膠原蛋白是由三條延伸的第二型聚脯胺酸螺旋結構 (PPⅡ) 沿一個軸向扭轉而成，藉氫鍵作用形成一個排列緊密的右旋三股螺旋結構。在膠原蛋白中最常見也最穩定的胜□序列是 Pro-Hyp-Gly，因此我們以胺基酸序列為 (Pro-Hyp-Gly)n 的模擬胜□來研究膠原蛋白的穩定性。合成一系列的主客模擬胜□ (Pro-Hyp-Gly)n-Pro-Hyp-Xaa-(Pro-Hyp-Gly)m，Xaa 為 β-丙胺酸 (β-Ala)、L-丙胺酸 (L-Ala)、D-丙胺酸 (D-Ala) 和肌胺酸 (Sar)，目的是分別探討 backbone 的延長、側鏈造成的立體效應以及氫鍵的破壞對膠原蛋白穩定性的影響。CD 及 NMR 實驗顯示胺基酸變異的胜□會形成比 wild-type 更不穩定的三股螺旋結構，但不同變異位置的胜□，其穩定性降低的程度也會不同，變異位置在胜□ C 端的結構穩定性比變異位置在 N 端的結構穩定性高。我們推測變異位置在 C 端時，由 C 端往 N 端的折疊會略過變異位置，再繼續折疊形成較大區域的 Pro-Hyp-Gly 三股螺旋結構，但當變異位置在 N 端，結構折疊到變異位置時會鬆開，且形成較小區域的 Pro-Hyp-Gly 序列重疊，使結構較不穩定。另外，D-丙胺酸取代在胜□ N 端及 C 端位置，其穩定性都比 L-丙胺酸的取代穩定性更低。因為 D-胺基酸的正 phi 角度使其不利於三股螺旋結構的形成。肌胺酸取代胜□中間位置的甘胺酸 (Gly) 仍可形成三股螺旋，表示氫鍵的破壞對結構穩定性的影響小於 backbone 改變或立體障礙對結構穩定性的影響。此外，動力學實驗顯示側鏈的立體障礙是影響三股螺旋結構折疊速率的主要因素。

Collagen is a right-handed triple helix consisting of three left-handed PPⅡ helices interacted by hydrogen bonding. It involves many repeats of the tripeptide sequence : Pro-Hyp-Gly, and thus we use it as the peptide model to investigate the stability of collagen. A series of host-guest collagen peptides, (Pro-Hyp-Gly)n-Pro- Hyp-Xaa-(Pro-Hyp-Gly)m, where Xaa is β-Ala, L-Ala, D-Ala, and sarcosine, were prepared to investigate the effects of a prolonged backbone, the side chain steric strain, and the hydrogen bond breaking on collagen stability. CD and NMR measurements indicated that the mutated peptides formed less stable triple helices compared to the wild type peptide, but the destabilizing effects at different sites were different. Our data have shown that the mutation at the N-terminus destabilized the triple helix more than that at the C-terminus. We suggest that coming across the N-terminal mutation while folding from the C- to N-terminus would significantly disperse the triple helix, but it could skip the C-terminal mutation site and then hold on folding at the other site to form a larger section of Pro-Hyp-Gly repeats. The substitution of a sarcosine at the middle site of a collagen peptide could still form a triple helix, suggesting that the hydrogen bond-breaking has less destabilization effects than the perturbation of backbone. Moreover, the kinetic studies have shown that steric effects of the side chains might be the main factor retarding the folding of a triple helix.

1.Marden, M. C.; Griffon, N.; Poyart, C. Transfus Clin. Biol. 1995, 2, 473.
2.Brodsky, B.; Persikov, A. V. Adv. Protein Chem. 2005, 70, 301.
3.陳柏翰、劉中行，科學發展期刊，2004年 8月 380期 pp. 4-35.
4.Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994, 266, 75.
5.Reyes, C. D.; Garc□a, A. J. J. Biomed. Mater. Res. 2003, 65A, 511.
6.Myllyharju, J.; Kivirikko, K. I. Trends Genet. 2004, 20, 33.
