Studying the photoconversion properties on the photoconvertible protein mEos2 S142E mutant under 405/450 nm irradiation

• Main Authors: Ho, Yu-Ming, 何育明
• Other Authors: Kao, Ya-Ting
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/q38fzd

碩士 === 國立交通大學 === 生物科技學系 === 106 === Green fluorescent protein (GFP) was first discovered from the Aequorea victoria and it has developed more than fifty years. As a powerful tool for biomedical sciences research, GFP exhibits many applications to biotechnology. Among GFP variants, there are some special variants which can be manipulated by light such as photoconvertible fluorescent proteins (PCFPs). PCFPs can be photoconverted when the violet light induced. The mEos2 is one of PCFPs that originally emits green fluorescence and could be converted to red-fluorescence emission under the 405 nm irradiation. We have found from previous studies that the mutant mEos2(S142E) behaves a special optical response under the 450 nm irradiation. Additional absorption and emission bands were detected after 405-nm photoconversion and were further confirmed as a neutral red-form of mEos2 S142E. This neutral red-form of mEos2 S142E could be further converted by 450-nm irradiation and a green emission emerged. Such a new appearing green emission could resulting from either photoconverting to fluorescent-less state or reversibly conversion to the original green form. To further confirm the structure of this new state, we incorporated HPLC/Mass spectrometer into experiments. The results showed that the chromophore’s structure is not similar the original green form but remains the red form structure. Under different oxidation conditions, our experimental results show that the 450-nm conversion rate become slower when the mEos2(S142E) red form is under the oxidizing condition but accelerated when it is in the reducing environments. Therefore, a photo-reduction process might occur upon 450-nm irradiation. However, the mass spectrometry results show that the chromophore of the two different types (before/after 450 nm conversion) is identical. Hence, the 450 nm irradiation does not change the structure of chromophore directly but forms a flurescence-less form. Since this form is highly affected by oxidation environment, 450-nm irradiation might trigger photoreduction reaction in the excited state of red-neutral form. Further experiments by time-resolved spectroscopy will provide more details to confirm this.