臺灣博碩士論文加值系統

本論文首次成功利用雷射捕陷誘發溶菌酶溶液高濃度叢集區域生成，並運用於控制其晶體之成長。在距離自發結晶之溶菌酶晶體邊緣10 微米之位置，聚焦近紅外光連續波雷射，當雷射捕陷焦點聚集溶液中的類液體溶菌酶叢集時，濃度將因此上升，同時引發液體－液體相分離，並且在焦點產生局部高濃度叢集區域；其中叢集間之相互作用力及排列通過叢集之間的氫鍵向焦點外擴展，最終誘發形成巨大高濃度叢集區域並且完全覆蓋包含溶菌酶晶體。此時晶體成長將完全取決於高濃度叢集區域中的叢集濃度以及排列。雷射捕陷侷限了初始高濃度叢集區域中的溶菌酶叢集之自由運動及排列方向，也因此抑制了晶體之成長。雷射捕陷焦點不斷捕捉初始叢集區域中的溶菌酶叢集，濃度持續增加使其結構及氫鍵聯結轉變。最終，初始高濃度叢集區域將轉變成具有較高濃度暨叢集排列性較低的叢集區域，晶體的成長速率也因此有顯著的提升。此外，叢集區域轉變前後均具有雷射極性依存之異相性特徵，此特性對於晶體成長具有密切關聯。本研究之成果不僅拓展了嶄新的蛋白質晶體成長控制方法，同時也發展了新的概念用於解釋雷射捕陷與蛋白質叢集間的交互作用。

We present the first demonstration of laser trapping-controlled crystal growth of protein, which is achieved for hen egg-white lysozyme (HEWL) through a highly concentrated cluster domain formation. A continuous-wave near-infrared laser beam is employed as a trapping light source and focused at a point 10 µm away from a target HEWL crystal formed spontaneously in solution. Laser trapping of HEWL liquid-like clusters in solution increases local concentration at the focus, where liquid-liquid phase separation should be considered to be triggered. The local association and orientation of the clusters expands from the focus to its outside through the hydrogen bonding network, and a large highly concentrated domain of the clusters is formed. The target HEWL crystal is totally covered with the domain and grown depending on orientation and concentration of the clusters in the domain. First, laser trapping restricts the free motion and reorientation of the clusters in the domain, which strongly suppresses crystal growth. Subsequent laser trapping of the clusters in the initial domain continuously increases local cluster concentration, leading to change in conformation of HEWL cluster structure and the hydrogen bonding network. Consequently, the domain is transformed into another domain with lower rigidity and ordering of the clusters, which surprisingly enhances crystal growth. Moreover, the clusters in both domains have anisotropic features reflecting laser polarization, which also contributes to crystal growth. These results will enable us not only to develop a new crystal growth method but also to understand novel interactions between focused laser light and protein clusters.

摘 要......i
Abstract......ii
Acknowledgements......iii
Table of Contents......iv
List of Figures......vii
List of Tables......xiii

Chapter 1. Introduction......1
1.1 Laser trapping......1
1.1.1 History of laser trapping......1
1.1.2 Principle of laser trapping......3
1.2 Protein crystallization......7
1.2.1 Structure of protein crystal......8
1.2.2 Phase diagram of protein crystallization......10
1.2.3 Crystallization theory......13
1.2.4 Crystal growth......18
1.2.4-1 Conventional methods......18
1.2.4-2 Dynamics and mechanism......21
1.2.4-3 Hen egg white lysozyme (HEWL) ......23
1.3 Light/laser-induced crystallization...... 29
1.3.1 Photochemical reaction-induced nucleation...... 29
1.3.2 Non-photochemical light-induced nucleation......31
1.3.3 Femtosecond laser-induced nucleation and crystal growth......33
1.4 Laser trapping-induced crystallization and liquid-liquid phase separation......37
1.4.1 Control of crystal nucleation and crystal polymorph......38
1.4.2 Assembling formation......45
1.4.3 Liquid-liquid phase separation......47
1.4.4 Control of crystal growth......54
1.4.5 Classification of laser trapping-induced phenomena......57
1.5 Motivation......59

Chapter 2. Experimental......62
2.1 Preparation of hen egg white lysozyme crystal......62
2.1.1 Hen egg white lysozyme solution...... 62
2.1.2 Chamber......63
2.1.3 Crystallization......64
2.2 Optical Setup......65
2.3 Conditions for laser trapping......66
2.3.1 Crystal growth......66
2.3.2 Liquid-liquid phase separation......69
2.4 Determination of crystal growth rate......70

Chapter 3. Results and discussion......71
3.1 Spontaneous crystal growth depending on solution concentration......71
3.2 Laser trapping-controlled crystal growth of hen egg white lysozyme......73
3.2.1 Irradiation position dependence......80
3.2.2 Effect of solution temperature and evaporation......80
3.2.3 Irradiation power dependence......85
3.3 Formation of laser trapping-induced highly concentrated domain......87
3.3.1 Formation of highly concentrated cluster domain......87
3.3.2 Laser trapping-induced liquid-liquid phase separation......89
3.3.3 Two different crystal growth behaviors......91
3.3.4 Two different highly concentrated domains......97
3.4 Laser polarization-dependent crystal growth of hen egg white lysozyme......99
3.5 Dynamics and mechanism......105

Chapter 4. Conclusion......109

References......111

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