A genetically encoded far-red fluorescent calcium ion biosensor derived from a biliverdin-binding protein

Authors

Rina Hashizume, Hajime Fujii, Sohum Mehta, Keisuke Ota, Yong Qian, Wenchao Zhu, Mikhail Drobizhev, Yusuke Nasu, Jin Zhang, Haruhiko Bito, Robert E. Campbell

Publication

Protein Science

Abstract

Far-red and near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for in vivo and multiplexed imaging of neural activity and cell signaling. Inspired by a previous report to engineer a far-red fluorescent protein (FP) from a biliverdin (BV)-binding NIR FP, we have developed a far-red fluorescent GECI, designated iBB-GECO1, from a previously reported NIR GECI. iBB-GECO1 exhibits a relatively high molecular brightness, an inverse response to Ca2+ with ?F/Fmin =??13, and a near-optimal dissociation constant (Kd) for Ca2+ of 105?nM. We demonstrate the utility of iBB-GECO1 for four-color multiplexed imaging in MIN6 cells and five-color imaging in HEK293T cells. Like other BV-binding GECIs, iBB-GECO1 did not give robust signals during in vivo imaging of neural activity in mice, but did provide promising results that will guide future engineering efforts.

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