Bioluminescence Reaction Research Articles

Light from the firefly Luciola praeusta at very low temperatures

Light emissions from a few Indian species of fireflies at different temperatures have been investigated in recent times. Changes observed in the steady-state and flash emissions in those investigations indicate interesting aspects of their light-emitting systems in the live condition. Here we report emission spectra and pulses from both male and female specimens of the species Luciola praeusta at temperatures much lower than the ones at which they normally emit. When the temperature is decreased to the region of 11.5–11 °C for males or 16.5–15 °C for females, the wavelength peak demonstrates a shift towards blue and the pulses show abnormal increase in the duration. These changes, which are reversible, indicate a slight change in the structure of the enzyme luciferase catalysing the bioluminescent reaction that produces the light of the firefly. This happening is proposed as the reason for the females generally disappearing a bit earlier than the males at the onset of the winter.

Quantitative Analysis of Bioluminescence Optical Signal.

Bioluminescence is light emission based on the luciferin-luciferase enzymatic reaction in living organisms. Optical signals from bioluminescence (BL) reactions are available for bioanalysis and bioreporters for gene expression, in vitro, in vivo, and ex vivo bioimaging, immunoassay, and other applications. Although there are numerous bioanalysis methods based on BL signal measurements, the BL signal is measured as a relative value, and not as an absolute value. Recently, some approaches have been established to completely quantify the BL signal, resulting in, for instance, the redetermination of the quantum yield of the BL reaction and counting the photon number of the BL signal at the single-cell level. Reliable and reproducible understanding of biological events in the bioanalysis and bioreporter fields can be achieved by means of standardized absolute optical signal measurements, which is described in an International Organization for Standardization (ISO) document.

Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features.

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering "character".

Biophysical analysis of Gaussia luciferase bioluminescence mechanisms using a non-oxidizable coelenterazine

Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but GLuc's fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine (Aza-CTZ), a non-oxidizable coelenterazine (CTZ) analog, as a probe to investigate its binding mode to GLuc. While analysing GLuc's activity, we unexpectedly found that salt and monovalent anions are absolutely required for Gluc's bioluminescence, which retrospectively appears reasonable for a sea-dwelling organism. The NMR-based investigation, using chemical shift perturbations monitored by 15N-1H HSQC, suggested that Aza-CTZ (and thus unoxidized CTZ) binds to residues in or near the hydrophobic cavity. These NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.

Crystal structure of semi-synthetic obelin-v after calcium induced bioluminescence implies coelenteramine as the main reaction product

Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.

Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

The bacterial bioluminescence system enables the generation of light by living cells without the requirement of an external luciferin. Due to the relatively low light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report, a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with sixfold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

Coelenterazine sulfotransferase from Renilla muelleri.

The luciferin sulfokinase (coelenterazine sulfotransferase) of Renilla was previously reported to activate the storage form, luciferyl sulfate (coelenterazine sulfate) to luciferin (coelenterazine), the substrate for the luciferase bioluminescence reaction. The gene coding for the coelenterazine sulfotransferase has not been identified. Here we used a combined proteomic/transcriptomic approach to identify and clone the sulfotransferase cDNA. Multiple isoforms of coelenterazine sulfotransferase were identified from the anthozoan Renilla muelleri by intersecting its transcriptome with the LC-MS/MS derived peptide sequences of coelenterazine sulfotransferase purified from Renilla. Two of the isoforms were expressed in E. coli, purified, and partially characterized. The encoded enzymes display sulfotransferase activity that is comparable to that of the native sulfotransferase isolated from Renilla reniformis that was reported in 1970. The bioluminescent assay for sensitive detection of 3’-phosphoadenosine 5’-phosphate (PAP) using the recombinant sulfotransferase is demonstrated.

14-3-3 proteins are luciferases candidate proteins from lanternfish Diaphus watasei.

The lanternfish is a deep-sea fish with ventral-lateral and head photophores. It uses its ventral-lateral photophores to camouflage its ventral silhouette, a strategy called counterillumination. The bioluminescent reaction of lanternfish involves coelenterazine as a substrate luciferin but the enzyme catalyzing the bioluminescent reaction has not been identified. We report a candidate enzyme of luciferase from lanternfish Diaphus watasei. We purified the luciferase and performed SDS-PAGE analysis resulted in two bands corresponding to the activity, and following mass spectrometry analysis detected three 14-3-3 proteins of which functions is known to exhibit protein-protein interactions. The molecular weights and isoelectric points of the 14-3-3 proteins were almost consistent with the luciferase properties. The addition of two 14-3-3 binding compounds, R18 peptide and fusicoccin, resulted in the inhibition of the luciferase activity. However, the two 14-3-3 recombinant proteins showed very slight luminescence activity. These results suggested that the 14-3-3 proteins are candidate luciferases of D. watasei.

