光致轉換螢光蛋白Dronpa與其變異種之光化學與光物理研究

Abstract

光轉換螢光蛋白Dronpa隸屬於光控螢光蛋白家族，能藉由照射特定波長的光而改變原本的光譜性質。Dronpa藉著光引發的構型重排於釋放強烈螢光的螢光態與不放光的非螢光態間進行往返，螢光態照射480nm光源後將轉換成非螢光態且發色團轉換為反式-質子化型;改以405nm光源激發非螢光態則會再轉變為含有順式-去子化型發色團的螢光態。根據其他的研究顯示，Dronpa的光轉換過程以光致異構化作用做為驅動力，而非早期認定的質子轉移行為。然而超快吸收光譜學與結晶學無法完整描整描述光轉換過程中的所有機制，有關Dronpa的光可逆調控機制仍待研究。 為了更加了解Dronpa受光激發時的光調控過程，本篇論文將使用穩態光譜、時間推延光譜與時間相關單光子計數技術探討調變發色團內外環境時，如何影響Dronpa與變異種的光物理以及光化學轉換過程。結果顯示調變外在環境酸鹼值時，Dronpa於pH = 5以上時仍有效的展示光轉換特性，而pH = 4的環境下改以時間推延螢光光譜也發現極緩慢的光轉換過程。經由時間相關單光子計數技術量測Dronpa的激發態螢光壽命時，其結果顯示所有酸鹼值下的螢光態Dronpa之螢光壽命約為3.3～3.5奈秒，我們推測外環境質子不與激發態當中的螢光態在進行異構化作用時相互競爭而影響其光轉換行為。實驗中取得的變異種S142A、S142Y、H193E在螢光光譜中顯示非螢光態發色團之放光能力獲得提升並於時間推延螢光光譜中量測到各自的光轉換過程。時間相關單光子計數技術的結果顯示，變異種S142A、S142Y、H193E、H193R之螢光態生命週期分別為2.5奈秒、3奈秒、2.2奈秒、2.7奈秒。根據我們的研究推測，突變種的螢光態發色團結構將呈現擾動與彈性增加，且發色團更難與第193號胺基酸維持共平面狀態並導致較不穩定的激發態傾向以非輻射性過程耗損能量，最終所有變異種的螢光生命週期呈現縮減。

Photoswichable fluorescence protein Dronpa, as an Optical Highlighter, has unique photochromic behavior that can change its spectral properties after excited by specific irradiations. The light-driven rearrangement in Dronpa is a switching process between a strong emission bright state and a weak emission dark state. Irradiating the bright state with 480 nm blue light covers it to the dark state leading to the trans-protonated chromophore. Moreover, the dark state can be turn back to the previous state by 405 nm light excitation and the chromophore becomes the cis-deprotonated form at the same time. According to other researches, the driven force of photoswitching process in Dronpa might depends on isomerization but not the proton transfer. However, previous studies by ultra-fast absorption spectra and crystallography did provide sufficient information about the detail mechanism of photoswitching processes. Hence, more investigations are indispensable to unravel the photoswitching mechanism of Dronpa. We use steady state spectra, time-lapse spectra and time-correlated single photon counting (TCSPC) to study the photophysical and photochemical behavior of the Dronpa and its variants when the external or internal environments are modulated. By changing the external conditions of pH value, our observations show that the Dronpa display normal switching capability under pH value of 5.0 and more. However, a relatively slow switching process in pH 4.0 conditions is also detected by fluorescence time-lapse spectra. Interestingly, the fluorescence lifetime of bright state Dronpa in various pH conditions exhibits similar results of 3.3 ~ 3.5 nanoseconds. We propose that the protons in environment are not competitive with isomerization process in excited bright state but effect the Dronpa photoswitching behavior.

The fluorescence spectra of Dronpa mutations (S142A, S142Y and H193E) show the fluorescent enhancement in dark form and the time-lapse studies display all mutants with the photoswitching capabilities. According to the results from TCSPC, S142A, S142Y, H193E and H193R exhibit different bright state lifetime of 2.5, 3.0, 2.2 and 2.7 nanoseconds, respectively. We propose that the mutations on 142 and 193 sites in our studies cause structural perturbation that weaken the stabilization of bright state chromophore and increase the local structure flexibility. It hinders the chromophore maintaining structural coplanarity to histidine 193 and leads to a less stable excited state resulting in dominant non-radiative processes.