Prion蛋白(PrP) 是一種能與銅離子(Cu2+ ) 結合的細胞表面糖蛋白，PrP有兩種不同結構區域(domain)：非結構化的氮端domain (residues 23-125) 和α-helix為主的碳端domain (residue 126-231)。當正常的PrP (PrPC) 錯誤摺疊轉變成異常的PrP (PrPSc) 時，部分的α-helices會轉變為β-sheets，這是引起一系列傳染性海綿狀腦病(TSEs) 的原因。常見的prion diseases包含狂牛症(BSE)、羊搔症、人類的庫魯症(kuru) 和庫賈氏症(CJD)。以人類的PrPC而言，氮端domain內位於residue 60-91有著一段重複的胺基酸序列：octarepeat (PHGGGWGQ)4，此片段是由八個胺基酸：octapeptide (PHGGGWGQ) 重複四次所組成，octapeptide片段與Cu2+ 有良好的親和力。這裡我們以octapeptide片段當作對象，研究proline在此胜肽片段結構和Cu2+ 結合中所扮演的角色。為證實靜電作用和立體電子效應對PrP與Cu2+ 的結合，我們設計並合成兩種不同組合的octapeptide系列胜肽：Ac-XaaHGGGWGQP-NH2 和 Ac-GQXaaHGGGWGQ-NH2之胜肽 (Xaa是proline在原生態的位置) 並以非極性胺基酸Ala和極性胺基酸帶正電的Lys、帶負電的Asp、不帶電的Ser和proline衍生物 (Flp、flp、Hyp、hyp) 取代proline。使用圓二色光譜儀(CD) 和螢光光譜儀(FL) 來測量octapeptide系列胜肽與Cu2+ 的結合力和與Cu2+ 結合後產生的結構變化。實驗結果顯示靜電作用和立體電子效應並非影響兩種octapeptide系列胜肽與Cu2+ 錯合物的二級結構轉變之主因。我們的結果亦說明研究proline在此蛋白片段的結構和Cu2+ 結合力中所扮演的角色時，Ac-GQXaaHGGGWGQ-NH2胜肽會是比較好的模型。

1.Nelson, D. L.; Cox, M. M., Lehninger principles of biochemistry. 4th ed.; W. H. Freeman and Company: New York, 2004.
2.Prusiner, S., Novel proteinaceous infectious particles cause scrapie. Science 1982, 216, 136-144.
3.Prusiner, S. B.; Groth, D. F.; Cochran, S. P.; Masiarz, F. R.; McKinley, M. P.; Martinez, H. M., Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent. Biochemistry 1980, 19, 4883-4891.
4.Prusiner, S. B., Prions. Proc. Natl. Acad. Sci. U.S A. 1998, 95, 13363-13383.
5.Prusiner, S. B., Prion biology and diseases. 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 2004.
6.Collinge, J., Prion diseases of humans and animals: their causes and molecular basis. Annu. Rev. Neurosci. 2001, 24, 519-550.
7.Pan, K. M.; Baldwin, M.; Nguyen, J.; Gasset, M.; Serban, A.; Groth, D.; Mehlhorn, I.; Huang, Z.; Fletterick, R. J.; Cohen, F. E., Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10962-10966.
8.Millhauser, G. L., Copper binding in the prion protein. Acc. Chem. Res. 2004, 37, 79-85.
9.Stahl, N.; Borchelt, D. R.; Hsiao, K.; Prusiner, S. B., Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 1987, 51, 229-240.
10.Sunyach, C.; Jen, A.; Deng, J.; Fitzgerald, K. T.; Frobert, Y.; Grassi, J.; McCaffrey, M. W.; Morris, R., The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein. EMBO. J. 2003, 22, 3591-3601.
11.Cobb, N. J.; Surewicz, W. K., Prion diseases and their biochemical mechanisms. Biochemistry 2009, 48, 2574-2585.
12.Riek, R.; Hornemann, S.; Wider, G.; Billeter, M.; Glockshuber, R.; Wuthrich, K., NMR structure of the mouse prion protein domain PrP(121-231). Nature 1996, 382, 180-182.
13.Donne, D. G.; Viles, J. H.; Groth, D.; Mehlhorn, I.; James, T. L.; Cohen, F. E.; Prusiner, S. B.; Wright, P. E.; Dyson, H. J., Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13452-13457.
14.Choi, C. J.; Kanthasamy, A.; Anantharam, V.; Kanthasamy, A. G., Interaction of metals with prion protein: possible role of divalent cations in the pathogenesis of prion diseases. Neurotoxicology 2006, 27, 777-787.
15.Miura, T.; Hori-i, A.; Mototani, H.; Takeuchi, H., Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence. Biochemistry 1999, 38, 11560-11569.
