Luciferin Molecule Research Articles

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.

IL-6 Mediates a Switch in Free Fatty Acid Metabolism from the Liver to the Bone Marrow to Support HSC Expansion during Infection

The hematopoietic stem cell (HSC) pool is tightly controlled in both normal and stressed conditions. Under normal conditions, HSCs reside quiescently in their hypoxic niche with minimal mitochondrial activity, instead favouring glycolysis to meet their energy needs. During acute infection HSCs switch to oxidative phosphorylation over glycolysis due to increased energy requirements. This metabolic switch enables HSCs to expand and differentiate into downstream progenitors to increase the immune cell pool. We recently showed that fatty acid (FA) uptake and metabolism play a central role in providing HSCs with sufficient energy for their expansion during acute infection. However, how infection drives changes in available FAs for HSC expansion is not known. This study investigated the role of liver in regulating lipid availability for HSC expansion. We have previously shown that circulating free fatty acids (FFAs) within the serum of mice increased in response to infection using both lipopolysaccharide (LPS) (6 hours) and S. Typhimurium (72 hours) models. This correlated with an expansion of bone marrow HSCs 16 hours after LPS and 72 hours after S. Typhimurium confirmed using flow cytometry. To investigate real-time fatty-acid uptake by hematopoietic cells in response to infection, an in vivo transplant model was used. CD45.1 lineage negative, CD117-positive cells tagged with firefly luciferase (LK+FF) were transplanted into CD45.2 mice. After transplantation was confirmed, mice were injected with LPS for 16 hours. Mice were then subsequently injected with a luciferin molecule conjugated to a long-chain FFA (FFA-luc), which is visible via bioluminescent imaging if the FFA-luc is taken up into the transplanted LK+FF engrafted cells. Live animal imaging using this method confirmed that long-chain FFA is taken up by hematopoietic cells in response to LPS in vivo. As the liver is the master regulator of FA in the serum we isolated livers from control and LPS (6 hour) treated mice. RNAseq revealed down-regulation of genes involved in FA uptake and metabolism as well genes involved in ketogenesis and up-regulation in genes involved in glucose uptake. Further kinetic studies showed that FA uptake and metabolism genes were downregulated within 2 hours of LPS treatment. To understand if this was a direct LPS effect on hepatocytes which express the receptor for LPS (TLR4) or mediated through unknown factors, we isolated primary hepatocytes and treated them with LPS or media conditioned with serum from control or LPS treated mice. Only the media conditioned with serum from LPS treated mice downregulated genes associated with FA uptake and metabolism. To identify the factors driving this metabolic switch in hepatocytes, a cytokine array of serum from control or LPS treated mice (90 minutes) was performed. This proteome cytokine array revealed a significant upregulation of several cytokines and chemokines, including IL-6, TNFα and CXCL1 and CXCL2. Primary mouse hepatocytes were then treated with factors identified in the array for 2 and 4 hours. RT-qPCR confirmed that IL-6 downregulated FA uptake and metabolism genes in primary hepatocytes. To understand if this was a LPS specific response or induce by infection mice were treated with S. Typhimurium for various time points and livers extracted. RT-qPCR confirmed that S. Typhimurium treated mice down-regulated genes associated with FA uptake and metabolism. To study the role of IL-6 in regulating liver FA metabolism in response to LPS, mice were treated with IL-6 neutralising antibody (MP5-20F3). Pre-treatment of mice with MP5-20F3 inhibited LPS induced downregulation of liver FA uptake and metabolic genes, which lead to reversing LPS-induced increase in serum FA. In conclusion we have shown a role for IL-6 in switching fatty acid metabolism from the liver to the bone marrow to allow HSC expansion during infection.

In silico Analysis of Renilla Muelleri, Photinus Pyralis and Metridia Longa Luciferase

Luciferase derived from different organism is widely used for various biological process in the cells or tissues. Current in silico study was done to investigate the protein-substrate interactions of Renilla muelleri, Metridia longa and Photinus pyralis luciferase with their respective substrates coelenterazine, Luciferin, and ATP. RaptorX which is an online tool was used for luciferase modeling. PyRx v9.0 was used for protein-substrate binding. Online server Cluspro was used for protein-protein docking. CASTp was used for protein active site pocket prediction. Photinus pyralis luciferase was bonded with ATP molecules through Glu83, Asp153, Asp44, Ser85, Gln87 and His171, while Photinus Pyralis luciferase was bonded with luciferin molecules through five different residues i.e. His171, Arg62, Met90, Leu63 and Val168. Photinus pyralis residues that were docked with ATP and luciferin molecules were present in N terminal domain of Photinus pyralis luciferase. In case of Renilla muelleri, catalytic residues, His285 was present in its all the docking complex. Renilla muelleri and Metridia longa luciferase were also docked with different substrate and found that efficiency of Renilla muelleri and Metridia longa luciferase was lower towards Photinus pyralis substrates as compared to their own substrate coelentereazine. According to the findings, it has been concluded that luciferase of every light emitting organism required specific (its own) substrate for proper light reaction. Keywords: Renilla muelleri; Metridia longa; Photinus pyralis; luciferase

Action spectroscopy of isomer-selected luciferin anions

Luciferin molecules are common luminophores found throughout the biological kingdoms. Here, electrospray ionization and tandem ion mobility spectrometry coupled with laser spectroscopy are used to demonstrate that D-luciferin and oxyluciferin deprotonated anions can be produced in two isomeric forms, which can be separated by virtue of their different collision cross sections with a buffer gas. The two isomers possess distinguishable but partially overlapping photodepletion action spectra over the visible range, implying distinct intrinsic absorption profiles. The site of deprotonation and tautomeric forms of the electrosprayed isomers are assigned through comparisons between experimental and calculated collision cross sections and electronic excitation energies. The study clearly shows that electrospray ionization of biochromophore molecules can generate multiple isomeric forms with distinct electronic spectra.

