簡易檢索 / 詳目顯示

回結果列表
研究生： 郭乃瑋
Nai-Wei Kuo
論文名稱： 研究轉彎序列對於beta-摺板型胜肽結構、穩定度及摺疊動力學的影響
Effects of turn mutation on the structure, stability and folding kinetics of a beta-sheet peptide
指導教授： 陳長謙教授
Sunney I. Chan Ph. D
口試委員:
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2004
畢業學年度： 92
語文別： 英文
論文頁數： 82
中文關鍵詞： 蛋白質摺疊 、貝他摺板型胜肽 、轉彎序列
外文關鍵詞： protein folding, beta-sheet, turn mutation
相關次數： 點閱：2  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 中文摘要
    氫鍵、疏水性作用、轉彎序列的影響是三個主要決定□□摺板穩定度的因素。一般來說，□-轉彎序列在影響□□摺板的穩定度上擔任一個重要角色。由我們實驗團隊之前以一個有三條反向平行長帶形成的□-摺板所做的結果顯示，將轉彎序列上的D-Pro以Asp代換後，可以降低原來□-摺板的穩定度。而且，在第一個轉彎序列上的這種突變，可以造成第一條與第二條長帶上相對應的胺基酸在骨架上往轉彎序列處移動一個胺基酸形成一個有TSDGK的轉彎序列，並且造成第一條胺基酸上的R基180度的翻轉至另一面。然而，這樣將第二個轉彎序列上的D-Pro以Asp代換的突變並沒有造成類似的胺基酸移動或是R基翻轉。於是，在這篇論文中我們想了解的是造成胺基酸往轉彎序列處移動的原因是因為受到新的轉彎序列影響還是新形成兩條長帶間疏水性作用的影響？我們將TSDGK的轉彎序列代換成在第二個轉彎序列突變後並沒有造成胺基酸移動的VDGO轉彎序列來探討這個作用力。
    此外，我們也利用這個□-摺板來做早期蛋白質摺疊的動力學研究。我們的方法是利用一個對光不穩定的結合物來環化這個□-摺板使其結構改變，再以308-366nm波長的雷射光照射使對光不穩定結合物斷裂，讓□-摺板重新摺疊而恢復其原來的結構。在此過程中，我們以光聲波熱卡計來做動力學部分的研究。

    Abstract
    The hydrogen bonding, hydrophobic interactions, and the turn effect are three factors affecting the stability of □-structures. Generally, the □-turn is thought to play an important role in stabilizing the □-structures. Previous studies in our group have disclosed that replacing DPro with Asp destabilizes the peptide whose DPG segment is in type II’ turn. More specifically, the DP to D mutation at DP-6 position caused an one amino-acid frameshift of the first □-strand toward the turn region and resulted in side-chain inversion of the first □-strand to give a five-residue TSDGK turn which is composed of a type I turn plus a □ bulge. However, a similar mutation at the DP-14 position did not change the hydrogen bond network and formed a VDGO turn instead of the VDPGO turn. The VDGO turn is in type II’ turn. The frameshift in the first hairpin raises the questions: whether the structural rearrangement is driven by the new turn sequence or by the hydrophobic interactions of new strand pairings. Here, we have replaced the TSDGK turn with the VDGO turn in the first hairpin to examine the driving force.
    In this work, we used this small □-sheet to study the early stage of protein folding. This method is based on photolysis of caged □-sheet. The photolabile linker caged the □-sheet to alter its structure. When peptide is exposed to the 308- and 366- nm light, the linker can be cleaved rapidly. The refolding begins as soon as the photolabile linker is cleaved. We used the technique of Photoacoustic Calorimetry to study the refolding kinetics of the □-sheet.

