螢光蛋白mEos2與其突變種之光致轉化研究

Abstract

綠色螢光蛋白（GFP）是一種會放出綠色螢光的螢光蛋白，其在生物相關的研究上常被用來標記其它種類之蛋白或是目標物，現今GFP已被改造出多種變異種，而在眾多變異種中有部分螢光蛋白在接受特定波長照射後會出現螢光特性改變的現象，故稱之為光控螢光蛋白，其根據發色團放光機制的不同可分為三類：光致活化螢光蛋白、光致轉化螢光蛋白與光致變色螢光蛋白。本實驗中所使用的螢光蛋白──mEos2是由EosFP突變而得，屬於光致轉化螢光蛋白，可被400 nm波長的光作光轉換，其放出之螢光會由綠色轉為紅色。mEos2綠螢光放光型態受到波長505 nm的光激發後會放出主波峰為515 nm的綠螢光，紅螢光放光型態受到波長570 nm的光激發後會放出主波峰為584 nm的紅螢光。對於光轉換反應機制，藉由轉換前後的蛋白質晶體結構，推測發色團會產生ESPT且進行發色團主鏈上β-elimination使共軛雙鍵變長，進而使得螢光放光紅移，但目前尚無直接反應動態實驗直接證明，因此仍待真相之探討。 本實驗將mEos2做外在環境與內在環境之調控，欲透過環境變化來觀察其光化學與光物理之差異，藉以找出影響mEos2光轉換之關鍵殘基。外在環境調控之變因為黏滯度與pH值，我們利用甘油濃度不同來調整黏滯度；內在環境調變則是進行mEos2單點突變。因單點突變之mEos2 Q38R、mEos2 Q38W、mEos2 T59R與mEos2 L210Y在可見光區沒有明顯之螢光變化，故我們推斷此四種突變種可能是其發色團未成熟所致。 根據實驗結果，mEos2 wildtype與mEos2 Q38E之光譜波形較為相似，但mEos2 Q38E光轉換效率不彰；mEos2 S142E光譜則有些許差異。在80%甘油濃度環境下，大體而言三者都會有較快的光轉換速率；mEos2 wildtype與mEos2 Q38E、mEos2 S142E在pH6環境下亦有較快之光轉換速率。因此當環境中黏滯度愈高、pH值愈低時會有較快之光轉換速率。此外，mEos2 S142E可被405 nm光轉換，亦可被450 nm光轉換。由實驗結果之觀察，我們推測450 nm之光轉換或許可將其從紅螢光放光型態轉換成為綠螢光放光型態。

Photoconvertible fluorescent proteins (PCFPs) are classified into an unique family of fluorescent proteins owing to their special light responses. When exposed with irradiation of a certain wavelength, the chromophores of PCFPs can be optically converted from one fluorescence color to another and such conversion processes is irreversible. mEos2 is a monomeric protein mutating from EosFP, which was extracted from the coral Lobophyllia hemprichii found in the Indo-Pacific Ocean, is a green-to-red PCFP. The monomeric Eos2 (mEos2) exhibits high quantum yield in both its green and red forms (0.88 and 0.66, respectively). Although the mechanism has been proposed, little is known on which residues play essential role on photoconversion process. In this research, we aim to figure out the effects of environmental modulation and conformational perturbation in converting the fluorescence emission in mEos2. We vary the viscosity and pH value of the buffer solution as two environmental modulation. We also mutate mEos2 into mEos2 T59R, mEos2 Q38R, mEos2 Q38E, mEos2 Q38W, mEos2 S142E and mEos2 L210Y as conformational perturbation, including mEos2 T59R, mEos2 Q38R, mEos2 Q38E, mEos2 Q38W, mEos2 S142E and mEos2 L210Y. However, only two of them exhibit absorption and fluorescence emission in the visible range. We suggest that the chromophore of mEos2 T59R, mEos2 Q38R, mEos2 Q38W and mEos2 L210Y did not mature. Therefore we carried out photoconversion investigation on mEos2 wildtype, mEos2 Q38E and mEos2 S142E. We used the solutions of 20%, 40%, 60% and 80% glycerol and we observed that mEos2 in the 80%-glycerol solution was converted most rapidly. We also changed the pH value of the solution from 8.0 to 6.0 and 10.0. mEos2 in the pH 6.0 buffer solution has the fastest conversion rate. In contrast, mEos2 in the pH 10.0 buffer solution has the slowest conversion rate. These results can also be discovered in mEos2 Q38E and mEos2 S142E photoconversion process. In a word, our observations show that the lower pH value and the higher viscosity environments, the faster green-to-red conversion rate. Surprisingly, we observed an additional photoconvertible form in mEos2 S142 after 405 nm photoconversion. Additional absorption and emission bands was detected after 405-nm photoconversion and was further confirmed as a neutral red-form of mEos2 S142E. This photoconverted form could be further converted by 450-nm irradiation and a green emission emerged. Such a new appearing green emission could resulting from either further oxidation of chromophore or reversibly conversion to the original green form. Similar results were also observed in mEos2 wildtype under strong acidic conditions of pH 4.