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

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 ΔF/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. Significance Genetically encoded calcium ion (Ca2+) indicators (GECIs) compatible with common far‐red laser lines (~630–640 nm) on commercial microscopes are of critical importance for their widespread application to deep‐tissue multiplexed imaging of neural activity. In this study, we engineered a far‐red excitable fluorescent GECI, designated iBB‐GECO1, that exhibits a range of preferable specifications such as high brightness, large fluorescence response to Ca2+, and compatibility with multiplexed imaging in mammalian cells.

It's a two‐way street: Photoswitching and reversible changes of the protein matrix in photoswitchable fluorescent proteins and bacteriophytochromes

“…In fact, calcium sensors were built by inserting a Calmodulin/RS20 fusion into this site in mIFP [93,183]. Also, a blue‐shifted version (~ 40 nm) of this sensor class has been produced by adding a second cysteine in the binding pocket creating two thioether linkages and thus shortening the π‐electron system [184]. The second entry site is found at the beginning of the α‐helix enclosing the chromophore at the opposite side from the β‐sheet (258, Fig.…”

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…97−99 The most recent NIR Ca 2+ sensor, iBB-GECO1 (from NIR-GECO2), has a good turn-off response to Ca 2+ and an ideal K d , and it was particularly useful for multiplexed imaging in cultured cells with three or four other different color fluorescent proteins. 99 Another series of single fluorescent protein-based sensors was developed using a minimal bacteriophytochrome GAF domain (GAF-FP), but these probes require cells to be supplemented with BV to observe fluorescence. 80,81 The first FRET-based NIR sensor, iGECI, utilizes miRFP670 and miRFP720 and a CaM/M13-containing linker and enabled imaging of Ca 2+ transients deep in the mouse cortex; however, it has a large molecular weight of 86 kDa.…”

“…FRET-based Ca 2+ sensors are less well-developed and not as widely used by comparison with single fluorescent protein-based Ca 2+ sensors . Most FRET-based Ca 2+ sensors utilize CFP and YFP or variants, although some green/red and NIR FRET-based Ca 2+ sensors have been recently developed. − Advanced Ca 2+ sensor designs in general have focused on improving the kinetics of the response to Ca 2+ and developing red and NIR sensors for multiplexing experiments. ,,,,− Some of the fastest Ca 2+ sensors come from the single fluorescent protein-based GCaMP family of sensors. − The latest GCaMPs, the jGCaMP8 series, have achieved ultrafast kinetics and high sensitivity as a result of screening various CaM-binding peptides and using optimized variants with a CaM-binding peptide from endothelial nitric oxide synthase . Another recent GCaMP-based sensor, GCaMP-X, was developed to reduce CaM-mediated toxicity that can occur when CaM-containing sensors like GCaMP are overexpressed. , GCaMP-X includes an additional apoCaM-binding motif at the N-terminus, which effectively prevents the GCaMP CaM domain from interfering with Ca 2+ signaling pathways that rely on CaM.…”

“…Another limitation of green fluorescent Ca 2+ sensors is the limited depth of imaging, which has motivated the development of red and NIR Ca 2+ sensors. ,,,,− ,− The first red fluorescent genetically encoded Ca 2+ sensor was R-GECO, which combines circularly permuted mApple and CaM/RS20 . Engineering R-GECO led to blue- and red-shifted variants, including blue-shifted O-GECO, which has a high dynamic range (41 in HeLa cells) but low affinity ( K d = 1.5 μM), and red-shifted CAR-GECO1 and REX-GECO1, which have dynamic ranges of ∼10–13 in HeLa cells and higher affinities. , The reliance of these sensors and the improved jrGECO1a on mApple, however, limits their utility in combination with blue light-activated optogenetic tools because they can be photoactivated by blue light. , mRuby-based RCaMP Ca 2+ sensors were developed in parallel with the R-GECO series, and while the original variants had relatively low dynamic ranges and weak Ca 2+ affinities, the optimized jRCaMPs have higher affinities and are not photoactivated by blue light. , Useful red and far-red Ca 2+ sensors have also been developed from different fluorescent proteins, including FusionRed (K-GECO1) and mKelly (FR-GECO series), but extending Ca 2+ sensors into the NIR region of the light spectrum has not been possible using GFP-like β-barrel fluorescent proteins. , Genetically encoded NIR Ca 2+ sensors have instead utilized phytochrome-derived biliverdin IXα (BV)-binding fluorescent proteins (Figure C). ,,− Since the first NIR single fluorescent protein-based Ca 2+ sensor (NIR-GECO1) was developed using monomeric infrared fluorescent protein (mIFP), several variants have been engineered. − The most recent NIR Ca 2+ sensor, iBB-GECO1 (from NIR-GECO2), has a good turn-off response to Ca 2+ and an ideal K d , and it was particularly useful for multiplexed imaging in cultured cells with three or four other different color fluorescent proteins . Another series of single fluorescent protein-based sensors was developed using a minimal bacteriophytochrome GAF domain (GAF-FP), but these probes require cells to be supplemented with BV to observe fluorescence. , The first FRET-based NIR sensor, iGECI, utilizes miRFP670 and miRFP720 and a CaM/M13-containing linker and enabled imaging of Ca 2+ transients deep in the mouse cortex; however, it has a large molecular weight of 86 kDa .…”

Zinc-Induced Fluorescence Turn-On in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins

“…Fluorescent protein-based sensors can be genetically encoded into living cells or organisms, allowing for species-specific, cell-specific, or subcellular imaging of metal ions. – Fluorescent proteins can be used as sensors by attaching or embedding a metal binding site in a single fluorescent protein that modulates fluorescence in response to the metal ion. ,, Multifluorescent protein-based probes can also be prepared by employing a FRET approach where two proteins are linked by a metal site that undergoes a conformational change upon metal binding. ,,, Given that most genetically encoded metal sensors are based on GFPs and related mFruits, – here, we sought to evaluate the PEB-binding orange fluorescent All1280g2 protein as a platform for developing a single protein or FRET-based sensor. Most bilin-binding fluorescent protein-based sensor development thus far has focused on designing NIR Ca 2+ sensors using bacteriophytochrome proteins. – Few examples of GAF or other bilin-binding proteins have been used as transition metal ion sensors. ,, In one example, bacteriophytochrome-derived miRFPs can be inherently quenched by Cu 2+ ions with picomolar dissociation constants . In another example, a PEB-bound GAF domain from Spirulina subsalsa (NCBI WP_017306776.1) was reported to be quenched by Cu 2+ ions with an apparent K D of ∼15 μM .…”