蛋白質結構對於維持其功能是互相影響的，但在特定條件下，蛋白質會產生變性，進而誘發一些疾病，因此研究蛋白質早期變性的過程是非常重要的，本篇研究，利用低濃度的尿素(Urea)使泛素(Ubiquitin) 結構不穩定，產生早期展開之結構，之後我們使用一系列NMR光譜技術去研究蛋白質在早期展開狀態中的蛋白質骨幹動態。
首先，分析T1, T2,弛豫以及NOE數值之測量，將其測量結果套用至無模型分析(ModelFree method)方法，結果顯示發現泛素(Ubiquitin)在早期展開條件之下，特定區域的S2 (Order Parameter) 數值下降，顯示區域性的動態增加，同時構型交換(Rex)也在更多的胺基酸被偵測到，同時我們也利用新型NMR光譜技術，場循環核磁共振(Field cycling NMR)，去測量從磁場範圍20 Telsa (850 Hz)~1 Telsa (42.5 Hz)中個別骨幹的縱向弛豫 (T1)，結果反應出內分子運動的時間尺度，而此結果與T1, T2, NOE弛豫之測量與無模型方法(ModelFree method)互補，可以提供更精準的測量。接著我們也做了泛素(Ubiquitin)在早期展開條件之下，二級結構預測以及氫鍵實驗，其結果顯示結構並無太大改變。
總結上述，藉由NMR技術搭配場循環核磁共振(Field cycling NMR)技術可以精準反映內分子運動的尺度，提供蛋白質早期展開時，更完整的結構以及動態描述。

The protein unfolding is highly related with the maintenance of protein structures and functions in certain content. Therefore, to study protein early-stage unfolding process would be critical in understanding the diseases induced by protein unfolding. Here, low Urea concentration (< 1.5 M) is used to destabilize Ubiquitin and derive a structure representing the early-unfolding state. We used a series of NMR methods to detect protein backbone dynamics at the early-unfolding state.
In the first examination, we measured T1, T2, NOE of individual residues and employed ModelFree method to analyze the dynamic parameters. Low concentration Urea led reduced S2 and increased number of residues with conformational exchange (Rex). Meanwhile, we also use a modern field-cycling NMR (FCNMR) method to detect the longitudinal relaxation rates (T1) of the individual Ubiquitin backbone amides under the magnetic field strengths from 20 Telsa (850 MHz) to ~1 Telsa (42.5 MHz). The field-dependent relaxations precisely reported backbone internal motion with wider time scale (nanosecond to picosecond). The result well corresponded the result derived from T1, T2, NOE measurements and ModelFree analysis. We also performed secondary structure prediction and hydrogen bond detection by NMR and the results showed less significant change in structure.
In summary, NMR approaches including FCNMR technology could report a wider range of time-scale for molecular dynamics, providing a more complete insight and dynamic description for studying protein early-stage unfolding process.

中文摘要 I
Abstract II
縮寫(Abbreviations) IV
中英對照表 V
目錄 VI
第一章 前言 1
第二章 材料與方法 7
2.1 蛋白質表現純化 7
2.2 核磁共振 (Nuclear magnetic resonance, NMR) 8
2.2.1 NMR樣品備製 8
2.2.2 泛素骨幹標定(Backbone Assignment) 9
2.2.3 蛋白質化學位移擾動(chemical shift perturbation) 10
2.3 蛋白質動態(dynamic)分析 11
2.3.1 核磁共振自旋弛豫理論(NMR spin relaxation) 11
2.3.2 Nuclear Overhauser Effect (NOE) 13
2.3.3 無模型分析(Model-free formalism) 13
2.3.4 氫鍵實驗 16
2.3.5 場循環核磁共振(Field cycling NMR) 17
第三章 結果 22
3.1 化學位移擾動分析 22
3.2 Ubiquitin動力學分析 23
3.3 氫鍵分析 26
3.4 場循環分析 27
第四章 討論 43
參考文獻 46

