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

Genetically encoded fluorescent metal ion sensors are powerful tools for elucidating metal dynamics in living systems. Over the last 25 years since the first examples of genetically encoded fluorescent protein-based calcium indicators, this toolbox of probes has expanded to include other essential and non-essential metal ions. Collectively, these tools have illuminated fundamental aspects of metal homeostasis and trafficking that are crucial to fields ranging from neurobiology to human nutrition. Despite these advances, much of the application of metal ion sensors remains limited to mammalian cells and tissues and a limited number of essential metals. Applications beyond mammalian systems and in vivo applications in living organisms have primarily used genetically encoded calcium ion sensors. The aim of this Perspective is to provide, with the support of historical and recent literature, an updated and critical view of the design and use of fluorescent protein-based sensors for detecting essential metal ions in various organisms. We highlight the historical progress and achievements with calcium sensors and discuss more recent advances and opportunities for the detection of other essential metal ions. We also discuss outstanding challenges in the field and directions for future studies, including detecting a wider variety of metal ions, developing and implementing a broader spectral range of sensors for multiplexing experiments, and applying sensors to a wider range of single- and multi-species biological systems.

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

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 µM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.

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

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. GAF2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that, unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent GAF2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here we show that GAF2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn 2+ significantly enhances GAF2-PEB fluorescence across a biologically relevant pH range from 6.0-9.0 and with pH-dependent µM to nM dissociation constants. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement, respectively. Mutation of the cysteine residue within the DXCF motif to alanine abolishes zinc-induced fluorescence enhancement. Collectively, these results support the presence of a fluorescence enhancing Zn 2+ binding site in GAF2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.

A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.

A Single Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.

Cyanobacteriochrome (CBCR) GAF domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, becoming fluorescent. The third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803 binds phycocyanobilin (PCB) natively, yielding red/green photoswitching properties but also binds phycoerythrobilin (PEB). GAF3-PCB has low quantum yields but non-photoswitching GAF3-PEB is brighter, making it a promising platform for new genetically encoded fluorescent tools. GAF3, however, shows low PEB binding efficiency (chromophorylation) at ∼3% compared to total protein expressed in E. coli . Here we explored site-directed mutagenesis and plasmid-based methods to improve GAF3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. We found that a single mutation improved chromophorylation while tuning the emission over ∼30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised by ∼7-fold. Moreover, we show that protein-chromophore interactions can tune autoisomerization of PEB to PUB in a GAF domain, which will facilitate future engineering of similar GAF domain-derived fluorescent proteins.