Self-Training Strategy Based on Finite Element Method for Adaptive Bioluminescence Tomography Reconstruction.

Bioluminescence tomography (BLT) is a promising pre-clinical imaging technique for a wide variety of biomedical applications, which can non-invasively reveal functional activities inside living animal bodies through the detection of visible or near-infrared light produced by bioluminescent reactions. Recently, reconstruction approaches based on deep learning have shown great potential in optical tomography modalities. However, these reports only generate data with stationary patterns of constant target number, shape, and size. The neural networks trained by these data sets are difficult to reconstruct the patterns outside the data sets. This will tremendously restrict the applications of deep learning in optical tomography reconstruction. To address this problem, a self-training strategy is proposed for BLT reconstruction in this paper. The proposed strategy can fast generate large-scale BLT data sets with random target numbers, shapes, and sizes through an algorithm named random seed growth algorithm and the neural network is automatically self-trained. In addition, the proposed strategy uses the neural network to build a map between photon densities on surface and inside the imaged object rather than an end-to-end neural network that directly infers the distribution of sources from the photon density on surface. The map of photon density is further converted into the distribution of sources through the multiplication with stiffness matrix. Simulation, phantom, and mouse studies are carried out. Results show the availability of the proposed self-training strategy.

Metabolic labeling and bioluminescent imaging of nascent peptidoglycan

Peptidoglycan (PG) is the main substance for cell wall synthesis and plays an essential role in bacterial physiological activities such as proliferation and division. It protects bacterial from external environment and resists pressure generated by bacterial internal expansion. Despite the incredible importance, there is still lack of effective tools for direct imaging and quantitative analysis of PG. Herein, we designed a D-luciferin/luciferase bioluminescent system to visualize and quantify PG levels in living bacterial cells. In our system, we metabolically label PG through aryl azide modified D-amino acid (DAz). Afterwards, TL-PDC, in which the phenolic hydroxyl of D-luciferin is initially protected through “caged” strategy, is introduced to react with DAz through Staudinger ligation reaction and releases the free D-luciferin. This will initiate luciferase catalyzed bioluminescence reaction and generates a strong luminescence signal. It is demonstrated that TL-PDC has good selectivity and sensitivity towards DAz and DAz-labeled PG. Moreover, the system was used to evaluate PG level in living mice by bioluminescent imaging. Most importantly, this method has potential value in biological experiments related to bacterial detection.

Comparative Investigation of the Chemiluminescent Properties of a Dibrominated Coelenterazine Analog.

Chemi- and bioluminescence are remarkable light-emitting phenomena, in which thermal energy is converted into excitation energy due to a (bio)chemical reaction. Among a wide variety of chemi-/bioluminescent systems, one of the most well-known and studied systems is that of marine imidazopyrazinones, such as Coelenterazine and Cypridina luciferin. Due to the increasing usefulness of their chemi-/bioluminescent reactions in terms of imaging and sensing applications, among others, significant effort has been made over the years by researchers to develop new derivatives with enhanced properties. Herein, we report the synthesis and chemiluminescent characterization of a novel dibrominated Coelenterazine analog. This novel compound consistently showed superior luminescence, in terms of total light output and emission lifetime, to natural imidazopyrazinones and commercially available analogs in aprotic media, while being capable of yellow light emission. Finally, this new compound showed enhanced chemiluminescence in an aqueous solution when triggered by superoxide anion, showing potential to be used as a basis for optimized probes for reactive oxygen species. In conclusion, bromination of the imidazopyrazinone scaffold appears to be a suitable strategy for obtaining Coelenterazines with enhanced properties.

Bioluminescent test systems based on firefly luciferase for studying stress effects on living cells.

The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly luciferase. The possibilities of using this reaction to study the influence of external effectors of a physical and chemical nature (temperature exposure, additions of drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes) are considered. Examples of the use of test systems based on living cells producing thermostable firefly luciferase for monitoring cellular homeostasis are given. The study of the kinetics of changes in the concentration of ATP and luciferase inside and outside cells made it possible to determine in dynamics the metabolic activity, cytotoxicity, and survival of cells under conditions of cellular stress, to study the processes of ATP synthesis/hydrolysis, and to evaluate the effectiveness of lytic agents in changing the permeability of the cell membrane.

