To monitor the calcium dynamics in cells, various genetically encoded calcium indicators (GECIs) based on FRET between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, the authors developed a GECI with 1.5-times higher dynamic range, Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the calcium dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.
Tomoki Matsuda, Shinya Sakai, Kei-Ichi Okazaki, Takeharu Nagai (2024) Improvement of the Green-Red Forster Resonance Energy Transfer-Based Ca(2+) Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor., ACS sensors
Published in 2024 Mar 21 (Electronic publication in March 21, 2024, midnight )
(Abstract) To monitor the Ca(2+) dynamics in cells, various genetically encoded Ca(2+) indicators (GECIs) based on Forster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca(2+) dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.