16.Bonomo, R. P.; Impellizzeri, G.; Pappalardo, G.; Rizzarelli, E.; Tabbì, G., Copper(II) binding modes in the prion octapeptide PHGGGWGQ: a spectroscopic and voltammetric study. Chem. Eur. J. 2000, 6, 4195-4202.
17.Shyng, S. L.; Heuser, J. E.; Harris, D. A., A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J. Cell. Biol. 1994, 125, 1239-1250.
18.Brown, L. R.; Harris, D. A., Copper and zinc cause delivery of the prion protein from the plasma membrane to a subset of early endosomes and the Golgi. J. Neurochem. 2003, 87, 353-363.
19.Pauly, P. C.; Harris, D. A., Copper stimulates endocytosis of the prion protein. J. Biol. Chem. 1998, 273, 33107-33110.
20.Burns, C. S.; Aronoff-Spencer, E.; Dunham, C. M.; Lario, P.; Avdievich, N. I.; Antholine, W. E.; Olmstead, M. M.; Vrielink, A.; Gerfen, G. J.; Peisach, J.; Scott, W. G.; Millhauser, G. L., Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry 2002, 41, 3991-4001.
21.Westergard, L.; Christensen, H. M.; Harris, D. A., The cellular prion protein (PrPC): its physiological function and role in disease. Biochim. Biophys. Acta. 2007, 1772, 629-644.
22.Brown, D. R.; Wong, B. S.; Hafiz, F.; Clive, C.; Haswell, S. J.; Jones, I. M., Normal prion protein has an activity like that of superoxide dismutase. Biochem. J. 1999, 344, 1-5.
23.Brown, D. R.; Clive, C.; Haswell, S. J., Antioxidant activity related to copper binding of native prion protein. J. Neurochem. 2001, 76, 69-76.
24.Kozlowski, H.; Luczkowski, M.; Remelli, M., Prion proteins and copper ions. Biological and chemical controversies. Dalton Trans. 2010, 39, 6371-6385.
25.Halliwell, B., Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 2006, 97, 1634-1658.
26.Gabriel, J. M.; Oesch, B.; Kretzschmar, H.; Scott, M.; Prusiner, S. B., Molecular cloning of a candidate chicken prion protein. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9097-9101.
27.Wopfner, F.; Weidenhöfer, G.; Schneider, R.; von Brunn, A.; Gilch, S.; Schwarz, T. F.; Werner, T.; Schätzl, H. M., Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. J. Mol. Biol. 1999, 289, 1163-1178.
28.Calzolai, L.; Lysek, D. A.; Pérez, D. R.; Güntert, P.; Wüthrich, K., Prion protein NMR structures of chickens, turtles, and frogs. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 651-655.
29.Smith, C. J.; Drake, A. F.; Banfield, B. A.; Bloomberg, G. B.; Palmer, M. S.; Clarke, A. R.; Collinge, J., Conformational properties of the prion octa-repeat and hydrophobic sequences. FEBS Lett. 1997, 405, 378-384.
30.Sulkowski, E., Spontaneous conversion of PrPC to PrPSc. FEBS Lett. 1992, 307, 129-130.
31.Brown, P., Spongiform encephalopathies: B lymphocytes and neuroinvasion. Nature 1997, 390, 662-663.
32.Hornshaw, M. P.; McDermott, J. R.; Candy, J. M., Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein. Biochem. Biophys. Res. Commun. 1995, 207, 621-629.
33.Hornshaw, M. P.; McDermott, J. R.; Candy, J. M.; Lakey, J. H., Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochem. Biophys. Res. Commun. 1995, 214, 993-999.
34.Brown, D. R.; Qin, K.; Herms, J. W.; Madlung, A.; Manson, J.; Strome, R.; Fraser, P. E.; Kruck, T.; von Bohlen, A.; Schulz-Schaeffer, W.; Giese, A.; Westaway, D.; Kretzschmar, H., The cellular prion protein binds copper in vivo. Nature 1997, 390, 684-687.
35.Walter, E. D.; Stevens, D. J.; Spevacek, A. R.; Visconte, M. P.; Rossi, A. D.; Millhauser, G. L., Copper binding extrinsic to the octarepeat region in the prion protein. Curr Protein Pept Sci. 2009, 10, 529-535.
36.Viles, J. H.; Cohen, F. E.; Prusiner, S. B.; Goodin, D. B.; Wright, P. E.; Dyson, H. J., Copper binding to the prion protein: Structural implications of four identical cooperative binding sites. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2042-2047.
37.Aronoff-Spencer, E.; Burns, C. S.; Avdievich, N. I.; Gerfen, G. J.; Peisach, J.; Antholine, W. E.; Ball, H. L.; Cohen, F. E.; Prusiner, S. B.; Millhauser, G. L., Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy. Biochemistry 2000, 39, 13760-13771.