Recombinant light-sensitive photoprotein berovin from ctenophore Beroe abyssicola: Bioluminescence and absorbance characteristics

The bright bioluminescence of ctenophores inhabiting the oceans worldwide is caused by light-sensitive Ca2+-regulated photoproteins. By now, the cDNAs encoding photoproteins from the four different ctenophore species have been cloned and the recombinant proteins have been characterized to some extent. In this work, we report on the specific activity and the quantum yield of bioluminescence reaction as well as the absorbance characteristics of high-purity recombinant berovin. To determine those, we applied the amino acid composition analysis to accurately measure berovin concentration and the recombinant aequorin as a light standard to convert relative light units to quanta. The extinction coefficient of 1% berovin solution at 435 nm was found to be 1.82. The one can be employed to precisely determine the protein concentration of active photoproteins from other ctenophore species. The specific activity and the bioluminescence quantum yield were respectively found to be 1.98 × 1015 quanta/mg and 0.083. These values appeared to be several times lower than those of the cnidarian photoproteins, which is obviously due to differences in amino acid environments of the substrate in active sites of these photoproteins.

Droplet Microfluidic Device for Chemoenzymatic Sensing.

The rapid detection of pollutants in water can be performed with enzymatic probes, the catalytic light-emitting activity of which decreases in the presence of many types of pollutants. Herein, we present a microfluidic system for continuous chemoenzymatic biosensing that generates emulsion droplets containing two enzymes of the bacterial bioluminescent system (luciferase and NAD(P)H:FMN–oxidoreductase) with substrates required for the reaction. The developed chip generates “water-in-oil” emulsion droplets with a volume of 0.1 L and a frequency of up to 12 drops per minute as well as provides the efficient mixing of reagents in droplets and their distancing. The bioluminescent signal from each individual droplet was measured by a photomultiplier tube with a signal-to-noise ratio of up to 3000/1. The intensity of the luminescence depended on the concentration of the copper sulfate with the limit of its detection of 5 M. It was shown that bioluminescent enzymatic reactions could be carried out in droplet reactors in dispersed streams. The parameters and limitations required for the bioluminescent reaction to proceed were also studied. Hereby, chemoenzymatic sensing capabilities powered by a droplet microfluidics manipulation technique may serve as the basis for early-warning online water pollution systems.

(Digital Presentation) Wavelength Controllable Multi-Color Firefly Luciferin Analogues

I will show you the multi-color luciferin analogues, and in particular the usefulness of near-infrared(NIR) luciferin analogues.Firefly bioluminescent reaction requires luciferin, luciferase, Mg2+, ATP and O2, and emits light at 560 nm (green-yellow light). As the light emission efficiency of the firefly bioluminescence system is extremely high(ΦBL=41%), this system is attracting widespread attention as a bioimaging tool.We have studied the relationship between structure modification and the firefly luminescence wavelength, where the luciferase of Photinus pyralis (Ppy) was used. As a result, we are able to control the wavelength of the luciferin analogues to some extent. An analog having the thiazoline ring connected directly to p-(dimethylamino)phenyl group exhibited blue shifted luminescence maximum. Furthermore, replacement of the dimethylamino group into a hydroxyl group caused blue shift of luminescence wavelength. On the other hand, Introduction of a double bond between the thiazoline ring and the benzene ring caused large red shift of luminescence wavelength.Based on these structure-activity relationships, we have synthesized a number of luciferin analogs. The shortest emission wavelength of luciferin analogs is 430 nm, and the longest is 760 nm by using Ppy luciferase. Among them, near-infrared(NIR) light is expected to be applied to in vivo imaging because of its high living body permeability.Therefore, Thus, AkaLumine, a luciferin analogue that produces light at 675 nm wavelength, was developed. This compound was shown to yield useful results when applied to the in vivo imaging of mice. We have improved the characteristics of AkaLumine to optimize this compound for use in in vivo imaging, thus obtaining the compounds dubbed TokeOni and seMpai. In this way, bioluminescence imaging technology is undergoing constant innovation, and we continue to develop NIR luciferin analogues that can be used as imaging tools.In this conference, I will present the characteristics of recently developed multi-color luciferin analogues, include NIR luciferin analogues. Figure 1

Tuning the Intramolecular Chemiexcitation of Neutral Dioxetanones by Interaction with Ionic Species.

