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Review
. 2022 Jun 10;12(6):400.
doi: 10.3390/bios12060400.

Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing

Affiliations
Review

Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing

Vadim R Viviani et al. Biosensors (Basel). .

Abstract

Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical purposes for over 5 decades. Under acidic conditions, high temperatures and the presence of heavy metals, they produce red light, a property that is called pH-sensitivity or pH-dependency. Despite the demand for physiological intracellular biosensors for pH and heavy metals, firefly luciferase pH and metal sensitivities were considered drawbacks in analytical assays. We first demonstrated that firefly luciferases and their pH and metal sensitivities can be harnessed to estimate intracellular pH variations and toxic metal concentrations through ratiometric analysis. Using Macrolampis sp2 firefly luciferase, the intracellular pH could be ratiometrically estimated in bacteria and then in mammalian cells. The luciferases of Macrolampis sp2 and Cratomorphus distinctus fireflies were also harnessed to ratiometrically estimate zinc, mercury and other toxic metal concentrations in the micromolar range. The temperature was also ratiometrically estimated using firefly luciferases. The identification and engineering of metal-binding sites have allowed the development of novel luciferases that are more specific to certain metals. The luciferase of the Amydetes viviani firefly was selected for its special sensitivity to cadmium and mercury, and for its stability at higher temperatures. These color-tuning luciferases can potentially be used with smartphones for hands-on field analysis of water contamination and biochemistry teaching assays. Thus, firefly luciferases are novel color-tuning sensors for intracellular pH and toxic metals. Furthermore, a single luciferase gene is potentially useful as a dual bioluminescent reporter to simultaneously report intracellular ATP and/or luciferase concentrations luminometrically, and pH or metal concentrations ratiometrically, providing a useful tool for real-time imaging of intracellular dynamics and stress.

Keywords: bioimaging; bioluminescence; cadmium; heavy metals; mercury; pH indication; ratiometric biosensors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The Macrolampis sp2 firefly-induced bioluminescence display time-dependent color change after stimulation by anesthesia/adrenalin: (A) in the beginning just after adrenalin injection the color is yellow-green; (B) after 5 min, a subtle color change to yellow-orange is observable. Continuous glow stimulation may induce lantern acidification, causing the color change.
Figure 2
Figure 2
Effect of pH on bioluminescence spectra of four firefly luciferases displaying distinct pH sensitivities. Reprinted with permission from ref. [29]. Copyright 2011 Royal Society of Chemistry. (a) Amydetes viviani; (b) Cratomorphus distinctus; (c) Photinus pyralis, and (d) Macrolampis sp2. (A) pH 6.5; (B) pH 7.0; (C) pH 7.5, and (D) pH 8.0.
Figure 3
Figure 3
The residue E354 is important for luciferase sensitivity to Zinc. Effect of 2 mM zinc on the bioluminescence spectra of: (A) Macrolampis sp2 wild-type; (B) C. distinctus wild-type; (C) C. distinctus E354N, and (D) Macrolampis sp2 N354E mutant (black line) without zinc, and (gray lines) in the presence of zinc. Reprinted with permission from ref. [33]. Copyright 2016 Springer.
Figure 4
Figure 4
Structure of the metal-binding site in Amydetes viviani firefly luciferase: (A) Zoom showing the metal-binding site residues (yellow: H310; green: E311; magenta: E354) and oxyluciferin phenolate binding site (deep blue); (B) Metal-binding site showing zinc in red, being coordinated by the side chains of H310, E311 and E354. Reprinted from ref. [31].
Figure 5
Figure 5
The proposed mechanism of pH and metal sensitivity in firefly luciferases involves the oxyluciferin phenol/phenolate group excited state proton transfer and electrostatic interactions between residues E311 and R337, and H310 and E354, which keep the active site closed. Whereas the keto form of excited oxyluciferin was considered the most likely emitter in this figure for simplicity, the process of keto-enol tautomerization in bioluminescence color determination can not be ruled out. Reprinted from ref. [31].
Figure 6
Figure 6
(Left graph) ratiometric analysis of pH in bacteria using: (A) Macrolampis and (B) Cratomorphus firefly luciferases; (right image) effect of pH on in vivo bioluminescence of E. coli colonies expressing Macrolampis and Cratomorphus firefly luciferases; (t0–t3) represent the time (minutes) when the bioluminescence image was taken after addition of acidic D-luciferin. Reprinted with permission from ref. [32]. Copyright 2014 Royal Society of Chemistry.
Figure 7
Figure 7
Bioluminescence spectra in COS-1 cells transfected with Macrolampis firefly luciferase carrying vector, pCMV-Mac, and ratiometric curves. Effect of pH in cells transfected with: (A) pCMV-Mac;cytoplasm; (B) pCMV-Mac-Nucleus; (C) pCMV-Mac with calibration buffer containing nigericin at different pHs (pH 6.0, 6.5, 7.0, 7.5 and 8.0); (D) ratiometric analysis between R (Igreen/Ired) and pH. Reprinted with permission from ref. [34]. Copyright 2019 Royal Society of Chemistry.
Figure 8
Figure 8
Firefly luciferase color reporting of intracellular pH change in mammalian cells. In vivo bioluminescence color change of COS-1 cells transfected with pCMV-Mac expressing Macrolampis firefly luciferase at different pH in calibration buffer containing nigericin. Reprinted with permission from ref. [34]. Copyright 2019 Royal Society of Chemistry. (left panels: ad) luciferase directed to cytoplasm; (right panel: eh) luciferase directed to nucleus; (a,e) pH 6.5; (b,f) pH 7.0; (c,g) pH 7.5; and (d,h) pH 8.0. The reddish bioluminescence indicates acidic pH, whereas the orange-yellow indicates more alkaline pH.
Figure 9
Figure 9
Comparison of the effect of temperature on the ratio of bioluminescence color of beetle luciferases: (left graph) ratiometric estimation of temperature using distinct firefly luciferases. (red) Macrolampis sp2; (black) Photinus pyralis; (green) Cratomorphus distinctus, and (blue) Amydetes viviani; (right images) in vivo bioluminescence color of E.coli colonies expressing Macrolampis sp2 (Mac-Luc) and Amydetes viviani (Amy-Luc) luciferases, after spraying D-luciferin, at 22 and 40 °C. Reprinted with permission from ref. [85]. Copyright 2014 Royal Society of Chemistry. One can see that the luciferases with steeper curves such as Macrolampis luciferase (red) are the most sensitive to temperature, and those with less steep curves, such as Amydetes viviani luciferase (blue), are the least sensitive.
Figure 10
Figure 10
Bioluminescence spectra of Macrolampis sp2 firefly luciferase and its mutants showing spectral change at different concentrations of ZnSO4: (A) Wild-type; (B) N354H; (C) H310C; (D) N354C; (E) H310C/N315C; Reprinted with permission from ref. [33]. Copyright 2016 Springer.
Figure 11
Figure 11
Use of Amydetes viviani firefly luciferase as cadmium- and mercury-selective luciferase: (A,B) bioluminescence spectra in presence of mercury and cadmium; (C,D) bioluminescence activity in presence of mercury and cadmium; (E,F) ratio of luminescence intensities in green and red regions in presence of mercury and cadmium. Reprinted with permission from ref. [84]. Copyright 2019 National Library of Medicine.
Figure 12
Figure 12
Smartphone detection of bioluminescence color tuning by cadmium, using Amydetes viviani firefly luciferase.

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