人類S100A4蛋白質為S100蛋白質家族一份子，這個蛋白質家族在結構上具有高度相似性，它們皆具有EF-hand motif且會與鈣離子結合，在與鈣離子結合後會改變其構型並與目標蛋白交互作用，而EGF蛋白為其中一種會與S100A4結合的目標蛋白。人類表皮生長因子( EGF )可促使多種生物細胞( 包括腫瘤細胞 )的增生分化，而此生物活性是透過EGF蛋白與細胞膜表面上的特定受體( EGFR )結合進而誘導產生一系列訊息傳導所致。在近期的研究中，科學家利用西方點墨法指出S100A4蛋白會與EGF蛋白作反應。
在本篇論文中，我們希望更進一步了解S100A4與EGF蛋白之間的反應，以及研究所形成之複合物。為了配合S100A4在水溶液條件中的最佳狀態，我們將EGF蛋白溶於PH6.0的溶液中，再利用三維核磁共振實驗及軟體計算解出EGF蛋白在PH6.0之三維結構後，以二維核磁共振滴定之方法，找出S100A4與EGF蛋白作用位置；另外結合恆溫滴定熱卡計、螢光等數據，來推測S100A4與EGF蛋白之結合比例與解離常數。而從實驗結果推測出，S100A4與EGF蛋白大概以1：1之比例結合形成複合物，而其解離常數為 μM等級。本研究幫助我們更了解S100A4與EGF蛋白反應之情形，而仍需更多延伸研究來分析這個反應，探討此反應是否與實際生理情形相關。

Human S100A4 protein belongs to S100 protein family which shares structure similarities with each other: they all have EF-hand motifs and could bind calcium. When S100A4 binds with calcium, it will change its conformation and interact with their target protein. Human epidermal growth factor ( EGF ) is the target protein which is also one of the high affinity ligands of EGFR. EGF/EGFR system promotes cell survival, growth and differentiation via the activation of several integrated signaling pathways. In recent studies, scientists used western blotting to show that S100A4 could interact with EGF.
In this thesis, we study the interaction between S100A4 and EGF, and we’d like to understand the complex’s characteristics. In order to meet the conditions of S100A4 NMR buffer, we dissolved EGF protein in PH6.0 solution. Using 3D NMR experiments and software calculation, we solved the structure of EGF. The binding region was characterized using 1H-15N HSQC perturbation experiments. Furthermore, we determined the binding ratio ( 1:1 ) and dissociation constant ( ~μM range ) by ITC and Fluorescent. Further studies would be necessary to characterize this interaction in more detail and to investigate if this interaction is physiologically relevant.

(1) Marenholz, I.; Heizmann, C. W.; Fritz, G. Biochemical and biophysical research communications 2004, 322, 1111–22.
(2) Marenholz, I.; Lovering, R. C.; Heizmann, C. W. Biochimica et biophysica acta 2006, 1763, 1282–3.
(3) Donato, R.; Heizmann, C. W. Cardiovascular psychiatry and neurology 2010, 2010, 929712.
(4) Bhattacharya, S.; Bunick, C. G.; Chazin, W. J. Biochimica et biophysica acta 2004, 1742, 69–79.
(5) Vallely, K. M.; Rustandi, R. R.; Ellis, K. C.; Varlamova, O.; Bresnick, A. R.; Weber, D. J. 2002, 1, 12670–12680.
(6) Ikura, M.; Ames, J. B. Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 1159–64.
(7) Wright, N. T.; Cannon, B. R.; Wilder, P. T.; Morgan, M. T.; Varney, K. M.; Zimmer, D. B.; Weber, D. J. Journal of Molecular Biology 2009, 386, 1265–1277.
(8) Ostendorp, T.; Leclerc, E.; Galichet, A.; Koch, M.; Demling, N.; Weigle, B.; Heizmann, C. W.; Kroneck, P. M. H.; Fritz, G. The EMBO journal 2007, 26, 3868–78.
(9) Kiryushko, D.; Novitskaya, V.; Soroka, V.; Klingelhofer, J.; Lukanidin, E.; Berezin, V.; Bock, E. Molecular and cellular biology 2006, 26, 3625–38.
