Unravel the key residues in converting the fluorescence emission in photoconvertible fluorescence protein mEos2

• Main Authors: Hsu, Chen-Yin, 許甄听
• Other Authors: Kao, Ya-Ting
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/78124031367905218602

碩士 === 國立交通大學 === 生物科技學系 === 104 === 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.