in converting the fluorescence emission in photoconvertible fluorescence protein mEos2), NCTU, 碩士論文, 2016.7
[31] Nienhaus, G.U., et al., Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications. Photochemistry and Photobiology, 2006. 82(2): p. 351-358.
[32] Wachter, R.M., Photoconvertible Fluorescent Proteins and the Role of Dynamics in Protein Evolution. International Journal of Molecular Sciences, 2017. 18(8): p. 1792.
[33] Pouwels, L.J., et al., Kinetic isotope effect studies on the de novo rate of chromophore formation in fast-and slow-maturing GFP variants. Biochemistry, 2008. 47(38): p. 10111-10122.
[34] Zhang, L., et al., Reaction progress of chromophore biogenesis in green fluorescent protein. Journal of the American Chemical Society, 2006. 128(14): p. 4766-4772.
[35] Lelimousin, M.l., et al., Photoconversion of the fluorescent protein EosFP: a hybrid potential simulation study reveals intersystem crossings. Journal of the American Chemical Society, 2009. 131(46): p. 16814-16823.
[36] Kim, H., et al., A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins. Structure, 2015. 23(1): p. 34-43.
[37] Perozzo, M., et al., X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals. Journal of Biological Chemistry, 1988. 263(16): p. 7713-7716.
[38] Khan, F., et al., 19F NMR studies of the native and denatured states of green fluorescent protein. J. Am. Chem. Soc., 2006. 128(33): p. 10729-10737.
[39] Brakemann, T., et al., A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nature biotechnology, 2011. 29(10): p. 942.
[40] Mizuno, H., et al., Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Molecular cell, 2003. 12(4): p. 1051-1058.

[41] Shevchenko, A., Wilm, M., Vorm, O and Mann, M. (1996). Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal. Chem. 68, 850-858. Bruker, Proteomic protocols for mass spectrometry (2008 Edition) ,In-gel and In-Solution Trypsin Digestion
[42] Xin X. Zhou and Michael Z. Lin, Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications, Curr Opin Chem Biol. 2013 August ; 17(4): 682–690.
[43] Sean A. McKinney1, et al., A bright and photostable photoconvertible fluorescent protein for fusion. tags.Published as: Nat Methods. 2009 February ; 6(2): 131–133.
[44] Jo rg Wiedenmann1., et al. From EosFP to mIrisFP: structure-based development of advanced photoactivatable marker proteins of the GFP-family. J. Biophotonics 4, No. 6, 377–390 (2011)
[45] 徐佑武, 光致轉化螢光蛋白mEos2之光轉化動力學機制研究 (The photoconversion Dynamic of fluorescent protein mEos2 and its Variant), NCTU, 碩士論文,2016.8.
[46] Pierre Bouguer, Essai d'optique sur la gradation de la lumière (Paris, France: Claude Jombert, 1729) pp. 16–22.
[47] Beer (1852). Bestimmung der Absorption des rothen Lichts in farbigen Flüssigkeiten Determination of the absorption of red light in colored liquid. Annalen der Physik und Chemie. 86: 78–88
[48] Antonie J.W.G. Visser and Olaf J. Rolinski (12/03/10) BASIC PHOTOPHYSICS
[49] Nathan Shaner et al (2004) Nature Biotech. 22:1567-1572 Lei Wang et al (2004)Proc. Natl. Acad. Sci. USA 101:16745-16749
[50] C. Shaner1, George H. Patterson2 and Michael W. Davidson3 Advances in fluorescent protein technology. 2007Journal of Cell Science 120, 4247-4260
[51] Dominique Bourgeois1 , Aline Regis-Faro and Virgile Adam . Photoactivated structural dynamics of fluorescent proteins Biochemical Society Annual Symposium No. 79