The intramolecular chemiexcitation of high-energy peroxide intermediates, such as dioxetanones, is an essential step in different chemi- and bioluminescent reactions. Here, we employed the Time-Dependent Density Functional Theory (TD-DFT) methodology to evaluate if and how external stimuli tune the intramolecular chemiexcitation of model dioxetanones. More specifically, we evaluated whether the strategic placement of ionic species near a neutral dioxetanone model could tune its thermolysis and chemiexcitation profile. We found that these ionic species allow for the “dark” catalysis of the thermolysis reaction by reducing the activation barrier to values low enough to be compatible with efficient chemi- and bioluminescent reactions. Furthermore, while the inclusion of these species negatively affected the chemiexcitation profile compared with neutral dioxetanones, these profiles appear to be at least as efficient as anionic dioxetanones. Thus, our results demonstrated that the intramolecular chemiexcitation of neutral dioxetanones can be tuned by external stimuli in such a way that their activation barriers are decreased. Thus, these results could help to reconcile findings that neutral dioxetanones could be responsible for efficient chemi-/bioluminescence, while being typically associated with high activation parameters.

Morphological changes in the tracheal system associated with light organs of the firefly Photinus pyralis (Coleoptera: Lampyridae) across life stages.

Oxygen is an important and often limiting reagent of a firefly's bioluminescent chemical reaction. Therefore, the development of the tracheal system and its subsequent modification to support the function of firefly light organs are key to understanding this process. We employ micro-CT scanning, 3D rendering, and confocal microscopy to assess the abdominal tracheal system in Photinus pyralis from the external spiracles to the light organ's internal tracheal brush, a feature named here for the first time. The abdominal spiracles in firefly larvae and pupae are of the biforous type, with a filter apparatus and appear to have an occlusor muscle to restrict airflow. The first abdominal spiracle in the adult firefly is enlarged and bears an occlusor muscle, and abdominal spiracles two through eight are small, with a small atrium and bilobed closing apparatus. Internal tracheal system features, including various branches, trunks, and viscerals, were homologized across life stages. In adults, the sexually dimorphic elaboration and increase in volume associated with tracheal features of luminous segments emphasizes the importance of gas exchange during the bioluminescent process.

Theoretical Study of the Thermolysis Reaction and Chemiexcitation of Coelenterazine Dioxetanes.

Coelenterazine and other imidazopyrazinones are important bioluminescent substrates widespread in marine species and can be found in eight phyla of luminescent organisms. Light emission from these systems is caused by the formation and subsequent thermolysis of a dioxetanone intermediate, whose decomposition allows for efficient chemiexcitation to singlet excited states. Interestingly, some studies have also reported the involvement of unexpected dioxetane intermediates in the chemi- and bioluminescent reactions of Coelenterazine, albeit with little information on the underlying mechanisms of these new species. Herein, we have employed a theoretical approach based on density functional theory to study for the first time the thermolysis reaction and chemiexcitation profile of two Coelenterazine dioxetanes. We have found that the thermolysis reactions of these species are feasible but with relevant energetic differences. More importantly, we found that the singlet chemiexcitation profiles of these dioxetanes are significantly less efficient than the corresponding dioxetanones. Furthermore, we identified triplet chemiexcitation pathways for the Coelenterazine dioxetanes. Given this, the chemiexcitation of these dioxetanes should lead only to minimal luminescence. Thus, our theoretical investigation of these systems indicates that the thermolysis of these dioxetanes should only provide "dark" pathways for the formation of nonluminescent degradation products of the chemi- and bioluminescent reactions of Coelenterazine and other imidazopyrazinones.

Cryo-EM structure of the fatty acid reductase LuxC–LuxE complex provides insights into bacterial bioluminescence

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.

Rationalizing the role of electron/charge transfer in the intramolecular chemiexcitation of dioxetanone-based chemi-/bioluminescent systems

The thermolysis of dioxetanones is a key process in the intramolecular chemiexcitation step of several chemi- and bioluminescent reactions. This step is generally explained with mechanisms based on either electron transfer (ET), such as the Chemically Initiated Electron-Exchange Luminescence (CIEEL) mechanism, or charge transfer (CT), such as the Charge Transfer-Initiated Luminescence (CTIL) mechanism. Here, we have used a TD-DFT approach to characterize the thermolysis and chemiexcitation steps of model dioxetanones, to rationalize the role of ET/CT in those intramolecular processes. Our results showed that ET/CT can reduce the activation barrier of the thermolysis reaction, by reducing the repulsion between the reacting fragments (ketone and CO2 moieties) that originate during peroxide bond breaking. However, in terms of singlet chemiexcitation profiles, those of non-CIEEL/CTIL-based dioxetanones appear to be more efficient than of CIEEL/CTIL-based ones. Furthermore, the ground state to singlet excited state transitions were found to be local excitations, without CT between the peroxide ring and the electron-rich moiety. So, ET/CT appear to be responsible for tuning the activation barrier of the thermolysis reaction, without playing a role in efficient singlet chemiexcitation itself.