Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62302
Title: | HmBRI 突變設計蛋白質之光學特性與應用研究 Photonic properties and application studies of an engineered HmBRI protein |
Authors: | Yi-Chung Shen 沈宜中 |
Advisor: | 楊?伸(Chii Shen, Yang) |
Keyword: | Highly-Expressed Bacteriorhodopsin,光週期,pH 值-光強度依賴顏色變化,無電池光強度感測器。, Highly-Expressed Bacteriorhodopsin (HEBR),photocycle,pH-light intensity dependent color changing,cell-free light intensity indicator, |
Publication Year : | 2013 |
Degree: | 碩士 |
Abstract: | 維他命A 醛結合蛋白質又稱為視紫紅質,結構由七個穿膜的α螺旋組成,並在結構的中央有個活性袋狀區,與具感光功能的視黃醛結合,在生物體中多作為感光受體蛋白質。視紫紅質在吸收一定波長的光源被激發後,分子結構將會從基態進入激發態,在微結構上有連續的改變,而最終能行使一次功能,此過程稱為光週期。本實驗室在嗜鹽古細菌 Haloarucla marismortui 當中分離純化出了六種不同的rhodopsin,並且將其中一種被稱為細菌視紫紅質 (Bacteriorhodopsin,BR)的光驅動外向氫離子幫浦,以點突變的方式建構出了一種具有高的表現量及穩性,且有較緩慢光週期的細菌視紫紅質突變蛋白質,命名為HEBR(Highly-Expressed Bacteriorhodopsin)。我們觀察到 HEBR 在光照下會產生顏色的變化(紫色變為黃色),也發現其變色的程度與溶劑環境的 pH 值正相關。利用光週期測試的方式,我們證實 BR 光週期中一重要的中間態:M 態存在的時間,會隨著 pH 值上升延長而累積;而這個具有不同特徵吸收值的中間態累積,正是 HEBR 在照光時顏色變化的原因。另外,利用化學反應平衡的方法推定光週期反應,解釋 M 態累積與 pH 值的關係,並在 HEBR 變色反應中引進了光照強度這個影響因子,更以時間解析吸收光譜加以證實且進行量化。本研究除確實觀察到 HEBR 蛋白質在不同 pH 值以及不同光照強度下的不同變色情形之外,亦以標準比色卡對真實視覺顏色變化做校正,並以 RGB 數量化所有顏色變化。利用此特殊的變色性質,HEBR 蛋白質的光色特性可被應用在無電池光強度感測系統中,作環境光強度的感測;亦可根據特定光強度下的顏色變化,投入在 pH 量測的應用。 Retinal-binding proteins (RBPs) are composed of seven-transmembrane α helix, which form a binding pocket to secure a photosensing retinal molecule. RBPs response to light illumination after abosorbing maximally at a specific wavelength and exhibit different visual colors. Our laboratory reported six new RBPs from a halophilic archaea, Haloarucla marismortui, and constructed a mutated protein named Highly-Expressed Bacteriorhodopsin (HEBR) from of one of the RBPs, HmBRI, and found it to have exceptional high expression efficiency and high stability, as well as a slower photocycle recovery rate. In addition, HEBR showed purple to yellow color changing when exposure to light, and the degree of such color changing was pH-dependent. Photocycle measurement unveiled that the accumulation of an important intermediate state, M state, is proportional to the increasing of pH value and consequently the color changing. In addition, by chemical reaction equilibrium, we introduced light intensity as another variable to describe such pheromone. Furthermore, this study calibrated the visual color changing with a professional standard color card with software, and quantified them as RGB values. Based on thecorrelation between HEBR color changing, pH value and light intensity, a series of HEBR under different pH was shown to report different light intensity and we proposed to use it as a battery-free light intensity indicator. Furthermore, HEBR also could serve as pH indicator by the color changing under certain light intensity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62302 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科技學系 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-102-1.pdf Restricted Access | 10.86 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.