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

Abstract

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.

Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins

“…On the other hand, binding titrations for copper reveal picomolar dissociation constants (13.5–20.5 pM; Table ) for all proteins when fitted to single-site binding models accounting for the presence of 1 mM NTA (Figure and Table ), and the quenching induced by Cu 2+ is much more significant than that observed for Ni 2+ , Co 2+ , and Zn 2+ (Table ). These Cu 2+ affinities are stronger than those reported for GFP, mFruit, and flavin-based fluorescent proteins that are also quenched by Cu 2+ and often measured in the micromolar range. ,,− We initially measured similar micromolar binding affinities for the miRFPs when titrating with Cu 2+ in the absence of a chelator; however, copper has low solubility (∼2 μM) in aqueous buffer at neutral pH. Given that these previous studies measuring Cu 2+ affinities were typically carried out in the absence of a chelator and that we observed a significant difference in K d upon adding a chelator, it is worth reconsidering these values.…”

“…Five milliliters of growth culture was centrifuged, and then the cell pellets were collected. The cell pellets were washed twice with MOPS minimal medium (pH 7.4) , and incubated at 30 °C for 2 to 3 doubling times to reach an OD 600 of 3 to 5. Resuspended cells (200 mL) were added to EDTA (1 mM) in metal-free MOPS buffer (10 mM MOPS and 2.5 mM NaOAc, pH 7.0) in a 1.5 mL metal-free centrifuge tube and incubated at room temperature for 10 min.…”

Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions

“…Several fluorescent proteins, including green fluorescent proteins (GFPs), Discosoma sp. red fluorescent protein (DsRed), and flavin-binding fluorescent proteins (FbFPs), have been investigated for metal-induced fluorescence quenching and have been suggested as optical biosensors for metal quantitation [18][19][20]. However, few biosensors based on biliproteins such as CBCRs with intense fluorescence emission are available for the detection of metal ions.…”

Characterization of flavin binding in oxygen‐independent fluorescent reporters

“…19 We have also discovered brighter LOV reporters from algae by implementing a genome mining-based approach, 20 which was recently extended to identify a new and highly thermostable LOV reporter from thermophilic bacteria. 21 Taken together with additional benefits such as small size (~12 kDa), acid tolerance, 22 and metal-responsive fluorescence, [23][24][25] fluorescence imaging to several anaerobes, including gut bacteria, oral flora, parasitic protists, and pathogenic fungi. [26][27][28][29][30][31][32] As the fluorescence properties of LOV reporters are determined by protein-bound FMN, it would be useful to develop a method for separating FMN from the protein and reconstituting the apo protein with FMN (or other flavins) in order to characterize the equilibrium dissociation constant (that is, K d ), specificity, reaction kinetics, binding energetics, and related thermodynamic properties.…”

“…We have also discovered brighter LOV reporters from algae by implementing a genome mining‐based approach, 20 which was recently extended to identify a new and highly thermostable LOV reporter from thermophilic bacteria 21 . Taken together with additional benefits such as small size (~12 kDa), acid tolerance, 22 and metal‐responsive fluorescence, 23‐25 LOV reporters are making it possible to extend fluorescence imaging to several anaerobes, including gut bacteria, oral flora, parasitic protists, and pathogenic fungi 26‐32 …”