雞細胞激素介白素-1β (Interleukin-1β, IL-1β) 為一個 162 個胺基酸（18KD) 組成，具有多樣生物活性的訊息傳遞分子，在宿主免疫反應、組織損害、微生物入侵及發炎反應中都扮演重要的介質角色。先前的研究指出介白素-1β 的蛋白質結構內部包含有一個疏水性的孔洞，但其重要性至今仍不明確。我們有興趣知道的是，疏水性孔洞如何影響蛋白質結構的穩定性。雞介白素-1β 野生型與突變型 (Y157F) 在研究中指出其結構非常的相似。將胺基酸 157 位置的酪胺酸 (Tyr) 突變成苯丙胺酸 (Phe) 後其側鏈的鍵角與整體蛋白質結構構型皆無相異，且具有高相似性的疏水性孔洞大小及形狀。兩者結構不同的地方在於野生型蛋白質 157 位置親水性胺基酸 Tyr 側鏈上的羥基 (-OH) 可能與 133 位置疏水性胺基酸 Ile 骨架上的羰基 (C=O) 形成氫鍵。令人驚訝的是，藉由圓二色偏極 (CD) 光譜發現，突變的蛋白質結構較為穩定，Tm 值高了約 10 OC。在結構生物學的觀念裡，我們大多認為氫鍵的形成可以穩定蛋白質的結構，但此觀念不能適用在這特定的氫鍵上。本篇論文，我們利用多維核磁共振技術偵測蛋白質動態情形來解釋此現象。藉由 HNCO 與氫氘交換光譜，野生與突變種的比較，我們得到一個直接的證據，證明 I133 與 Y157 胺基酸間具有氫鍵，並且會造成 β11 與 β13 間氫鍵作用力的減弱，然而突變種的此一區域趨於穩定。我們推測，雞介白素-1β 野生種 157 位置的親水性羥基會產生不利的能量存在於疏水性孔洞內，導致羥基為了趨於穩定而與鄰近胺基酸 I133的羰基形成氫鍵，因此會產生相反方向的作用力，將 I133 所在的二級結構位置 β11 拉向中心的疏水性孔洞（或 157 酪胺酸所在位置）遠離相配對的 β13。此作用降低 β11 和 β13 間結構的穩定性，進而影響整體蛋白質結構的穩定。

Protein IL-1β plays a key role in the innate immunity and development of the host reaction to microbial invasion and tissue injury. In structure, it has been reported to contain a hydrophobic cavity in protein interior, however the importance has not been clarified. We intend to realize how protein stability correlates with hydrophobicity on inside of cavity. The structures of chicken IL-1β and the variant, Y157F, have been reported to be nearly identical. The replacement of phenylalanine at residue 157 makes no structural difference in its side chain orientation. Two structures own highly similar hydrophobic cavities in both size and shape. A visible difference is an existing hydrogen bond between tyrosine side chain hydroxyl group and I133 carbonyl group in native IL-1β, where mutant Y157F lacks the hydrogen bonding. Surprisingly, melting temperature of Y157F is 10-degree higher than native IL-1β, reflected by a circular dichroism measurement. To resolve the contradiction of that hydrogen bond usually stabilizes macromolecular folding, we probe protein backbone dynamics by NMR. NMR H/D exchange experiment reveals that contact between sheets β11 and β13 is weakened in the presence of hydrogen bond between I133 CO and Y157 OH, meanwhile the corresponding region is relatively stable in Y157F. We purpose that Y157 OH group introduces an unfavorable energy in the hydrophobic cavity because of its polarity, thus hydroxyl sequesters itself on forming a hydrogen bond with the proximate residue, I133. The structural element β11, where I133 is located at, therefore exerts a force to move toward to the cavity (or Y157) and away from the paired □-sheet, β13. The effect might destabilize the pair-wise □ sheets and further perturbs the overall protein stability.