1. Whitford, D., Proteins:struture and function. 2013.
2. Hopkins, A.L., Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol, 2008. 4(11): p. 682-90.
3. Kanehisa, M., et al., KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res, 2010. 38(Database issue): p. D355-60.
4. Takano, T., Structure of myoglobin refined at 2-0 A resolution. I. Crystallographic refinement of metmyoglobin from sperm whale. J Mol Biol, 1977. 110(3): p. 537-68.
5. Takano, T., Structure of myoglobin refined at 2-0 A resolution. II. Structure of deoxymyoglobin from sperm whale. J Mol Biol, 1977. 110(3): p. 569-84.
6. Ison, R.E., S. Hovmoller, and R.H. Kretsinger, Proteins and their shape strings. An exemplary computer representation of protein structure. IEEE Eng Med Biol Mag, 2005. 24(3): p. 41-9.
7. Ma, J., New advances in normal mode analysis of supermolecular complexes and applications to structural refinement. Curr Protein Pept Sci, 2004. 5(2): p. 119-23.
8. Pusey, P.N. and W. Vanmegen, Phase-Behavior Of Concentrated Suspensions Of Nearly Hard Colloidal Spheres. Nature, 1986. 320(6060): p. 340-342.
9. Ackerson, B.J. and P.N. Pusey, Shear-Induced Order In Suspensions Of Hard-Spheres. Physical Review Letters, 1988. 61(8): p. 1033-1036.
10. Rizzuti, B. and V. Daggett, Using simulations to provide the framework for experimental protein folding studies. Arch Biochem Biophys, 2013. 531(1-2): p. 128-35.
11. McKinley, M.P., D.C. Bolton, and S.B. Prusiner, A protease-resistant protein is a structural component of the scrapie prion. Cell, 1983. 35(1): p. 57-62.
12. Syme, C.D., et al., Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques. J Biol Chem, 2004. 279(18): p. 18169-77.
13. Stefani, M. and C.M. Dobson, Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med (Berl), 2003. 81(11): p. 678-99.
14. Dobson, C.M., Protein folding and misfolding. Nature, 2003. 426(6968): p. 884-90.
15. Vijay-Kumar, S., C.E. Bugg, and W.J. Cook, Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol, 1987. 194(3): p. 531-44.
16. Cole, R. and J.P. Loria, FAST-Modelfree: a program for rapid automated analysis of solution NMR spin-relaxation data. J Biomol NMR, 2003. 26(3): p. 203-13.
17. John Cavanagh, W.J.F., Arthur G. Palmer III, Mark Rance and Nicholas J. Skelton, Protein NMR Spectroscopy
18. Palmer, A.G., 3rd, et al., Improved resolution in three-dimensional constant-time triple resonance NMR spectroscopy of proteins. J Biomol NMR, 1992. 2(1): p. 103-8.
19. Bax, A. and M. Ikura, An efficient 3D NMR technique for correlating the proton and 15N backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15N/13C enriched proteins. J Biomol NMR, 1991. 1(1): p. 99-104.
20. Wishart, D.S., B.D. Sykes, and F.M. Richards, The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry, 1992. 31(6): p. 1647-51.
21. Bloch, F., The Principle of Nuclear Induction. Science, 1953. 118(3068): p. 425-30.
22. Kay, L.E., D.A. Torchia, and A. Bax, Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry, 1989. 28(23): p. 8972-9.
23. Meiboom, S. and D. Gill, Modified Spin-Echo Method for Measuring Nuclear Relaxation Times. Review Of Scientific Instruments, 1958. 29(8): p. 688-691.
24. Levitt, M.H., Spin Dynamics: Basics of Nuclear Magnetic Resonance. 2001.
25. Lipari, G. and A. Szabo, Model-Free Approach To the Interpretation Of Nuclear Magnetic-Resonance Relaxation In Macromolecules .2. Analysis Of Experimental Results. Journal Of the American Chemical Society, 1982. 104(17): p. 4559-4570.
26. Lipari, G. and A. Szabo, Model-Free Approach To the Interpretation Of Nuclear Magnetic-Resonance Relaxation In Macromolecules .1. Theory And Range Of Validity. Journal Of the American Chemical Society, 1982. 104(17): p. 4546-4559.
27. Clore, G.M., et al., Deviations From the Simple 2-Parameter Model-Free Approach To the Interpretation Of N-15 Nuclear Magnetic-Relaxation Of Proteins. Journal Of the American Chemical Society, 1990. 112(12): p. 4989-4991.
28. Blumenschein, T.M., et al., Dynamics of the C-terminal region of TnI in the troponin complex in solution. Biophys J, 2006. 90(7): p. 2436-44.
29. d'Auvergne, E.J. and P.R. Gooley, Set theory formulation of the model-free problem and the diffusion seeded model-free paradigm. Molecular Biosystems, 2007. 3(7): p. 483-494.
30. Cordier, F., et al., Direct detection of N-H[...]O=C hydrogen bonds in biomolecules by NMR spectroscopy. Nat Protoc, 2008. 3(2): p. 235-41.
31. Dingley, A.J., et al., Direct detection of N-H[...]N hydrogen bonds in biomolecules by NMR spectroscopy. Nat Protoc, 2008. 3(2): p. 242-8.
32. Kay, L.E., et al., Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990. J Magn Reson, 2011. 213(2): p. 423-41.
33. Kimmich, R. and E. Anoardo, Field-cycling NMR relaxometry. 2004.
34. Redfield, A.G., Field-cycling NMR applied to macromolecular structure and dynamics. Nmr as a Structural Tool for Macromolecules, 1996: p. 123-132.
35. Huang, J.R., et al., Sequence-specific mapping of the interaction between urea and unfolded ubiquitin from ensemble analysis of NMR and small angle scattering data. J Am Chem Soc, 2012. 134(9): p. 4429-36.