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Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (20 December 2020) | Viewed by 76981

Special Issue Editor

Federal Research Center, Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia
Interests: bioluminescent and fluorescent proteins; molecular mechanism of bioluminescence; structure of bioluminescent proteins; bioluminescent analysis and imaging; BRET technologies
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Bioluminescence, a natural phenomenon of visible light emission by living organisms, has been found in numerous terrestrial and marine organisms. The study of bioluminescent reactions in organisms has revealed a specific feature—the mechanisms underlying light emission considerably differ in species of different taxa. Both the substrates and cofactors involved in bioluminescence reactions as well as the enzymes catalyzing these reactions have turned out to be peculiar. In addition to luciferases, many bioluminescence systems contain “antenna” proteins, such as Green fluorescent protein (GFP), which e.g., modulate the bioluminescence color by means of energy transfer. Although investigation of bioluminescence and fluorescence proteins is undoubtedly of fundamental interest, the main driving force that has attracted researchers to this topic is the broad analytical potential of bioluminescent proteins. Currently, there are no fields of biology or medicine where bioluminescence could not be widely used. This Special Issue of the International Journal of Molecular Science is devoted to experimental and theoretical studies on functioning mechanisms of bioluminescent proteins from various organisms, the proteins involved in bioluminescence (the lumazine protein, GFP and GPF-like proteins, and the enzymes participating in the synthesis of substrates), novel assays with the use of these proteins, and applications of these proteins in experimental biology and medicine. We invite authors to present their latest research in related fields.

Dr. Eugene S. Vysotski
Guest Editor

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Keywords

  • bioluminescence
  • luciferase
  • photoprotein
  • luciferin
  • coelenterazine
  • GFP
  • GFP-like proteins
  • immuno and hybridization assays
  • BRET and FRET assays
  • imaging

Published Papers (21 papers)

