臺灣博碩士論文加值系統

我們建立了光鉗微操控系統，藉由觀察被雷射光鉗捕捉下，聚苯乙烯微粒因熱擾動所造成的布朗運動並分析其微粒位置的機率分佈可進而推導出其光學彈性係數。在使用NA 1.24的油鏡及波長1064nm 的Nd: YVO4 雷射19mW下其所量測出的光鉗光學彈力常數約為115±1pN/μm。本篇論文旨在利用光鉗系統來探討Lon protease與HU protein的作用機制，希望藉由光鉗量測HUβ降解的辨識區域是否與結合作用區有關係，並希望可以區分出Lon protease與HU protein結合過程與降解作用不同階段作用力的變化，經統計分析後，實驗結果顯示HUα、HUβ、HUβA20D protein與Lon protease之間的作用力分別為24±6 (pN) 、28±5(pN) 、31±4(pN)。在加入ATP後它們之間的作用力均下降，分別為14±2 (pN) 、11±4(pN) 、15±2(pN) ，其中以HUβ最為明顯，其次是HUβA20D，最後為HUα，下降率分別為41％、60％、51％。我們將Lon protease的作用機制看成兩階段的受質選擇，第一階段是結合作用，區隔出Lon protease與HU protein、BSA作用力的不同，顯示酵素與其受質間演化出特殊的作用力，用以區隔受質與非受質，我們發現HUβ、HUβA20D之間作用力並無明顯差異，顯示Lon protease和HU protein 切點與結合區域並不是同一個地方。第二階段靠著ATP提供能量，推動將受質移位現象，我們推測這便是HUβ、HUα、HUβA20D作用力均下降的原因。

We setup an optical manipulation system to trap a polystyrene particle via optical tweezers and measured the transverse optical spring constant by analyzing the Brownian motion of the trapped particle driven by thermal fluctuation. In our experiments, the transverse optical spring constant was measured to be approximately 115±1pN/mm when a polystyrene particle (diameter =2.88μm) was trapped with a 19mW IR laser beam (wavelength = 1064nm) focused by an oil-immersion objective lens with a numerical aperture (NA) of 1.24. In biological application, we focused on the study of the interaction of Lon protease and HU protein by coating Lon on one polystyrene particle and HU on the other and measured their binding force by optical tweezers. From biochemical experiment data, we know Lon protease can degrade HUβ, but not HUα. To investigate the origin of this difference, we measure the binding force of Lon protease interacting with HUα、HUβ and the mutant protein, HUβ-A20D, in which. In experiments without energy supplement of ATP, the binding forces of Lon protease interacting with BSA, HUβ,HUα and HUβ-A20D were determined to be approximately 0(pN), 24±6(pN), 28±6(pN) and 31±4(pN), respectively. The lack of interaction of BSA with Lon protease indicates the specificity of the enzyme and the substrates, whereas the very small differences in the binding force of Lon-HUβ and Lon-HUβ-A20D indicate that the binding site and the cleavage site of Lon protease on HU protein may not be the same. However, with ATP supplement, the binding force of Lon protease interacting with HUα,HUβ and HUβ-A20D were measured to be approximately 14±2(pN), 11±4(pN) and 15±2(pN), respectively. The significant decrease in the binding force in all cases (in the presence of ATP) is speculated to be due to the conformation change of the Lon protease in the presence of ATP.

目錄
第1章 序論..........................................6
1.1 光學鉗住的歷史................................6
1.2 光學鉗住的基本原理 ...........................11
1.3 光學鉗住之發展與應用.........................13
1.3.1 藉由光鉗探討靈芝多醣體對於巨噬細胞的影響.....13
1.4 Lon protease 與 Hu Protein 之簡介..........17
1.4.1 E. coli Lon protease之特性與功能.............17
1.4.2 HU protein之特性與功能.......................19
1.5 實驗目的.....................................20
第2章 實驗架設和材料...............................21
2.1 Lon protease 與 Hu Protein之純化.............21
2.2 E. coli HUβ-A20D的突變.......................22
2.3 聚苯乙烯小球與Lon protease、Hu Protein間黏著製程 .............................................23
2.4 聚苯乙烯小球與Lon protease、Hu Protein間黏著確認 .............................................24
2.5 實驗架設.....................................26
2.6 QPD基本原理..................................29
2.7 利用布朗運動量測光鉗之彈力係數...............31
第3章 實驗結果與討論...............................34
3.1 Lon protease與HU protein之降解關係...........34
3.2 Lon protease與HUβ Protein、HUβA20Q Protein之降
解關係.......................................37
3.3 光鉗量測Lon protease與HU Protein之作用力.....40
3.3.1 Optical tweezers之作用力分析.................48
第4章 結論.........................................53
第5章 參考文獻.....................................55

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