    Table of contents Abstract I Abstract Chinese II Acknowledgements III Abbreviation V Table of Contents VII Chapter 1: Introduction 1 1.1 The general concepts of protein folding and folding problem 1 1.2 The role of □-turns on the stability and structure of □-sheets 6 1.3 The classes of the turn type 9 1.4 Previous studies on 20-mer 11 1.5 The development of cyclized strategy 14 1.6 The aim of this project 18 Chapter 2: Materials and Methods 23 2.1 Materials 23 2.1.1 Water 23 2.1.2 Chemicals 23 2.1.3 Chromatography column, membranes, filters 25 2.1.4 pH meter 25 2.1.5 Peptide automated synthesizer 25 2.1.6 Centrifuge 25 2.1.7 Reverse-phase high performance liquid chrometagraphy 26 2.1.8 Lyophilizer 26 2.1.9 Mass spectroscopy 26 2.1.10 Circular dicroism spectroscopy 27 2.1.11 Ultraviolet spectroscopy 27 2.1.12 Phtotchemical reactor 27 2.1.13 Nuclear magnetic resonance spectroscopy 27 2.2 Methods 28 2.2.1 Solid phase peptide synthesis 28 2.2.2 Purified the linker 28 2.2.3 Head-to-side chain cyclization of peptide with linker 29 2.2.4 Cleavage peptide from resin 30 2.2.5 Peptide purification and identification 30 2.2.6 NMR spectra 31 2.2.7 NMR assignment 31 2.2.8 Hydrogen exchange rate 32 2.2.9 Structure calculation 32 2.2.10 Circular dichroism spectroscopy 33 2.2.11 Ultraviolet spectroscopy 33 2.2.12 Photochemical experiment 34 2.2.13 Photoacoustic Calorimetry 34 Chapter 3: Results and Discussion (I): structure calculation 38 3.1 Peptide synthesis, purification, and identification 38 3.2 Aggregation studies 38 3.3 Estimation of peptide 19-mer adopt a three-stranded antiparallel □-sheet structure 40 3.4 19-mer adopted a left-handed twist 53 3.5 Structural stability of 19-mer by CD 54 Chapter 4: Results and Discussion (II): Cyclized peptide 59 4.1 Peptide synthesis 59 4.2 Purification of BrAc-CMB-OH 59 4.3 Synthesis of c-19merE11C (cyclized for 1 to 11) 60 4.4 Peptide purification, identification and CD data 61 4.5 The initial kinetic data of c-19merE11C 66 4.5.1 Introduction to Photoacousitc Calorimetry 66 4.5.2 The PAC signals of c-19merE11C 69 4.5.3 Introduction to Photothermal Beam Deflection 73 4.5.4 The PBD signals of c-19merE11C 74 Chapter 5: Conclusion and Future Plans 77 Reference 80