38.Millhauser, G. L., Copper and the prion protein: methods, structures, function, and disease. Annu. Rev. Phys. Chem. 2007, 58, 299-320.
39.Chattopadhyay, M.; Walter, E. D.; Newell, D. J.; Jackson, P. J.; Aronoff-Spencer, E.; Peisach, J.; Gerfen, G. J.; Bennett, B.; Antholine, W. E.; Millhauser, G. L., The octarepeat domain of the prion protein binds Cu(II) with three distinct coordination modes at pH 7.4. J. Am. Chem. Soc. 2005, 127, 12647-12656.
40.Walter, E. D.; Chattopadhyay, M.; Millhauser, G. L., The affinity of copper binding to the prion protein octarepeat domain: evidence for negative cooperativity. Biochemistry 2006, 45, 13083-13092.
41.Walter, E. D.; Stevens, D. J.; Visconte, M. P.; Millhauser, G. L., The prion protein is a combined zinc and copper binding protein: Zn2+ alters the distribution of Cu2+ coordination modes. J. Am. Chem. Soc. 2007, 129, 15440-15441.
42.Nadal, R. C.; Davies, P.; Brown, D. R.; Viles, J. H., Evaluation of copper2+ affinities for the prion protein. Biochemistry 2009, 48, 8929-8931.
43.Davies, P.; Marken, F.; Salter, S.; Brown, D. R., Thermodynamic and voltammetric characterization of the metal binding to the prion protein: insights into pH dependence and redox chemistry. Biochemistry 2009, 48, 2610-2619.
44.Kramer, M. L.; Kratzin, H. D.; Schmidt, B.; Römer, A.; Windl, O.; Liemann, S.; Hornemann, S.; Kretzschmar, H., Prion protein binds copper within the physiological concentration range. J. Biol. Chem. 2001, 276, 16711-16719.
45.Jackson, G. S.; Murray, I.; Hosszu, L. L. P.; Gibbs, N.; Waltho, J. P.; Clarke, A. R.; Collinge, J., Location and properties of metal-binding sites on the human prion protein. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8531-8535.
46.Wells, M. A.; Jackson, G. S.; Jones, S.; Hosszu, L. L. P.; Craven, C. J.; Clarke, A. R.; Collinge, J.; Waltho, J. P., A reassessment of copper(II) binding in the full-length prion protein. Biochem. J. 2006, 399, 435-444.
47.Speare, J. O.; Rush, T. S.; Bloom, M. E.; Caughey, B., The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein. J. Biol. Chem. 2003, 278, 12522-12529.
48.Ziegler, J.; Sticht, H.; Marx, U. C.; Muller, W.; Rosch, P.; Schwarzinger, S., CD and NMR studies of prion protein (PrP) Helix 1: novel implications for its role in the PrPC → PrPSc conversion process. J. Biol. Chem. 2003, 278, 50175-50181.
49.Eyles, S. J.; Gierasch, L. M., Multiple roles of prolyl residues in structure and folding. J. Mol. Biol. 2000, 301, 737-747.
50.Reiersen, H.; Rees, A. R., The hunchback and its neighbours: proline as an environmental modulator. Trends Biochem. Sci 2001, 26, 679-684.
51.Bhattacharyya, R.; Chakrabarti, P., Stereospecific interactions of proline residues in protein structures and complexes. J. Mol. Biol. 2003, 331, 925-940.
52.DeRider, M. L.; Wilkens, S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.; Raines, R. T.; Markley, J. L., Collagen stability: insights from NMR spectroscopic and hybrid density functional computational investigations of the effect of electronegative substituents on prolyl ring conformations. J. Am. Chem. Soc. 2002, 124, 2497-2505.
53.Park, S.; Radmer, R. J.; Klein, T. E.; Pande, V. S., A new set of molecular mechanics parameters for hydroxyproline and its use in molecular dynamics simulations of collagen-like peptides. J. Comput. Chem. 2005, 26, 1612-1616.
54.Hinderaker, M. P.; Raines, R. T., An electronic effect on protein structure. Protein Sci. 2003, 1188-1194.
55.Wolfe, S., Gauche effect. Stereochemical consequences of adjacent electron pairs and polar bonds. Acc. Chem. Res. 1972, 5, 102-111.
56.Improta, R.; Benzi, C.; Barone, V., Understanding the role of stereoelectronic effects in determining collagen stability. 1. A quantum mechanical study of proline, hydroxyproline, and fluoroproline dipeptide analogues in aqueous solution. J. Am. Chem. Soc. 2001, 123, 12568-12577.