7.Burjanadze, T. V. Biopolymers 2000, 53, 523.
8.Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777.
9.Vitagliano, L.; Berisio, R.; Mastrangelo, A.; Mazzarella, L.; Zagari, A. Protein Sci. 2001, 10, 2627.
10.Bella, J.; Berman, H. M. J. Mol. Biol. 1996, 264, 734.
11.Bhate, M.; Wang, X.; Baum, J.; Brodsky, B. Biochemistry 2002, 41, 6539.
12.McLaughlin, S. H.; Bulleid, N. J. Matrix Biol. 1998, 16, 369.
13.Ackerman, M. S.; Bhate, M.; Shenoy, N.; Beck, K.; Ramshaw, J. A. M.; Brodsky, B. J. Biol. Chem. 1999, 274, 7668.
14.Lamande, S. R.; Bateman, J. F. Cell DeV. Biol. 1999, 10, 455.
15.Buevich, A. V.; Dai, Q. H.; Liu, X.; Brodsky, B.; Baum, J. Biochemistry 2000, 39, 4299.
16.Buevich, A. V.; Silva, T.; Brodsky, B.; Baum, J. J. Biol. Chem. 2004, 279, 46890.
17.Hinderaker, M. P.; Raines, R. T. Protein Sci. 2003, 12, 1188.
18.Bella, J.; Brodsky, B.; Berman, H. M. Structure 1995, 3, 893.
19.Beck, K.; Chan, V. C.; Shenoy, N.; Kirkpatrick, A.; Ramshaw, J. A. M.; Brodsky, B. Proc. Natl. Acad. Sci. USA 2000, 97, 4273.
20.Wang, Q.; Orrison, B. M.; Marini, J. C. J. Biol. Chem. 1993, 268, 25162.
21.Butcher, D. J.; Luo, Z.; Huang, Z. Biochem. Biophys. Res. Commun. 1999, 265, 350.
22.Feng, Y.; Melacini, G.; Taulane, J. P.; Goodman, M. J. Am. Chem. Soc. 1996, 118, 10351.
23.Khew, S. T.; Tong Y. W. Biochemistry 2008, 47, 585.
24.Lelais, G.; Seebach, D. Biopolymers 2004, 76, 206.
25.Qiu, J. X.; Petersson, E. J.; Matthews, E. E.; Schepartz, A. J. Am. Chem. Soc. 2006, 128, 11338.
26.Goodman, J. L.; Petersson, E. J.; Daniels, D. S.; Qiu, J. X.; Schepartz, A. J. Am. Chem. Soc. 2007, 129, 14746.
27.Krause, E.; Bienert, M.; Schmieder, P.; Wenschuh, H. J. Am. Chem. Soc. 2000, 122, 4865.
28.Schumacher, M.; Mizuno, K.; B□chinger, H.P. J. Biol. Chem. 2005, 280, 20397.
29.Anil, B., Song, B.; Tang, Y.; Raleigh, D.P. J. Am. Chem. Soc. 2004, 126, 13194.
30.Valiyaveetil, F.I.; Sekedat, M.; MacKinnon, R.; Muir, T.W. Proc. Natl. Acad. Sci. USA 2004, 101, 17045.
31.Tsai, M.; Xu, Y.; Dannenberg, J. J. J. Am. Chem. Soc. 2005, 127, 14130.
32.Horng, J. C.; Kotch, F. W.; Raines, R. T. Protein Sci. 2007, 16, 208.
33.Chorghade, M. S.; Mohapatra, D. K.; Sahoo, G.; Gurjar, M. K.; Mandlecha, M. V.; Bhoite, N.; Moghe, S.; Raines, R. T. J. Fluorine Chem. 2008, 129, 781.
34.Melacini, G.; Feng, Y.; Goodman, M. J. Am. Chem. Soc. 1996, 118, 10359.
35.Brodsky, B.; Li, M.; Long, C. G.; Apigo, J.; Baum, J. Biopolymers 1992, 32, 447.
36.Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2007, 129, 15034.
37.Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2008, 130, 7509.
38.Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2007, 129, 2683.