| Summary: | 碩士 === 國立交通大學 === 生物科技學系 === 105 === 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.
|
|---|