1. Dinarello CA, E. S. (1989) Role for interleukin-1 in the pathogenesis of hypersensitivity diseases., J Cell Biochem.
2. Gabay, C., Lamacchia, C., and Palmer, G. (2010) IL-1 pathways in inflammation and human diseases, Nat Rev Rheumatol 6, 232-241.
3. Dinarello, C. A. (2009) Immunological and inflammatory functions of the interleukin-1 family, Annu Rev Immunol 27, 519-550.
4. Feghali, C. A., and Wright, T. M. (1997) Cytokines in acute and chronic inflammation, Front Biosci 2, d12-26.
5. Conti, P., Barbacane, R. C., Panara, M. R., Reale, M., Placido, F. C., Fridas, S., Bongrazio, M., and Dempsey, R. A. (1992) Human recombinant interleukin-1 receptor antagonist (hrIL-1ra) enhances the stimulatory effect of interleukin-2 on natural killer cell activity against MOLT-4 target cells, Int J Immunopharmacol 14, 987-993.
6. Economides, A. N., Carpenter, L. R., Rudge, J. S., Wong, V., Koehler-Stec, E. M., Hartnett, C., Pyles, E. A., Xu, X., Daly, T. J., Young, M. R., Fandl, J. P., Lee, F., Carver, S., McNay, J., Bailey, K., Ramakanth, S., Hutabarat, R., Huang, T. T., Radziejewski, C., Yancopoulos, G. D., and Stahl, N. (2003) Cytokine traps: multi-component, high-affinity blockers of cytokine action, Nat Med 9, 47-52.
7. Greenfeder, S. A., Nunes, P., Kwee, L., Labow, M., Chizzonite, R. A., and Ju, G. (1995) Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex, J Biol Chem 270, 13757-13765.
8. Korherr, C., Hofmeister, R., Wesche, H., and Falk, W. (1997) A critical role for interleukin-1 receptor accessory protein in interleukin-1 signaling, Eur J Immunol 27, 262-267.
9. Cullinan, E. B., Kwee, L., Nunes, P., Shuster, D. J., Ju, G., McIntyre, K. W., Chizzonite, R. A., and Labow, M. A. (1998) IL-1 receptor accessory protein is an essential component of the IL-1 receptor, J Immunol 161, 5614-5620.
10. Dunne, A., and O'Neill, L. A. (2003) The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense, Sci STKE 2003, re3.
11. van Oostrum J, P. J., Grütter MG, Schmitz A. (1991) The structure of murine interleukin-1 beta at 2.8A resolution., J Structure Biol 107, 189-195.
12. B. Yu, M. B., A. M. Gronenborn, G. M. Clore, and D. L. D. Caspar. (1999) Disordered water within a hydrophobic protein cavity visualized by X-ray crystallography., Proc Natl Acad Sci USA 96, 103-108.
13. Finzel BC, C. L., Holland DR, Muchmore SW, Watenpaugh KD, Einspahr HM. (1989) Crystal structure of recombinant human interleukin-1 beta at 2.0A resolution. , J Mol Biol 209, 779-791.
14. Cheng, C. S., Chen, W. T., Lee, L. H., Chen, Y. W., Chang, S. Y., Lyu, P. C., and Yin, H. S. (2011) Structural and functional comparison of cytokine interleukin-1 beta from chicken and human, Mol Immunol 48, 947-955.
15. Dongli Wang, S. Z., Liang Li, Xi Liu, Kunrong Mei & Xinquan Wang. (2010) Structural insights into the assembly and activation of IL-1 beta eith its receptor, Nature immunology 11, 905-912.
16. Chao-Sheng Cheng, W.-S. L., I-Fan Tu, Ping-Chiang Lyu and Hsien-Sheng Yin. (2010) Comparative analysis of receptor binding by chicken and human interleukin-1β, J Mol Model 10.
17. Covalt, J. C., Jr., Roy, M., and Jennings, P. A. (2001) Core and surface mutations affect folding kinetics, stability and cooperativity in IL-1 beta: does alteration in buried water play a role?, J Mol Biol 307, 657-669.
18. Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H., and Blow, D. M. (1974) Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-A resolution, Biochemistry 13, 4212-4228.