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19 pages, 26858 KiB  
Article
Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction
by Albert E. Lisitsa, Lev A. Sukovatyi, Sergey I. Bartsev, Anna A. Deeva, Valentina A. Kratasyuk and Elena V. Nemtseva
Int. J. Mol. Sci. 2021, 22(16), 8827; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168827 - 17 Aug 2021
Cited by 4 | Viewed by 1927
Abstract
Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was [...] Read more.
Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial bioluminescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. Moreover, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis. Full article
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19 pages, 3522 KiB  
Article
Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching
by Joanna Krasowska, Katarzyna Pierzchała, Agnieszka Bzowska, László Forró, Andrzej Sienkiewicz and Beata Wielgus-Kutrowska
Int. J. Mol. Sci. 2021, 22(16), 8565; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168565 - 09 Aug 2021
Cited by 4 | Viewed by 2928
Abstract
Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging [...] Read more.
Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO2, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers. Full article
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11 pages, 1995 KiB  
Article
Microfabrication of a Chamber for High-Resolution, In Situ Imaging of the Whole Root for Plant–Microbe Interactions
by Lauren K. Jabusch, Peter W. Kim, Dawn Chiniquy, Zhiying Zhao, Bing Wang, Benjamin Bowen, Ashley J. Kang, Yasuo Yoshikuni, Adam M. Deutschbauer, Anup K. Singh and Trent R. Northen
Int. J. Mol. Sci. 2021, 22(15), 7880; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22157880 - 23 Jul 2021
Cited by 6 | Viewed by 2743
Abstract
Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe [...] Read more.
Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe a new ‘Imaging EcoFAB’ and the use of this device to image the entire root system of growing Brachypodium distachyon at high resolutions (20×, 40×) over a 3-week period. The device is capable of investigating root–microbe interactions of multimember communities. We examined nine strains of Pseudomonas simiae with different fluorescent constructs to B. distachyon and individual cells on root hairs were visible. Succession in the rhizosphere using two different strains of P. simiae was examined, where the second addition was shown to be able to establish in the root tissue. The device was suitable for imaging with different solid media at high magnification, allowing for the imaging of fungal establishment in the rhizosphere. Overall, the Imaging EcoFAB could improve our ability to investigate the spatiotemporal dynamics of the rhizosphere, including studies of fluorescently-tagged, multimember, synthetic communities. Full article
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14 pages, 1821 KiB  
Article
Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor
by Longteng Tang, Shuce Zhang, Yufeng Zhao, Nikita D. Rozanov, Liangdong Zhu, Jiahui Wu, Robert E. Campbell and Chong Fang
Int. J. Mol. Sci. 2021, 22(1), 445; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010445 - 05 Jan 2021
Cited by 11 | Viewed by 3902
Abstract
Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm [...] Read more.
Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths. Full article
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13 pages, 2124 KiB  
Article
A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes
by Vadim R. Viviani, Vanessa R. Bevilaqua, Daniel R. de Souza, Gabriel F. Pelentir, Michio Kakiuchi and Takashi Hirano
Int. J. Mol. Sci. 2021, 22(1), 303; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010303 - 30 Dec 2020
Cited by 11 | Viewed by 3937
Abstract
Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is [...] Read more.
Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6′-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636–655 nm. Among them, the combination of PxRE-R215K mutant with 6′-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues. Full article
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10 pages, 1689 KiB  
Article
In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans
by Motomichi Doi, Megumi Sato and Yoshihiro Ohmiya
Int. J. Mol. Sci. 2021, 22(1), 119; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010119 - 24 Dec 2020
Cited by 7 | Viewed by 2455
Abstract
Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared [...] Read more.
Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions. Full article
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17 pages, 4618 KiB  
Article
Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc
by Yue Ding, Dimitra Apostolidou and Piotr Marszalek
Int. J. Mol. Sci. 2021, 22(1), 55; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010055 - 23 Dec 2020
Cited by 8 | Viewed by 2865
Abstract
NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no [...] Read more.
NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies. Full article
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18 pages, 1666 KiB  
Article
A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes
by Carina Höring, Ulla Seibel, Katharina Tropmann, Lukas Grätz, Denise Mönnich, Sebastian Pitzl, Günther Bernhardt, Steffen Pockes and Andrea Strasser
Int. J. Mol. Sci. 2020, 21(22), 8440; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21228440 - 10 Nov 2020
Cited by 19 | Viewed by 4515
Abstract
In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs [...] Read more.
In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z’ factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay. Full article
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8 pages, 1504 KiB  
Communication
High Sensitivity In Vivo Imaging of Cancer Metastasis Using a Near-Infrared Luciferin Analogue seMpai
by Jun Nakayama, Ryohei Saito, Yusuke Hayashi, Nobuo Kitada, Shota Tamaki, Yuxuan Han, Kentaro Semba and Shojiro A. Maki
Int. J. Mol. Sci. 2020, 21(21), 7896; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21217896 - 24 Oct 2020
Cited by 15 | Viewed by 4683
Abstract
Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The [...] Read more.
Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The AkaLumine-AkaLuc BLI system (Aka-BLI) can detect resolution at the single-cell level; however, it has a clear hepatic background signal. Here, to enable the highly sensitive detection of bioluminescence from the surrounding liver tissues, we focused on seMpai (C15H16N3O2S) which has been synthesized as a luciferin analogue and has high luminescent abilities as same as AkaLumine. We demonstrated that seMpai BLI could detect micro-signals near the liver without any background signal. The solution of seMpai was neutral; therefore, seMpai imaging did not cause any adverse effect in mice. seMpai enabled a highly sensitive in vivo BLI as compared to previous techniques. Our findings suggest that the development of a novel mutated luciferase against seMpai may enable a highly sensitive BLI at the single-cell level without any background signal. Novel seMpai BLI system can be used for in vivo imaging in the fields of life sciences and medicine. Full article
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20 pages, 4197 KiB  
Article
RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities
by Adil Bakayan, Sandrine Picaud, Natalia P. Malikova, Ludovic Tricoire, Bertrand Lambolez, Eugene S. Vysotski and Nadine Peyriéras
Int. J. Mol. Sci. 2020, 21(21), 7846; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21217846 - 22 Oct 2020
Cited by 3 | Viewed by 1845
Abstract
Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is [...] Read more.
Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice. Full article
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13 pages, 3926 KiB  
Communication
Luminescence of Cypridina Luciferin in the Presence of Human Plasma Alpha 1-Acid Glycoprotein
by Shusei Kanie, Mami Komatsu and Yasuo Mitani
Int. J. Mol. Sci. 2020, 21(20), 7516; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21207516 - 12 Oct 2020
Cited by 6 | Viewed by 2836
Abstract
The enzyme Cypridina luciferase (CLase) enables Cypridina luciferin to emit light efficiently through an oxidation reaction. The catalytic mechanism on the substrate of CLase has been studied, but the details remain to be clarified. Here, we examined the luminescence of Cypridina luciferin in [...] Read more.
The enzyme Cypridina luciferase (CLase) enables Cypridina luciferin to emit light efficiently through an oxidation reaction. The catalytic mechanism on the substrate of CLase has been studied, but the details remain to be clarified. Here, we examined the luminescence of Cypridina luciferin in the presence of several proteins with drug-binding ability. Luminescence measurements showed that the mixture of human plasma alpha 1-acid glycoprotein (hAGP) and Cypridina luciferin produced light. The total value of the luminescence intensity over 60 s was over 12.6-fold higher than those in the presence of ovalbumin, human serum albumin, or bovine serum albumin. In the presence of heat-treated hAGP, the luminescence intensity of Cypridina luciferin was lower than in the presence of intact hAGP. Chlorpromazine, which binds to hAGP, showed an inhibitory effect on the luminescence of Cypridina luciferin, both in the presence of hAGP and a recombinant CLase. Furthermore, BlastP analysis showed that hAGP had partial amino acid sequence similarity to known CLases in the region including amino acid residues involved in the drug-binding ability of hAGP. These findings indicate enzymological similarity between hAGP and CLase and provide insights into both the enzymological understanding of CLase and development of a luminescence detection method for hAGP. Full article
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22 pages, 8544 KiB  
Article
Mapping Calcium Dynamics in the Heart of Zebrafish Embryos with Ratiometric Genetically Encoded Calcium Indicators
by Jussep Salgado-Almario, Manuel Vicente, Pierre Vincent, Beatriz Domingo and Juan Llopis
Int. J. Mol. Sci. 2020, 21(18), 6610; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186610 - 10 Sep 2020
Cited by 10 | Viewed by 3226
Abstract
Zebrafish embryos have been proposed as a cost-effective vertebrate model to study heart function. Many fluorescent genetically encoded Ca2+ indicators (GECIs) have been developed, but those with ratiometric readout seem more appropriate to image a moving organ such as the heart. Four [...] Read more.
Zebrafish embryos have been proposed as a cost-effective vertebrate model to study heart function. Many fluorescent genetically encoded Ca2+ indicators (GECIs) have been developed, but those with ratiometric readout seem more appropriate to image a moving organ such as the heart. Four ratiometric GECIs based on troponin C, TN-XXL, Twitch-1, Twitch-2B, and Twitch-4 were expressed transiently in the heart of zebrafish embryos. Their emission ratio reported the Ca2+ levels in both the atrium and the ventricle. We measured several kinetic parameters of the Ca2+ transients: systolic and diastolic ratio, the amplitude of the systolic Ca2+ rise, the heart rate, as well as the rise and decay times and slopes. The systolic ratio change decreased in cells expressing high biosensor concentration, possibly caused by Ca2+ buffering. The GECIs were able to report the effect of nifedipine and propranolol on the heart, which resulted in changes in heart rate, diastolic and systolic Ca2+ levels, and Ca2+ kinetics. As a result, Twitch-1 and Twitch-4 (Kd 0.25 and 2.8 µM, respectively) seem the most promising GECIs for generating transgenic zebrafish lines, which could be used for modeling heart disorders, for drug screening, and for cardiotoxicity assessment during drug development. Full article
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11 pages, 1663 KiB  
Article
NanoBiT System and Hydrofurimazine for Optimized Detection of Viral Infection in Mice—A Novel in Vivo Imaging Platform
by Natasa Gaspar, Giorgia Zambito, Iris J. C. Dautzenberg, Steve J. Cramer, Rob C. Hoeben, Clemens Lowik, Joel R. Walker, Thomas A. Kirkland, Thomas P. Smith, Wytske M. van Weerden, Jeroen de Vrij and Laura Mezzanotte
Int. J. Mol. Sci. 2020, 21(16), 5863; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21165863 - 15 Aug 2020
Cited by 9 | Viewed by 6147
Abstract
Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing [...] Read more.
Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models. Full article
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21 pages, 2107 KiB  
Article
Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents
by Elena V. Eremeeva, Tianyu Jiang, Natalia P. Malikova, Minyong Li and Eugene S. Vysotski
Int. J. Mol. Sci. 2020, 21(15), 5446; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155446 - 30 Jul 2020
Cited by 6 | Viewed by 2250
Abstract
Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates [...] Read more.
Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues. Full article
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14 pages, 1973 KiB  
Article
An Intact Cell Bioluminescence-Based Assay for the Simple and Rapid Diagnosis of Urinary Tract Infection
by Sherwin Reyes, Nga Le, Mary Denneth Fuentes, Jonathan Upegui, Emre Dikici, David Broyles, Edward Quinto, Sylvia Daunert and Sapna K. Deo
Int. J. Mol. Sci. 2020, 21(14), 5015; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21145015 - 16 Jul 2020
Cited by 11 | Viewed by 4032
Abstract
Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an [...] Read more.
Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies—tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)—were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens—namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 105 CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 105–106 CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments. Full article
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16 pages, 2649 KiB  
Article
The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins
by Marina D. Larionova, Svetlana V. Markova, Nina V. Tikunova and Eugene S. Vysotski
Int. J. Mol. Sci. 2020, 21(14), 4971; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21144971 - 14 Jul 2020
Cited by 6 | Viewed by 2743
Abstract
Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of [...] Read more.
Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins. Full article
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13 pages, 3009 KiB  
Communication
NanoLuc Bioluminescence-Driven Photodynamic Activation of Cholecystokinin 1 Receptor with Genetically-Encoded Protein Photosensitizer MiniSOG
by Yuan Li and Zong Jie Cui
Int. J. Mol. Sci. 2020, 21(11), 3763; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21113763 - 26 May 2020
Cited by 8 | Viewed by 2972
Abstract
In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was [...] Read more.
In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOGPM in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm−2, 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOGPM via an internal ribosome entry site (IRES) sequence (pminiSOGPM-IRES-NanoLuc). The resultant miniSOGPM-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOGPM-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOGPM photodynamic CCK1R activation, laying the foundation for its future in vivo applications. Full article
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Review