(10) Srikrishna, G. Journal of innate immunity 2012, 4, 31–40.
(11) Moroz, O. V; Dodson, G. G.; Wilson, K. S.; Lukanidin, E.; Bronstein, I. B. Microscopy research and technique 2003, 60, 581–92.
(12) Sparvero, L. J.; Asafu-Adjei, D.; Kang, R.; Tang, D.; Amin, N.; Im, J.; Rutledge, R.; Lin, B.; Amoscato, A. A.; Zeh, H. J.; Lotze, M. T. Journal of translational medicine 2009, 7, 17.
(13) Kozlova, E. N.; Lukanidin, E. 1999, 258, 249–258.
(14) Leclerc, E.; Fritz, G.; Vetter, S. W.; Heizmann, C. W. Biochimica et biophysica acta 2009, 1793, 993–1007.
(15) Welch, D. R.; Wei, L. L. 1998, 155–197.
(16) Takenaga, K. British journal of cancer 1999, 80, 127–32.
(17) Garrett, S. C.; Varney, K. M.; Weber, D. J.; Bresnick, A. R. The Journal of biological chemistry 2006, 281, 677–80.
(18) Rudland, P. S.; Platt-higgins, A.; Renshaw, C.; West, C. R.; Winstanley, J. H. R.; Robertson, L.; Barraclough, R. 2000, 4, 1595–1603.
(19) Gongoll, S.; Peters, G.; Mengel, M.; Piso, P.; Klempnauer, J.; Kreipe, H.; Von Wasielewski, R. Gastroenterology 2002, 123, 1478–1484.
(20) Davies, B. R.; O’Donnell, M.; Durkan, G. C.; Rudland, P. S.; Barraclough, R.; Neal, D. E.; Mellon, J. K. The Journal of pathology 2002, 196, 292–9.
(21) Ismail, N. I.; Kaur, G.; Hashim, H.; Hassan, M. S. Cancer cell international 2008, 8, 12.
(22) CHO, Y. G.; NAM, S. W.; KIM, T. Y.; KIM, Y. S.; KIM, C. J.; PARK, J. Y.; LEE, J. H.; KIM, H. S.; LEE, J. W.; PARK, C. H.; SONG, Y. H.; LEE, S. H.; YOO, N. J.; LEE, J. Y.; PARK, W. S. APMIS 2003, 111, 539–545.
(23) Hernan, R.; Fasheh, R.; Calabrese, C.; Frank, A. J.; Maclean, K. H.; Allard, D.; Barraclough, R.; Gilbertson, R. J. 2003, 140–148.
(24) Rosty, C.; Ueki, T.; Argani, P.; Jansen, M.; Yeo, C. J.; Cameron, J. L.; Hruban, R. H.; Goggins, M. The American journal of pathology 2002, 160, 45–50.
(25) Fearon, E. F.; Vogelstein, B. 1990, 61, 759–767.
(26) Bienz, M.; Clevers, H. 2000, 103, 311–320.
(27) Polakis, P. Current opinion in genetics & development 2007, 17, 45–51.
(28) Klingelhöfer, J.; Møller, H. D.; Sumer, E. U.; Berg, C. H.; Poulsen, M.; Kiryushko, D.; Soroka, V.; Ambartsumian, N.; Grigorian, M.; Lukanidin, E. M. The FEBS journal 2009, 276, 5936–48.
(29) Teratani, T.; Watanabe, T.; Yamahara, K.; Kumagai, H.; Ishikawa, A.; Arai, K.; Nozawa, R. Biochemical and biophysical research communications 2002, 291, 623–7.
(30) Chan, W. Y.; Xia, C.-L.; Dong, D.-C.; Heizmann, C. W.; Yew, D. T. Microscopy research and technique 2003, 60, 600–13.
(31) Camby, I.; LeFranc, F.; Titeca, G.; Neuci, S.; Fastrez, M.; Dedecken, L.; Schafer, B. W.; Brotchi, J.; Heizmann, C. W.; Pochet, R.; Salmon, I.; Kiss, R.; Decaestecker, C. Neuropathology and Applied Neurobiology 2000, 26, 76–90.
