光致轉化螢光蛋白mEos2之光轉化動力學機制研究

Abstract

從光致轉化螢光蛋白EosFP改良而得到的光致轉化螢光蛋白mEos2，是個能由綠轉紅的螢光蛋白。在受到波長400 nm左右的光源照射時，mEos2的發色團會發生光轉化讓原本的綠色螢光態轉變成紅色螢光態，使蛋白的螢光放光改變。根據目前對mEos2的研究中，光轉化主要的過程涉及到了胺基酸的鍵與鍵之間的斷裂與發色團本身π電子共振延長這兩步驟，並由於其光轉化的機制是藉由轉化前與後的蛋白質晶體構型來推測中間可能發生的過程，因此激發態的發色團是如何進行光轉化改變發色團結構的以及推測光轉化較為可能的機制就成為本篇研究的主要目的。為了研究mEos2在光轉化過程時的機制，因此本篇研究利用穩態光譜、時間推移光譜與時間相關單光子計數技術，研究外部環境和內在因素於光致轉化螢光蛋白mEos2以及其突變種mEos2 S142E光化學與光物理之光致轉化機制。 外部環境的調控我們藉由改變pH值，從酸性的pH 4.0到鹼性的pH 10。在pH越低的情況下，mEos2的吸收光譜在390 nm處有著較明顯的吸收峰並且會造成發色團506 nm的吸收波峰逐漸下降。而偏鹼性的環境下，390 nm的吸收波峰幾乎消失不見，而506 nm的吸收波峰則會因pH值越高而增加。而螢光放光光譜也有同樣的特性，當環境越酸則螢光放光越弱，環境越鹼則螢光放光會增強。我們認為是pH值改變了發色團中性態與離子態的比例導致吸收光譜與螢光光譜隨pH值改變而變動。我們再透過點突變來進行內部因素探討，將Ser142突變成Glu142並觀察mEos2 S142E在不同pH值下的差異，並發現突變成讓發色團周圍具有較多負電的Glu142會讓發色團較傾向中性態，讓吸收光譜與螢光放光光譜偏向mEos2較酸性時的性質以及能在波長 450 nm照射下激發光譜有可逆的性質。 最後利用時間推移光譜的時相關單光子計數法來對mEos2與其突變種做激發態螢光生命期的量測，我們發現green form的激發態螢光生命期衰減與原本四聚體的EosFP相似約為3.6奈秒，並且生命期並不會受到影響不同pH值的影響，顯示出mEos2的離子態發色團螢光放光並不會受到環境中pH值的干擾而改變其放光的特性。並且發現在光轉化時的激發態螢光生命期會降低，與單純看green form的生命期衰減不同，有著較複雜的生命期衰減過程。

Our target mEos2 is a monomeric protein mutating from EosFP, which was extracted from the coral Lobophyllia hemprichii, and it is a photoconvertible fluorescent protein, under irradiation around 400 nm, mEos2 could change its fluorescent color from green to red. From the previous crystal structures studies, it is proposed that a β-elimination process occurred resulting in breaking the chromophore’s Cα-Cα bound with another residues causing conformational change of chromophore. Therefore, the length of the electron resonance is extended and the absorbance spectrum energy is lowered leading to fluorescent emission red shift led the green to red fluorescent photoconversion. Although the mechanism has been proposed, little is known that what is really happened in the photoconversion process, so our lab use stead state spectrum and time-lapse spectroscopy to study mEos2 to find out the photoconversion process mechanism and dynamics. By changing the pH value from pH 4.0 to 10 and observing the environmental modulation. We found that the absorbance spectrum at 390 nm will rise when pH value is getting low and the 506 nm of chromophore absorbance will descend. On the contrary, 390 nm will almost disappear and 506 nm will rise when pH value getting higher. We believed it is because the pH value controls the neutral form and anionic form ratio of mEos2 chromophore, higher the acidity leading more neutral form chromophore in mEos2 and higher the absorbance of 390 nm. This will also let the fluorescent intensity of mEos2 become weaker. But when alkalinity becomes higher the spectrum and fluorescent intensity of mEos2 will go completely opposite. Mutating Ser142 to Glu142 caused S142E has more neutral form chromophore suggests that electrostatic residues like Glu will stable chromophore and stay in neutral form. Finally, we used the technique of time-correlated single photon counting to investigate the photoconversion process of mEos2 and S142E to find out their excited state fluorescent lifetime. To our observation the excited state fluorescent lifetime of green form mEos2 is around 3.6 ~ 3.8 ns, it is similar to the tetramer EosFP. And changing the pH value won’t influence the excited state fluorescent lifetime of green form mEos2 and S142E. Detecting the excited state fluorescent lifetime while photoconversion process, the lifetime of photoconversion process will decrease a little bit and showing more complicated process.