19. Weining KC, S. C., Kaspers B, Staeheli P. (1998) A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and purification of active recombinant protein., Eur J Biochem 258, 994-1000.
20. Simon J. Hubbard, K.-H. G. a. P. A. (1994) Intramolecular cavities in globular proteins., Protein Eng 7, 613-626.
21. MARK A. WILLIAMS, J. M. G., JANET M. THORNTON (1994) Buried waters and internal cavities in monomeric proteins., Protein Sci 3, 1224-1235.
22. Brian W Matthews, L. L. (2009) A review about nothing: Are apolar cavities in protein really empty?, Protein Scoence 18, 494-502.
23. Adamek, D. H., Guerrero, L., Blaber, M., and Caspar, D. L. (2005) Structural and energetic consequences of mutations in a solvated hydrophobic cavity, J Mol Biol 346, 307-318.
24. Somani, S., Chng, C. P., and Verma, C. S. (2007) Hydration of a hydrophobic cavity and its functional role: a simulation study of human interleukin-1beta, Proteins 67, 868-885.
25. Huggins, M. L. (1971) 50 Years of Hydrogen Bond Theory, Angewandte Chemie International Edition 10, 147-208.
26. Greenfield, N. J. (2006) Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions, Nat Protoc. 1, 2527-1535.
27. Horowitz, M., Maddox, A. F., Wishart, J. M., Harding, P. E., Chatterton, B. E., and Shearman, D. J. (1991) Relationships between oesophageal transit and solid and liquid gastric emptying in diabetes mellitus, Eur J Nucl Med 18, 229-234.
28. Schwarzinger, S., Kroon, G. J., Foss, T. R., Chung, J., Wright, P. E., and Dyson, H. J. (2001) Sequence-dependent correction of random coil NMR chemical shifts, J Am Chem Soc 123, 2970-2978.
29. Silvia Spera, A. B. (1991) Empirical correlation between protein backbone conformation and C.alpha. and C.beta. 13C nuclear magnetic resonance chemical shifts, Journal of The American Chemical Society 113, 5490-5492.
30. Sue, S. C., Cervantes, C., Komives, E. A., and Dyson, H. J. (2008) Transfer of flexibility between ankyrin repeats in IkappaB* upon formation of the NF-kappaB complex, J Mol Biol 380, 917-931.
31. Wishart, M. V. B. a. D. S. (2005) A simple method to predict protein flexibility using secondary chemical shifts, J. Am. Chem. Soc. 127, 14970-14971.
32. Shen, Y., Delaglio, F., Cornilescu, G., and Bax, A. (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts, J Biomol NMR 44, 213-223.
33. Grzesiek, S., Stahl, S. J., Wingfield, P. T. & Bax, A. (1996) The CD4 determinant for sdown regulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR., Biochemistry 35, 10256-10261.
34. Sue, S. C., Chen, J. Y., Lee, S. C., Wu, W. G., and Huang, T. H. (2004) Solution structure and heparin interaction of human hepatoma-derived growth factor, J Mol Biol 343, 1365-1377.
35. Englarander, S. W. (1980) Internal motions in proteins and nucleic acids and their hydrogen exchange properties, Mol. Cell Biophys 1, 15-28.
36. Bai, Y., Milne, J. S., Mayne, L., and Englander, S. W. (1993) Primary structure effects on peptide group hydrogen exchange, Proteins 17, 75-86.
37. Englander, S. W., Sosnick, T. R., Englander, J. J., and Mayne, L. (1996) Mechanisms and uses of hydrogen exchange, Curr Opin Struct Biol 6, 18-23.
38. Fersht, A. Structure and mechanism in protein science, 563-567.
39. Naoki Asakawa, S. K., Hiromichi Kurosu, Isao Ando, Akira Shoji, Takuo Ozaki. (1992) Hydrogen-bonding effect on 13C NMR chemical shifts of l-Alanine residue carbonyl carbons of peptides in the solid state, J. Am. Chem. Soc. 114, 3261-3265.