57.Benzi, C.; Improta, R.; Scalmani, G.; Barone, V., Quantum mechanical study of the conformational behavior of proline and 4R-hydroxyproline dipeptide analogues in vacuum and in aqueous solution. J. Comput. Chem. 2002, 23, 341-350.
58.Mooney, S. D.; Kollman, P. A.; Klein, T. E., Conformational preferences of substituted prolines in the collagen triple helix. Biopolymers 2002, 64, 63-71.
59.Taylor, C. M.; Hardré, R.; Edwards, P. J. B., The impact of pyrrolidine hydroxylation on the conformation of proline-containing peptides. J. Org. Chem. 2005, 70, 1306-1315.
60.Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T., Conformational stability of collagen relies on a stereoelectronic effect. J. Am. Chem. Soc. 2001, 123, 777-778.
61.Kotch, F. W.; Guzei, I. A.; Raines, R. T., Stabilization of the collagen triple helix by O-methylation of hydroxyproline residues. J. Am. Chem. Soc. 2008, 130, 2952-2953.
62.Chiang, Y.-C.; Lin, Y.-J.; Horng, J.-C., Stereoelectronic effects on the transition barrier of polyproline conformational interconversion. Protein Sci. 2009, 18, 1967-1977.
63.Naduthambi, D.; Zondlo, N. J., Stereoelectronic tuning of the structure and stability of the trp cage miniprotein. J. Am. Chem. Soc. 2006, 128, 12430-12431.
64.Zheng, T.-Y.; Lin, Y.-J.; Horng, J.-C., Thermodynamic consequences of incorporating 4-substituted proline derivatives into a small helical protein. Biochemistry 2010, 49, 4255-4263.
65.Wu, S.-P.; Liu, S.-R., A new water-soluble fluorescent Cu(II) chemosensor based on tetrapeptide histidyl-glycyl-glycyl-glycine (HGGG). Sensors and Actuators B 2009, 141, 187-191.
66.Garnett, A. P.; Viles, J. H., Copper binding to the octarepeats of the prion protein. J. Biol. Chem. 2003, 278, 6795-6802.
67.Merrifield, B., Solid phase synthesis. Science 1986, 232, 341-347.
68.張湘戎, 體抑素胜肽分子內雙硫鍵建構之研究. 中原大學化學研究所碩士學位論文, 2003.
69.Chan, W. C.; White, P. D., Fmoc solid phase peptide synthesis: a practical approach. Oxford University Press: New York, 2000.
70.Berova, N.; Nakanishi, K.; Woody, R. W., Circular dichroism: principles and applications. VCH publishers: New York, 2000.
71.Velluz, L.; Legrand, M.; Grosjean, M., Optical circular dichroism. Academic Press: 1965.
72.Fasman, G. D., Circular dichroism and the conformational analysis of biomolecules. Plenum Press: New York, 1996.
73.Matsuo, K.; Yonehara, R.; Gekko, K., Improved estimation of the secondary structures of proteins by vacuum-ultraviolet circular dichroism spectroscopy. J. Biochem. 2005, 138, 79-88.
74.Skoog, D. A.; Holler, F. J.; Nieman, T. A., Principles of instrumental analysis. 5th ed.; Saunders College Publishers: Belmont, 1998.
75.Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T., How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4, 2411-2423.
76.Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M., Data for Biochemical Research. 1986.
77.MacCarthy, P., Simplified experimental route for obtaining Job's curves. Anal. Chem. 1978, 50, 2165-2165.
78.Di Natale, G.; Pappalardo, G.; Milardi, D.; Sciacca, M. F. M.; Attanasio, F.; La Mendola, D.; Rizzarelli, E., Membrane interactions and conformational preferences of human and avian prion N-terminal tandem repeats: the role of copper(II) ions, pH, and membrane mimicking environments. J. Phys. Chem. B 2010, 114, 13830-13838.
79.Klewpatinond, M.; Davies, P.; Bowen, S.; Brown, D. R.; Viles, J. H., Deconvoluting the Cu2+ binding modes of full-length prion protein. J. Biol. Chem. 2008, 283, 1870-1881.
80.Daniele, P. G.; Prenesti, E.; Ostacoli, G., Ultraviolet-circular dichroism spectra for structural analysis of copper(II) complexes with aliphatic and aromatic ligands in aqueous solution. J. Chem. Soc., Dalton Trans. 1996, 3269-3275.
81.Kowalik-Jankowska, T.; Rajewska, A.; Wiśniewska, K.; Grzonka, Z.; Jezierska, J., Coordination abilities of N-terminal fragments of α-synuclein towards copper(II) ions: a combined potentiometric and spectroscopic study. J. Inorg. Biochem. 2005, 99, 2282-2291.