矽質中孔洞材料具有高表面積、狹窄的孔洞尺寸分布以及高穩定性等特色。這類的材料在過往被證實於生物醫藥應用上相當有用，例如藥物輸送及釋放、固定蛋白質於孔洞內以增強蛋白質的再利用性以及蛋白質活性恢復。儘管應用如此廣泛，目前對於被裝填的蛋白質與中孔洞材料表面間的交互作用仍舊十分不明瞭。因此，更深入的瞭解蛋白質如何受孔洞內微觀環境影響、以及固定/裝填的過程是相當重要的。
近年來，定位自旋標記電子自旋共振(SDSL-ESR)技術被使用於研究—受矽質中孔洞材料的奈米通道侷限的胜肽分子動力學。先前的研究顯示，將胜肽(或小自由基分子)裝填於奈米通道內能有效的降低分子翻滾速率。因此，目標分子的動力學仍保持在電子自旋共振的靈敏區間內，使得可以於室溫並且無黏滯劑如丙三醇或蔗糖的環境下，研究生物分子的動力學。
然而，ESR技術結合奈米通道的方法目前尚未被應用於研究真正的蛋白質動力學上。本篇論文記述如何透過結合ESR及奈米孔洞材料研究蛋白質在孔洞材料內的分子動力學。我們製備了T4溶菌酶以及一系列含有單一半胱胺酸變體的T4溶菌酶，以進行自旋標記ESR的研究。結果指出當蛋白質溶液體積在MSU-H的孔洞體積以下時，ESR光譜對於T4溶菌酶的水合程度相當敏感。而當蛋白質溶液體積大過可佔據的孔洞體積時，隨著溶液量的增加，光譜幾乎沒有變化。根據此結果，我們選定的最佳比例為1/2(溶液體積/孔洞體積)。在這樣的水合程度下，不只使蛋白質足以保有其結構，並且也適合自旋探子反映局部環境的差異。
我們收集了一系列裝填於奈米孔洞內的T4溶菌酶之變溫ESR光譜，並且使用Stochastic Liouville Equation(SLE)的線形理論分析。此分析方法提供了定量的描述，描述著自旋標記位點於不同溫度下的局部移動性以及有序性。理論分析的結果顯示兩個光譜成分的存在，並且由於移動性的差異，將兩個成分分別標示為快運動(mobile, Mb)及慢運動成分(immobile, Im)。我們發現快運動成分移動性與骨架原子的B-factor (PDB: 3LZM)呈現正向關係，而慢運動成分移動性則與溫度有明顯的關聯。因此，我們認為快、慢運動成分分別與骨架運動以及側鏈內部運動有關。根據獲得的動力學參數，可以求得各個自旋標記位點的活化能，該值的大小顯示著局部的移動性。結果顯示，位在solvent-exposed site具有大於buried site的活化能。這樣的結果指出，當位於蛋白質表面側鏈的運動受奈米侷限水合環境限制時，蛋白質內部的微觀環境因三級結構的保護，而受到較少的影響。由於未使用奈米通道下，透過ESR研究蛋白質動力學是無法如此清楚地區分骨架運動及側鏈運動，因此透過奈米孔洞材料及ESR實驗的方法，對蛋白質動力學研究是相當重要的改善。
總結來說，我們從實驗及理論的ESR結果，詳盡地探討MSU-H中孔洞材料下的T4溶菌酶的局部動力學及有序性。裝填於奈米通道內時，T4溶菌酶於室溫下的翻滾運動大幅降低，因此 ESR光譜才更清楚地反應T4溶菌酶於微觀環境下的動力學差異。本篇研究證實了使用奈米通道於蛋白質動力學研究上的可行性。