    Reference

    1. Levinthal, C. (1968) J. Chim. Phys. 65, 44-45.
    2. Anfisen, C. B. (1973) Science 181, 223-230.
    3. Ptitsin, O. B. (1973) Doklady Akademii Nauk SSSR 210, 1213-1215.
    4. Kim, P. S. & Baldwin, R. L. (1990) Annual review of biochemistry 59, 631-660.
    5. Dyson, H. J. & Wright, P. E. (1993) Current opinion in structure biology 3, 60-65.
    6. Jackson, S. E. & Fersht, A. R. (1991) Biochemistry
    30, 10436-10443.
    7. Volk, M. (2001) European Journal of Organic Chemistry, 2605-2621.
    8. Dill, K. A., Bromberg, S., Yue, K., Fiebig, K. M., Yee, D. P., Thomas, P. D. & Chan, H. S. (1995) Protein Sci 4, 561-602.
    9. Ptitsyn, O. B. (1996) nature structural biology 3, 488-490.
    10. Leopold, P. E., Montal, M. & Onuchic, J. N. (1992) Proc Natl Acad Sci U S A 89, 8721-5.
    11. Davin, L. N. & Michael, M. C. (2000) Lehninger Principles of Biochemistry.
    12. Dobson, C. M. (2003) Nature 426, 884-890.
    13. Dinner, A. R., Sali, A., Smith, L. J., Dobson, C. M. & Karplus, M. (2000) Trends in Biochemical Sciences 25, 331-339.
    14. Scholtz, J. M. & Baldwin, R. L. (1993) Biochemistry 32, 4604-8.
    15. Lyu, P. C., Wemmer, D. E., Zhou, H. X., Pinker, R. J. & Kallenbach, N. R. (1993) Biochemistry 32, 421-425.
    16. Chakrabartty, A. & Baldwin, R. L. (1995) Adv Protein Chem 46, 141-76.
    17. Munoz, V. & Serrano, L. (1997) Biopolymers 41, 495-509.
    18. Aurora, R. & Rose, G. D. (1998) Protein Science 7, 21-38.
    19. Santiveri, C. M., Santoro, J., Rico, M. & Jimenez, M. A. (2004) Protein Science 13, 1134-1147.
    20. De Alba, E., Rico, M. & Jimenez, M. A. (1999) Protein Science 8, 2234-2244.
    21. Kortemme, T., Ramirez-Alvarado, M. & Serrano, L. (1998) Science 281, 253-256.
    22. Struthers, M. D., Cheng, R. P. & Imperiali, B. (1996) Science 271, 342-345.
    23. Kelly, J. W. (2000) Nature Structural Biology 7, 824-826.
    24. Gorman, P. M. & Chakrabartty, A. (2001) Biopolymers 60, 381-394.
    25. Minor, D. L. & Kim, P. S. (1994) Nature 367, 660-663.
    26. Griffiths-Jones, S. R., Sharman, G. J., Maynard, A. J. & Searle, M. S. (1998) Journal of Molecular Biology 284, 1597-1609.
    27. Das, C., Raghothama, S. & Balaram, P. (1999) Chemical Communications, 967-968.
    28. Das, C., Nayak, V., Raghothama, S. & Balaram, P. (2000) Journal of Peptide Research 56, 307-317.
    29. Blanco, F. J., Jimenez, M. A., Herranz, J., Rico, M., Santoro, J. & Nieto, J. L. (1993) Journal of American Chemistry Society 115, 5887-5888.
    30. de Alba, E., Jimenez, M. A. & M., R. (1997) Journal of American Chemistry Society 119, 175-183.
    31. Sibanda, B. L., Blundell, T. L. & Thornton, J. M. (1989) J Mol Biol 206, 759-77.
    32. Chou, P. Y. & Fasman, G. D. (1977) Journal of Moleclar Biology 115, 135-175.
    33. Rose, G. D., Gierasch, L. M. & Smith, J. A. (1985) Advances in Protein Chemistry 37, 1-109.
    34. Sibanda, B. L. & Thornton, J. M. (1985) Nature 316, 170-4.
    35. Schenck, H. L. & Gellman, S. H. (1998) Journal of the American Chemical Society 120, 4869-4870.
    36. Chen, P. Y., Lin, C. K., Lee, C. T., Jan, H. & Chan, S. I. (2000) Protein Science 10, 1794-1800.
    37. Bieri, O. & Kiefhaber, T. (1999) Biol Chem 380, 923-9.
    38. Jones, C. M., Henry, E. R., Hu, Y., Chan, C. K., Luck, S. D., Bhuyan, A., Roder, H., Hofrichter, J. & Eaton, W. A. (1993) Proceedings of the National Academy of Sciences of the United States of America 90, 11860-11864.
    39. Pascher, T., Chesick, J. P., Winkler, J. R. & Gray, H. B. (1996) Science 271, 1558-1560.
    40. Williams, S., Causgrove, T. P., Gilmanshin, R., Fang, K. S., Callender, R. H., Woodruff, W. H. & Dyer, R. B. (1996) Biochemistry 35, 691-697.
    41. Ballew, R. M., Sabelko, J. & Gruebele, M. (1996) Nature Structural Biology 3, 923-926.
    42. Rock, R. S. & Chan, S. I. (1996) Journal of Organic Chemistry 61, 1526-1529.
    43. Sheehan, J. C., Wilson, R. M. & Oxford, A. W. (1971) Journal of the American Chemistry Society 93, 7222-7228.
    44. Stowell, M. H. B., Rock, R. S., Rees, D. C. & Chan, S. I. (1996) Tetrahedron Letters 37, 307-310.
    45. Rock, R. S. & Chan, S. I. (1998) Journal of the American Chemical Society 120, 10766-10767.
    46. Chen, P.-Y., Huang, J.-T., Chen, H.-L., Jan, H., Velusamy, M., Lee, C.-T., Fann, W., Larsen, R. W. & Chan, S. I. (2004) Proceedings of the national academy of sciences of the United States of America 101, 7305-7310.
    47. Hansen, K. C., Rock, R. S., Larsen, R. W. & Chan, S. I. (2000) Journal of the American Chemical Society 122, 11567-11568.
    48. Sibanda, B. L. & Thornton, J. M. (1991) Methods Enzymol 202, 59-82.
    49. Bernstein, F. C., Koetzle, T. F., Williams, G. J., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977) J Mol Biol 112, 535-42.
    50. Griffiths-Jones, S. R., Maynard, A. J. & Searle, M. S. (1999) Journal of Molecular Biology 292, 1051-1069.
    51. Ramirez-Alvarado, M., Blanco, F. J., Niemann, H. & Serrano, L. (1997) J Mol Biol 273, 898-912.
    52. Ramirez-Alvarado, M., Blanco, F. J. & Serrano, L. (1996) Nat Struct Biol 3, 604-12.
    53. Johnson JR., W. C. (1988) Annual Review of Biophysics and Biophysical Chemistry 17, 145-166.
    54. Woody, R. W. (1995) Methods Enzymol 246, 34-71.
    55. Woody, R. W. & Dunker, A. K. (1996) in Circular dichroism and the conformational analysis of biomolecules, ed. Fasman, G., New York), pp. 109-158.
    56. Strickland, E. H. (1974) CRC Critical reviews in biotechnology 2, 113-175.
    57. Gensch, T. & Viappiani, C. (2003) Photochemical & Photobiological Sciences 2, 699-721.
    58. Miksovska, J. & Larsen, R. W. (2003) Journal of Protein Chemistry 22, 387-394.
    59. Abbruzzetti, S., Crema, E., Masino, L., Vecli, A., Viappiani, C., Small, J. R., Libertini, L. J. & Small, E. W. (2000) Biophysical Journal 78, 405-415.

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)

    QR CODE