(32) Myosin-iia, N.; Malashkevich, V. N.; Varney, K. M.; Garrett, S. C.; Wilder, P. T.; Knight, D.; Charpentier, T. H.; Ramagopal, U. A.; Almo, S. C.; Weber, D. J.; Bresnick, A. R. 2008, 5111–5126.
(33) Otterbein, L. R.; Kordowska, J.; Witte-Hoffmann, C.; Wang, C.-L. A.; Dominguez, R. Structure 2002, 10, 557–567.
(34) Cohen, S. The Journal of biological chemistry 1962, 237, 1555–62.
(35) Cohen, S.; Carpenter, G. 1975, 72, 1317–1321.
(36) Bielický, T. The Journal of Investigative Dermatology 1963, 41, 1–2.
(37) Venturi, S.; Venturi, M. Nutrition and Health 2009, 20, 119–134.
(38) Massagué, J.; Pandiella, A. Annual review of biochemistry 1993, 62, 515–41.
(39) Montelione, G. T.; Wuethrich, K.; Burgess, A. W.; Nice, E. C.; Wagner, G.; Gibson, K. D.; Scheraga, H. A. Biochemistry 1992, 31, 236–249.
(40) Fujiuchi, S.; Ohsaki, Y.; Kikuchi, K. Oncology 54, 134–40.
(41) Lu, H. S.; Chai, J. J.; Li, M.; Huang, B. R.; He, C. H.; Bi, R. C. The Journal of biological chemistry 2001, 276, 34913–7.
(42) Lemmon, M. A.; Bu, Z.; Ladbury, J. E.; Zhou, M.; Pinchasi, D.; Lax, I.; Engelman, D. M.; Schlessinger, J. The EMBO journal 1997, 16, 281–94.
(43) Ogiso, H.; Ishitani, R.; Nureki, O.; Fukai, S.; Yamanaka, M.; Kim, J.-H.; Saito, K.; Sakamoto, A.; Inoue, M.; Shirouzu, M.; Yokoyama, S. Cell 2002, 110, 775–787.
(44) Ullrich, A.; Coussens, L.; Hayflick, J. S.; Dull, T. J.; Gray, A.; Tam, A. W.; Lee, J.; Yarden, Y.; Libermann, T. A.; Schlessinger, J.; Downward, J.; Mayes, E. L. V.; Whittle, N.; Waterfield, M. D.; Seeburg, P. H. Nature 1984, 309, 418–425.
(45) Ward, C. W.; Hoyne, P. A.; Flegg, R. H. Proteins 1995, 22, 141–53.
(46) Jura, N.; Endres, N. F.; Engel, K.; Deindl, S.; Das, R.; Lamers, M. H.; Wemmer, D. E.; Zhang, X.; Kuriyan, J. Cell 2009, 137, 1293–307.
(47) Kriajevskas, M. V; Cardenas, M. N.; Grigorian, M. S.; Ambartsumian, N. S.; Georgievg, G. P.; Lukanidin, E. M. 1994, 4, 1–4.
(48) Takenaga, K.; Nakamura, Y.; Sakiyama, S.; Hasegawa, Y.; Sato, K.; Endo, H. The Journal of cell biology 1994, 124, 757–68.
(49) Kriajevska, M.; Fischer-Larsen, M.; Moertz, E.; Vorm, O.; Tulchinsky, E.; Grigorian, M.; Ambartsumian, N.; Lukanidin, E. The Journal of biological chemistry 2002, 277, 5229–35.
(50) Grigorian, M.; Andresen, S.; Tulchinsky, E.; Kriajevska, M.; Carlberg, C.; Kruse, C.; Cohn, M.; Ambartsumian, N.; Christensen, A.; Selivanova, G.; Lukanidin, E. The Journal of biological chemistry 2001, 276, 22699–708.
(51) Arnesano, F.; Banci, L.; Bertini, I.; Fantoni, A.; Tenori, L.; Viezzoli, M. S. Angewandte Chemie (International ed. in English) 2005, 44, 6341–4.
(52) Drohat, a C.; Baldisseri, D. M.; Rustandi, R. R.; Weber, D. J. Biochemistry 1998, 37, 2729–40.
(53) Huang, H.-W.; Mohan, S. K.; Yu, C. Biochemical and biophysical research communications 2010, 402, 705–10.