Mesoporous silica materials are characterized by a large surface area, a narrow pore size distribution and a high stability. The materials have been demonstrated useful in many biomedical applications, such as drug release and delivery, as well as immobilization of enzyme to enhance the reuse and recovery of protein activity. Despite the variety of the application, molecular interactions between the encapsulated protein and mesoporous surface remain largely unclear. It is crucial to better understand how the protein is affected by the microenvironment inside the pores and to gain more knowledge about the immobilization/encapsulation process.
Site-directed spin-labeling (SDSL) electron spin resonance (ESR) techniques have, in the recent years, been used to study the dynamics of peptides when confined in the nanochannels of mesoporous silica materials. The previous studies showed that the encapsulation of spin-labeled peptides (or small radical molecules) in nanochannels is effective to reduce the molecular tumbling. As a result, molecular dynamics of the target molecules remains within the ESR sensitive regime, making it possible to investigate the biomolecular dynamics in the absence of viscous agents such as glycerol and sucrose at room temperature. However, this combined approach of ESR with nanochannels has not been applied to studying dynamics of real proteins. 
In this thesis, we report a comprehensive study of protein dynamics can be improved with the combined approach of ESR and nanochannels. T4 lysozyme (T4L) and a variety of single-cysteine variants of T4L mutants, corresponding to various solvent-exposed or buried sites, were prepared for the spin-label ESR study. We show that ESR spectra are sensitive to the hydration level of the encapsulated T4L when the volume of protein solution is less than the pore volume of the MSU-H materials. When the protein solution is greater than the available pore volume, ESR spectra change little with the amount of the added solution. As such, this study has determined an optimal ratio of 1/2 (v/v for solution/pore) in which, the hydration is not only sufficient to retain the protein structure but also adequate for the spin probe to reflect the differences in the local environment.
A series of temperature-dependent ESR spectra of the encapsulated T4L have been collected and analyzed using the lineshape theory based on the stochastic Liouville equation (SLE). The analysis provides a quantitative description for the local mobility and ordering of the spin-labeled sites as a function of temperature. Two spectral components are identified from the theoretical analyses. The components are best characterized by high and low mobility, hence denoted by mobile (Mb) and immobile (Im) components, respectively. While the mobility of the Mb of the studied sites was found to show a positive correlation with the B-factors of the backbone atoms (PDB: 3LZM), the mobility of the Im exhibits a clear connection with temperature. The Mb and Im components are, therefore, assigned to the dynamics corresponding to the backbone and the internal motions of side chain, respectively. Based on the dynamical parameters obtained, the activation energy of the spin-labeled sites, which provides characterization for the local mobility, is determined. The solvent-exposed sites are substantially greater in the activation energy than the buried sites. The result indicates that while the mobility of the side chains at the protein surface is restricted by the nanoconfined hydration, the microenvironment within the protein is less affected due to the protection from the tertiary structure. Perhaps this is the most important improvement for protein dynamics by the  combined approach of ESR and nanochannels, as such a clear separation of the two dynamical motions is not possible when studying protein dynamics by ESR without nanochannels.
In conclusion, this study reports the experimental and theoretical ESR results that reveal details of the local dynamics and ordering of T4L in the MSU-H mesoporous materials. When encapsulated in nanochannels, molecular tumbling motions of T4L are largely slowed down even at room temperatures. As a result, dynamical differences between the microenvironments in T4L are better reported on the ESR lineshape. This study has demonstrated the feasibility of the study approach for protein dynamics.

目錄
謝誌 I
摘要 II
Abstract IV
目錄 VI
圖目錄 VIII
表目錄 X

第一章 緒論 1
1.1 電子自旋共振光譜於蛋白質動力學之應用 1
1.2 溶劑水分子對蛋白質之重要性 1
1.3 奈米孔洞材料以及奈米侷限空間應用之簡介 2
1.4 T4溶菌酶之簡介 2
1.5 研究動機與目的 3

第二章 儀器介紹與原理 4
2.1 電子自旋共振光譜儀 4
2.1.1 歷史發展 4
2.1.2 連續波型電子自旋共振光譜原理 4
2.1.2.1 賽曼效應(Zeeman interaction) 5
2.1.2.2 超微細偶合作用(Hyperfine interaction) 6
2.1.2.3 定位自旋標記(Site-Directed Spin Label, SDSL) 7
2.1.2.4 ESR光譜線形與分子的轉動情形 8
2.1.3 儀器裝置 10

第三章 光譜模擬 13
3.1 光譜模擬原理 13
3.1.1 Stochastic Liouville equation(SLE) 13
3.1.2 硝基氧化標記物的物理參數 14
3.1.2.1 磁性參數(magnetic parameters) 14
3.1.2.2 轉動擴散模型(Rotational Diffusion Model) 17
3.1.2.3 指向有序性(Orientational Ordering) 18
3.1.3 微觀有序巨觀無序模型(MOMD) 22
3.2 光譜模擬分析 24

第四章 樣品製備與儀器測量方法 25
4.1 樣品製備 25
4.1.1 T4溶菌酶之純化 25
4.1.2 定位自旋標記樣品之製備 26
4.1.3 孔洞材料裝填T4溶菌酶樣品之製備 26
4.2 連續波型電子自旋共振光譜量測 27
4.3 藥品與儀器 27
4.3.1 藥品 27
4.3.2 儀器 29