Jump to: Research

37 pages, 3036 KiB  
Review
Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow
by Ekaterina S. Potekhina, Dina Y. Bass, Ilya V. Kelmanson, Elena S. Fetisova, Alexander V. Ivanenko, Vsevolod V. Belousov and Dmitry S. Bilan
Int. J. Mol. Sci. 2021, 22(1), 148; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010148 - 25 Dec 2020
Cited by 12 | Viewed by 6648
Abstract
Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and [...] Read more.
Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca2+ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening. Full article
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29 pages, 2820 KiB  
Review
Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications
by Vasilisa V. Krasitskaya, Eugenia E. Bashmakova and Ludmila A. Frank
Int. J. Mol. Sci. 2020, 21(20), 7465; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21207465 - 10 Oct 2020
Cited by 24 | Viewed by 5115
Abstract
The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common [...] Read more.
The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases. Full article
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19 pages, 2894 KiB  
Review
Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission
by Mizuki Endo and Takeaki Ozawa
Int. J. Mol. Sci. 2020, 21(18), 6538; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186538 - 07 Sep 2020
Cited by 25 | Viewed by 4354
Abstract
In vivo bioluminescence imaging (BLI), which is based on luminescence emitted by the luciferase–luciferin reaction, has enabled continuous monitoring of various biochemical processes in living animals. Bright luminescence with a high signal-to-background ratio, ideally red or near-infrared light as the emission maximum, is [...] Read more.
In vivo bioluminescence imaging (BLI), which is based on luminescence emitted by the luciferase–luciferin reaction, has enabled continuous monitoring of various biochemical processes in living animals. Bright luminescence with a high signal-to-background ratio, ideally red or near-infrared light as the emission maximum, is necessary for in vivo animal experiments. Various attempts have been undertaken to achieve this goal, including genetic engineering of luciferase, chemical modulation of luciferin, and utilization of bioluminescence resonance energy transfer (BRET). In this review, we overview a recent advance in the development of a bioluminescence system for in vivo BLI. We also specifically examine the improvement in bioluminescence intensity by mutagenic or chemical modulation on several beetle and marine luciferase bioluminescence systems. We further describe that intramolecular BRET enhances luminescence emission, with recent attempts for the development of red-shifted bioluminescence system, showing great potency in in vivo BLI. Perspectives for future improvement of bioluminescence systems are discussed. Full article
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17 pages, 1385 KiB  
Review
Back to the Future: Genetically Encoded Fluorescent Proteins as Inert Tracers of the Intracellular Environment
by Francesco Cardarelli
Int. J. Mol. Sci. 2020, 21(11), 4164; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21114164 - 11 Jun 2020
Cited by 14 | Viewed by 3490
Abstract
Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their [...] Read more.
Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging. Full article
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