以核磁共振研究細菌視紫質之暗與光適應態之結構差異

Abstract

膜蛋白細菌視紫質在吸收綠光之後，產生一系列構形變化，將氫離子運輸至細胞膜外，並在細胞膜內外產生氫離子梯度，最終轉換為生物可利用能量三磷酸腺苷。膜蛋白的研究雖然重要，但是因為樣品製備等問題，造成研究上的困難。在降低了純化用緩衝溶液的酸鹼度以及加入了離子交換層析法之後，不只提升了純化步驟的產率，更使單體與三聚體的分離更有效率。我們更發現單體以及三聚體的比例可以透過人為的方式進行調控。經過介面活性劑與緩衝溶液種類的篩選，我們得到不錯的氫氮二維異核化學位移相關光譜。我們測量了樣品亮適應態、激發態的動力學數據以及光強度對亮適應的影響。將以上數據與四條光纖以及兩根核磁共振樣品管結合後，我們在原位液態核磁共振實驗中觀察到了亮適應態與暗適應態的差異。

A proton pump that builds up a proton gradient across a biological membrane is a membrane protein. A typical example is Haloarcula marismortui bacteriorhodopsin (HmbRI) that achieves its functionality as a proton pump by changing its conformation in the presence of green light. The study of the structural changes in membrane protein is crucial but difficult because of the problems such as sample preparation. In this thesis, we report that the purification of monomeric D94N HmbRI was improved by lowering the pH of the purification buffers and including an additional step of ionic-exchange chromatography, which can successfully and efficiently separate the monomeric and trimeric form of D94N HmbRI. Moreover, we found that the ratio between monomer and trimer can be manually controlled. With extensive detergent and buffer screening, we were able to obtain the 1H-15N TROSY HSQC spectrum of D94N HmbRI with good quality. By incorporating four optical fibers into a double layer NMR tube, the in-situ light coupling solution state NMR measurements were conducted. The lifetime of the light-adapted state and the M-state decay of D94N HmbRI was measured. The correlation between light intensity and the duration of light adaptation was further investigated. Using this device, the differences between the 1H-15N TROSY HSQC spectra for the dark-adapted state and the light-adapted state of D94N HmbRI were observed.