第五章 結果與討論 30
5.1 不同水合程度下T4溶菌酶於奈米孔洞內的運動情形 30
5.1.1 Solvent-exposed site T4L131R1 cw-ESR光譜分析 31
5.1.2 Buried site T4L99R1 cw-ESR光譜分析 35
5.1.3 奈米侷限空間下T4溶菌酶受水合程度的影響 37
5.2 侷限效應對T4溶菌酶運動模式影響之研究 38
5.3 探討溫度變化對奈米孔洞內T4溶菌酶運動模組的影響 47

第六章 結論 60

參考文獻 62

1. Barnes JP, Liang Z, McHaourab HS, Freed JH, & Hubbell WL (1999) A multifrequency electron spin resonance study of T4 lysozyme dynamics. Biophys. J. 76(6):3298-3306.
2. Fleissner MR, et al. (2011) Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy. Proc. Natl. Acad. Sci. USA 108(39):16241-16246.
3. Zhang Z, et al. (2010) Multifrequency electron spin resonance study of the dynamics of spin labeled T4 lysozyme. J. Phys. Chem. B 114(16):5503-5521.
4. McHaourab HS, Oh KJ, Fang CJ, & Hubbell WL (1997) Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. Biochemistry 36(2):307-316.
5. Columbus L & Hubbell WL (2004) Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA. Biochemistry 43(23):7273-7287.
6. Rupley JA & Careri G (1991) Protein hydration and function. Adv. Protein Chem. 41:37-172.
7. Frauenfelder H & Gratton E (1986) Protein dynamics and hydration. Methods Enzymol. 127:207-216.
8. Rupley JA, Gratton E, & Careri G (1983) Water and globular proteins. Trends Biochem. Sci 8(1):18-22.
9. Fenimore PW, Frauenfelder H, McMahon BH, & Young RD (2004) Bulk-solvent and hydration-shell fluctuations, similar to alpha- and beta-fluctuations in glasses, control protein motions and functions. Proc. Natl. Acad. Sci. USA 101(40):14408-14413.
10. Swenson J, Jansson H, Hedström J, & Bergman R (2007) Properties of hydration water and its role in protein dynamics. J. Phys.: Condens. Matter 19(20):205109.
11. Frauenfelder H, Fenimore P, Chen G, & McMahon B (2006) Protein folding is slaved to solvent motions. Proc. Natl. Acad. Sci. USA 103(42):15469-15472.
12. Slowing II, Trewyn BG, Giri S, & Lin VY (2007) Mesoporous silica nanoparticles for drug delivery and biosensing applications. Adv. Funct. Mater. 17(8):1225-1236.
13. Vallet‐Regí M, Balas F, & Arcos D (2007) Mesoporous materials for drug delivery. Angew. Chem. Int. Ed. 46(40):7548-7558.
14. Kahse M, et al. (2014) Stability, Hydration, and Thermodynamic Properties of RNase A Confined in Surface-Functionalized SBA-15 Mesoporous Molecular Sieves. J. Phys. Chem. C 118(37):21523-21531.
15. Kao K-C, Lin T-S, & Mou C-Y (2014) Enhanced activity and stability of lysozyme by immobilization in the matching nanochannels of mesoporous silica nanoparticles. J. Phys. Chem. C 118(13):6734-6743.
16. Ravindra R, Zhao S, Gies H, & Winter R (2004) Protein encapsulation in mesoporous silicate: the effects of confinement on protein stability, hydration, and volumetric properties. J. Am. Chem. Soc. 126(39):12224-12225.
17. Huang Y-W & Chiang Y-W (2011) Spin-label ESR with nanochannels to improve the study of backbone dynamics and structural conformations of polypeptides. Phys. Chem. Chem. Phys. 13(39):17521-17531.
18. Sung T-C & Chiang Y-W (2010) Identification of complex dynamic modes on prion protein peptides using multifrequency ESR with mesoporous materials. Phys. Chem. Chem. Phys. 12(40):13117-13125.
19. Gallo P, Rovere M, & Spohr E (2000) Glass transition and layering effects in confined water: a computer simulation study. J. Chem. Phys 113(24):11324-11335.
20. Harrach MF, Drossel B, Winschel W, Gutmann T, & Buntkowsky G (2015) Mixtures of Isobutyric Acid and Water Confined in Cylindrical Silica Nanopores Revisited: A Combined Solid-State NMR and Molecular Dynamics Simulation Study. J. Phys. Chem. C 119(52):28961-28969.
21. Yoshida K, Yamaguchi T, Kittaka S, Bellissent-Funel M-C, & Fouquet P (2008) Thermodynamic, structural, and dynamic properties of supercooled water confined in mesoporous MCM-41 studied with calorimetric, neutron diffraction, and neutron spin echo measurements. J. Chem. Phys 129(5):054702.
