光控螢光蛋白PSmOrange與PSmOrange2之光物理與光化學特性研究

Abstract

PSmOrange是由紅螢光蛋白DsRed改良而來，具有光致轉化特性的螢光蛋白，其能通過照射藍綠光使得螢光基團結構發生改變，進而放出波長更長的紅色螢光。由先前的文獻中，我們知道當PSmOrange照射藍綠光後，螢光基團與其連結的胺基酸上的主鏈會斷裂，使得原本的共軛雙鍵結構延長，但過程的反應機制目前還不得而知。 在本篇論文當中，我們利用穩態光譜與時間解析光譜，並調控外部環境和內在因素，來研究紅螢光蛋白PSmOramge以及其變異種PSmOrange2 (以下簡稱PS1及PS2) 之光化學與光物理光學特性。探討改變外部環境影響時，我們將紅螢光蛋白置於不同pH值和黏滯度的溶液中，來進行測試。我們發現紅螢光蛋的光譜特性對於pH值有高度的敏感度。由於其在不同溶液酸鹼性下，展現的光譜特性的不同，我們可以運用紅螢光蛋白PS1以及PS2作為測量生物體內pH值的指示劑，進而從標記在細胞內的螢光蛋白性質改變，可以得知周遭環境pH值的改變。當我們從內在因素探討時，我們比較紅螢光蛋白PS1以及其變異種PS2，於不同環境下的特性，探討特定胺基酸的結構如何影響螢光基團。從我們的實驗數據中可以看到，PS1及PS2皆在約560 nm左右的波長有一個主放光波段，在507 nm的位置有一個次放光波段，兩者主、次放光波段的比例有所不同，PS2的次放光波段強度較高，但兩者的次放光波段強度皆會因環境的pH值降低以及在氧化劑環境下照射藍綠光後而大幅下降。我們推論發光基團於紅螢光蛋白中有不同的結構，507 nm波段放光為共振較短的結構，而560 nm左右之波段為共振較長的結構。 由蛋白質晶體的立體結構圖來看，PS1及PS2的6個突變位點 (PS1/R17H/R36H/F65I/Q188L/A217S/G219A/PS2) 皆與螢光基團在立體位置上相隔一段距離，值得注意的是，PS2的S217會與E215形成以氫鍵鍵結及不鍵結兩種不同的立體構形，若S217與E215彼此以氫鍵鍵結 (2.6 Å)，則E215另一側的羥基會與螢光基團形成氫鍵 (3.1 Å)，由於在PS1中217的位置為Ala，使得E215由於距離過遠而無法與螢光基團形成氫鍵。我們推測兩構形的平衡影響了PS1及PS2在主、次放光波段的比例，也穩定了PS2在507 nm的放光結構，使得507 nm放光的螢光壽命相較於PS1來得更長。 當PS1及PS2在氧化劑環境下照光之後，507 nm的放光波段大幅下降，由此可知在加入氧化劑照光後兩結構的氧化還原平衡受到影響，因此我們推測560 nm放光與507 nm放光結構互為氧化還原態。而在不同pH值環境下，PS1及PS2會由於不放光的neutral form與放光的anionic form的結構平衡有不同的傾向，而導致螢光強度的消長。在鹼性環境下，溶液中放光的anionic form比例較高，使得560 nm放光以及507 nm放光強度皆上升；在酸性環境下，溶液中不放光的neutral form比例較高使得560 nm放光以及507 nm放光強度皆下降。

PSmOrange is a phototransformable fluorescence protein which derivative from DsRed. Its conformation could be photoconverted with violet light which alters its emission from red fluorescence to far-red fluorescence. In previous literatures, it is proposed that with violet light irradiation, PSmOrange exhibits an extra double-bond structure. However, the detail mechanism is still unknown. In our proposed study, we utilize steady-state microscopy and time correlated single photon counting to study photophysical and photochemical properties of PSmOrange (PS1) and its variant PSmOrange2 (PS2) by changing the pH value and the percetage of glycerol in buffer. Our observations show that the fluorescence intensity of both PS1 and PS2s are sensitive to the change of pH value. Due to its difference of spectral properties of different pH value, we could further utilize PS1 and PS2 as intracellular pH-sensitive indicators. We observed that PS1 and PS2 both have a major emission light at 560 nm and a minor emission light at 507 nm. 507 nm emission intensity substantially decrease in lower pH scale or irradiation with violet light in the presence of oxidants. We propose that two different chromophore structures are existing. According to the crystals structure, PS2 differs from PS1 by six points mutations, only two of which, F65I and A217S, are located in proximity to the chromophore. Notably, S217 in PSmOrange2, forms a strong H-bond with E215 and non-bonding, adopt two conformations. In bonding conformation, S217 and E215 form a strong H-bond (2.6 Å) with its side chain and push E215 closer to the chromophore, facilitating H-bonding between E215 and nitrogen of imidazolinone ring (3.1 Å) that connects it with the protein matrix. In PS1, Ala is in position 217, as a result, E215 moves away from the chromophore forming no H-bond in between these two residues. We speculate the balance of two conformations not only affect the ratio between major and minor emission light, but also stabilize the 507 nm chromophore structure resulting in a longer 507 nm fluorescence lifetime in PS2. After irradiation with violet light in the presence of oxidants, 507 nm emission intensity of PSmOrange and PSmOrange2 substantially decrease. We think that there is an equilibrium between 560 nm chromophore structure and 507 nm chromophore structure and they are in an oxidation-reduction relation (redox). Furthermore, in the different pH scale environment, due to the different tendency of equilibrium of non-luminescent neutral form and luminescent anionic form, making the fluorescence intensity different. In the base environment, equilibrium tend to the luminescent anionic form, making emission intensity of 560 nm and 507 nm increase. On the contrary, in the acid environment, equilibrium tend to the non-luminescent neutral form, making emission intensity of 560 nm and 507 nm decrease.