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.

Mutant Flavin-Based Fluorescent Protein Sensors for Detecting Intracellular Zinc and Copper in Escherichia coli.

Flavin-based fluorescent proteins (FbFPs) are a class of fluorescent reporters that undergo oxygen-independent fluorophore incorporation, which is an important advantage over green fluorescent proteins (GFPs) and mFruits. A FbFP derived from Chlamydomonas reinhardtii (CreiLOV) is a promising platform for designing new metal sensors. Some FbFPs are intrinsically quenched by metal ions, but the question of where metals bind and how to tune metal affinity has not been addressed. We used site-directed mutagenesis of CreiLOV to probe a hypothesized copper(II) binding site that led to fluorescence quenching. Most mutations changed the fluorescence quenching level, supporting the proposed site. One key mutation introducing a second cysteine residue in place of asparagine (CreiLOVN41C) significantly altered metal affinity and selectivity, yielding a zinc sensor. The fluorescence intensity and lifetime of CreiLOVN41C were reversibly quenched by Zn2+ ions with a biologically relevant affinity (apparent dissociation constant, Kd, of 1 nM). Copper quenching of CreiLOVN41C was retained but with several orders of magnitude higher affinity than CreiLOV (Kd = 0.066 fM for Cu2+, 5.4 fM for Cu+) and partial reversibility. We also show that CreiLOVN41C is an excellent intensity- and lifetime-based zinc sensor in aerobic and anaerobic live bacterial cells. Zn2+-induced fluorescence quenching is reversible over several cycles in Escherichia coli cell suspensions and can be imaged by fluorescence microscopy. CreiLOVN41C is a novel oxygen-independent metal sensor that significantly expands the current fluorescent protein-based toolbox of metal sensors and will allow for studies of anaerobic and low oxygen systems previously precluded by the use of oxygen-dependent GFPs.

Direct Synthesis of Partially Chain-Straightened Propylene Oligomers and P-MA Co-Oligomers Using Axially Flexible Shielded Iminopyridyl Palladium Complexes.

In this study, a series of partially chain-straightened propylene oligomers and functional propylene−methyl acrylate (P-MA) co-oligomers were synthesized with 8-alkyl-iminopyridyl Pd(II) catalysts. The molecular weight and polar monomer incorporation ratio could be tuned by using Pd(II) catalysts with various 8-alkyl-naphthyl substituents (8-alkyl: H, Me, and n-Bu). In propylene oligomerization, all the 8-alkyl-iminopyridyl Pd(II) catalysts convert propylene to partially chain-straightened (119−136/1000 C) oligomers with low molecular weights (0.3−1.5 kg/mol). Among the catalysts, Pd1 with non-substituent (H) on the ligand showed the highest activity of 5.4 × 104 g/((mol of Pd) h), generating oligomers with the lowest molecular weight (Mn: 0.3 kg/mol). Moreover, polar-functionalized propylene-MA co-oligomers with very high incorporation ratios (22.8−36.5 mol %) could be obtained in the copolymerization using these 8-alkyl-iminopyridyl Pd(II) catalysts. Additionally, Pd1 exhibited the best performance in propylene-MA copolymerization as it displayed the highest MA incorporation ratio of up to 36.5 mol%. All the three catalysts are capable of generating partially chain-straightened P-MA co-oligomers and the activities decrease gradually while the molecular weight increases with the increasing steric hindrance of the alkyl substituent (H < Me < n-Bu). Compared to Pd4 with the rigid 8-aryl substituent, the flexible 8-alkyl-iminopyridyl Pd(II) catalysts (Pd1-3) not only showed much higher activities in the propylene oligomerization, but also yielded P-MA co-oligomers with significantly higher incorporation ratios in the propylene co-oligomerization.

Live-Cell Copper-Induced Fluorescence Quenching of the Flavin-Binding Fluorescent Protein CreiLOV.

CreiLOV is a flavin-binding fluorescent protein derived from the blue-light photoreceptor protein family that contains light-oxygen-voltage (LOV) sensing domains. Flavin-binding fluorescent proteins represent a promising foundation for new fluorescent reporters and biosensors that can address limitations of the well-established green fluorescent protein (GFP) family. Flavin-binding fluorescent proteins are smaller than GFPs, are stable over a wider pH range, offer rapid chromophore incorporation, and are oxygen-independent so can be applied to live anaerobic organisms. Among the flavin-binding fluorescent proteins, CreiLOV has a high quantum yield and excellent photophysical properties, making it promising for cellular applications. Here, we investigated the suitability of CreiLOV as an intensity- and fluorescence-lifetime-based metal sensor. CreiLOV selectively binds copper(II) over other biologically relevant metals with low-micromolar affinity, resulting in fluorescence quenching and a decrease in the fluorescence lifetime that can be observed in cuvettes and live bacterial cells.