22. Baase WA, Liu L, Tronrud DE, & Matthews BW (2010) Lessons from the lysozyme of phage T4. Protein Sci. 19(4):631-641.
23. McHaourab HS, Lietzow MA, Hideg K, & Hubbell WL (1996) Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry 35(24):7692-7704.
24. Columbus L, Kálai T, Jekö J, Hideg K, & Hubbell WL (2001) Molecular motion of spin labeled side chains in α-helices: analysis by variation of side chain structure. Biochemistry 40(13):3828-3846.
25. Mchaourab HS, Kalai T, Hideg K, & Hubbell WL (1999) Motion of spin-labeled side chains in T4 lysozyme: Effect of side chain structure. Biochemistry 38(10):2947-2955.
26. Zavoisky E (1944) The paramagnetic absorption of a solution in parallel fields. J. Phys. USSR 8:377-380.
27. Feher G (1956) Method of polarizing nuclei in paramagnetic substances. Phys. Rev. 103(2):500.
28. Hyde JS, Chien JC, & Freed JH (1968) Electron–electron double resonance of free radicals in solution. J. Chem. Phys 48(9):4211-4226.
29. Eliav U & Freed JH (1984) The oscillatory nature of polarization evolution in CIDEP. J. Phys. Chem. 88(7):1277-1280.
30. Stone T, Buckman T, Nordio P, & McConnell H (1965) Spin-labeled biomolecules. Proc. Natl. Acad. Sci. USA 54(4):1010-1017.
31. Altenbach C, Flitsch SL, Khorana HG, & Hubbell WL (1989) Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines. Biochemistry 28(19):7806-7812.
32. Brustolon M, Maniero AL, & Corvaja C (1984) EPR and ENDOR investigation of tempone nitroxide radical in a single crystal of tetramethyl-1, 3-cyclobutanedione. Mol. Phys. 51(5):1269-1281.
33. Ding Z, Gullá AF, & Budil DE (2001) Ab initio calculations of electric field effects on the g-tensor of a nitroxide radical. J. Chem. Phys 115(23):10685-10693.
34. Budil DE, Lee S, Saxena S, & Freed JH (1996) Nonlinear-least-squares analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg–Marquardt algorithm. J. Magn. Reson. , Series A 120(2):155-189.
35. Dzikovski B, Tipikin D, & Freed J (2012) Conformational distributions and hydrogen bonding in gel and frozen lipid bilayers: a high frequency spin-label ESR study. J. Phys. Chem. B 116(23):6694-6706.
36. Owenius R, Engström M, Lindgren M, & Huber M (2001) Influence of solvent polarity and hydrogen bonding on the EPR parameters of a nitroxide spin label studied by 9-GHz and 95-GHz EPR spectroscopy and DFT calculations. J. Phys. Chem. A 105(49):10967-10977.
37. Guo Z, Cascio D, Hideg K, Kalai T, & Hubbell WL (2007) Structural determinants of nitroxide motion in spin-labeled proteins: tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme. Protein Sci. 16(6):1069-1086.
38. Lietzow MA & Hubbell WL (2004) Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet. Biochemistry 43(11):3137-3151.
39. Langen R, Oh KJ, Cascio D, & Hubbell WL (2000) Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure. Biochemistry 39(29):8396-8405.
40. Jansson H, Bergman R, & Swenson J (2011) Role of solvent for the dynamics and the glass transition of proteins. J. Phys. Chem. B 115(14):4099-4109.
41. Mallamace F, et al. (2007) Role of the solvent in the dynamical transitions of proteins: the case of the lysozyme-water system. J. Chem. Phys. 127(4):045104.
42. Han YJ, Stucky GD, & Butler A (1999) Mesoporous silicate sequestration and release of proteins. J. Am. Chem. Soc. 121(42):9897-9898.
43. Vinu A, Murugesan V, & Hartmann M (2004) Adsorption of lysozyme over mesoporous molecular sieves MCM-41 and SBA-15: Influence of pH and aluminum incorporation. J. Phys. Chem. B 108(22):7323-7330.
44. Katiyar A, Ji L, Smirniotis P, & Pinto NG (2005) Protein adsorption on the mesoporous molecular sieve silicate SBA-15: effects of pH and pore size. J. Chromatogr. A 1069(1):119-126.