- 學位論文
Haloarcula marismortui 中 HmBRI 及 HmBRII 蛋白質特性及功能研究
Functional analysis of HmBRI and HmBRII from Haloarcula marismortui
摘要
光對於生物體是重要的能量來源，在過去於嗜鹽古生菌的研究中，鑑定出四種的光受體 (photoreceptor) 能夠使用外界光源調控生理現象。這類受體都屬於視紫紅質 (rhodopsin) 的構形，主要能分成兩種離子幫浦及兩種調控光趨性的感光受體，其中細菌視紫紅質( Bacteriorhodopsin, BR ) 是第一個所被發現也徹底研究的古生菌視紫紅質，其被鑑定為一個光趨動的氫離子幫浦，具有重要的生理功能。 在2004年完成的Haloarcula marismortui 基因體解序中1，總共預測出六個視紫質的基因，其中兩個基因序列bop和xop1，被預測為是接近細菌視紫質的蛋白質。在本實驗室先前的研究中，將其命名為HmBRI和HmBRII。本研究將其選殖入E. coli中，進行表達重組蛋白質，試圖了解在H. marismotrui中，為何同時存在兩個細菌視紫質基因的生理意義，因為這是從未在任何古生菌發現的 首先，在嘗試進行蛋白質表現及純化後，得到最適化條件，並且利用熱處理的方式，改進原本的純化方式。其次，將所得蛋白質經由吸收光譜、光週期 (photocycle) 測定和氫離子幫浦 (proton pump) 能力測試後，確定其光譜特性和過去研究的結果類似，並且同樣具有氫離子幫浦功能，初步證實兩者皆具有細菌視紫質的特性和功能；接著利用特定胺基酸位置突變，得到與過去研究的結果具有相同的性質；並進行蛋白質結晶實驗，提供結構上的瞭解。再其次，本研究以序列分析、吸收光譜、光週期、幫浦能力和結構上的各種觀點，分析BRI和BRII兩者在蛋白質特性的異同，探討其生理功能，雖然未能完全解釋其可能的差異，但藉由了解其蛋白質性質，提供深入研究和工業發展上特性的基礎背景之建立。
並列摘要
Light represents one of the most important energy sources for life. Previous studies had identified photoreceptors from halophilic archaea that mediate four different physiological functions when responding to light. These photoreceptors belong to the rhodopsin family and can be classified as ion transporter or sensory receptor for phototaxis responses. Bacteriorhodopsin (BR) is the first well studied archaeal rhodopsin, which was identified as a light-driven proton pump responsible for energy generation under high intensity light illumination or under low oxygen condition. Haloarcula marismortui genome sequencing was completed in 2004 and a total of six rhodopsin genes were predicted, the most numerous in a single archaeon. Two genes－bop and xop1, were identified and determined to encode proteins called HmBRI and HmBRII, respectively, which formed a unique two-proton pump system. The goal of this thesis was set to compare protein features and functions between HmBRI and II to further understand the possible explanation why there are two BRs in the same cell. First, the HmRBI and II genes were expressed in E. coli and the optimum conditions were obtained and a new heat treatment procedure was introduced. Secondly, several assays for protein function probing were performed, including UV-vis absorption spectrum scanning, duration of photocycle and direct proton pump measurements, both HmBRI and HmBRII were proton pump and response to the same wavelength were thus concluded. Thirdly, protein sequence analysis, absorption spectra, photocycle measurements, proton pump activity and protein structure prediction analysis were carried out to extend our understanding of these two proteins. Finally, protein crystallization was performed to study the protein structures and needle-shaped crystals were successfully obtained. Even no clear conclusion was obtained; this study established the basic characteristics of those two BR proteins for any further study or development of industry applications.