Genomic and Transcriptomic Analyses of Bioluminescence Genes in the Enope Squid Watasenia scintillans

Watasenia scintillans, a sparkling enope squid, has bioluminescence organs to illuminate its body with its own luciferase activity. To clarify the molecular mechanism underlying its scintillation, we analysed high-throughput sequencing data acquired previously and obtained draft genome sequences accomplished with comparative genomic data among the cephalopods. The genome mapped by transcriptome data showed that (1) RNA editing contributed to transcriptome variation of lineage specific genes, such as W. scintillans luciferase, and (2) two types of luciferase enzymes were characterized with reasonable 3D models docked to a luciferin molecule. We report two different types of luciferase in one organism and possibly related to variety of colour types in the W. scintillans fluorescent organs.

Resurrecting the Ancient Glow

The color of firefly bioluminescence is primarily determined by the structure of the enzyme luciferase. To date, firefly luciferase genes have been isolated from over 30 extant species producing light ranging in color from deep-green to orange-yellow. We have reconstructed seven ancestral firefly luciferase genes, and characterised the enzymatic properties and crystal structures of the recombinant proteins in order to predict the evolution of firefly light emission. Results showed that the synthetic luciferase for the last common firefly ancestor exhibited blue-shifted emission achieving bright green light caused by a spatial constraint on luciferin molecule in the enzyme, while the fatty acyl-CoA synthetic activity, an original function of firefly luciferase, was diminished in exchange. All known firefly species are bioluminescent in the larval stages, with a common ancestor arising approximately 100 Mya. Combined, our findings propose that within the mid-Cretaceous forest the common ancestor of contemporary fireflies evolved green light luciferase via trade-off of original function, most likely for aposematic display from nocturnal predation.

НОВАЯ БИОЛЮМИНЕСЦЕНТНАЯ СИСТЕМА ГРИБОВ: ПЕРСПЕКТИВЫ ИСПОЛЬЗОВАНИЯ В МЕДИЦИНСКИХ ИССЛЕДОВАНИЯХ

Bioluminescence is chemical oxidation of a small luciferin molecule by air catalyzed by luciferase and accompanied by the emission of photons in the visible spectrum. This reaction is used in bioluminescent bioimaging, the method for the visualization of organism’s interior. Bioimaging is a popular tool used in medical research. However, it has an unfortunate drawback: it requires introduction of external luciferin to the system before every experiment. In this work we discuss a possibility of developing an autonomous luminescent system in eukaryotes based on the bioluminescent system of higher fungi.

Glowing mushrooms’ mechanism unmasked

Scientists have discovered the key molecule involved in the glow of bioluminescent fungi (Sci. Adv. 2017, DOI: 10.1126/sciadv.1602847). Numerous plants, animals, and insects exhibit bioluminescence. The biochemical pathways involved in this glow phenomenon have been, for the most part, well characterized. But even though scientists recently identified one of the components involved in fungal bioluminescence, the full mechanism has remained a mystery. Now, a large international team led by Ilia V. Yampolsky of the Russian Academy of Sciences in Moscow has identified and characterized fungal oxyluciferin, the molecule in glowing mushrooms that emits photons of a characteristic energy (or color) as it relaxes from an excited state to the ground state. Bioluminescence generally arises from the interaction of a species’ unique luciferin molecules with an enzyme known as luciferase, plus oxygen and light. The reactions produce species-specific oxyluciferin in its excited state, which exhibits a species-specific...

Functionalized Luciferins for Applications in Supramolecular Chemistry: In Vivo Light Switches

Sensing of supramolecular assembly in vivo has been a struggle for current research because a limited number of assays can probe this effectively. Firefly luciferin has been widely used as an in vivo reporter of adenosine triphosphate, magnesium cations, and esterase activity because of its highly selective enzyme catalysis and high quantum yield when oxidized in the presence of adenosine triphosphate and magnesium cations. The emitted photon is of a yellow-green color thus detection has been possible in vivo from cells to mammals by a simple luminescence assay. Caging, or rendering the firefly luciferin molecule incompatible for luciferase enzyme, has been accomplished most commonly by appending electron withdrawing groups to the 6’-hydroxyl position or esterification of the thiazole moiety. The caged luciferin then undergoes a reaction to yield its native form, resulting in emission of a photon upon oxidation and reporting the desired reaction or process. The goal of our research is to use firefly luciferin as a reporter of supramolecular assembly by a novel method of caging, reversibly with a host molecule (cyclodextrins and cucurbiturils). This talk will describe the synthesis and characterization of a novel set of firefly luciferin derivatives for use in the luciferin-luciferase type assays with the goal of detecting host-guest interactions. This talk will illustrate how supramolecular assembly and its predictability can be used to design novel biocompatible systems to overcome the challenges of a highly competitive cellular environment.

Designer molecule tracks copper’s role in fatty liver disease

Copper imbalances are suspected of playing a role in a number of diseases. But tools for detecting the metal in living organisms have been lacking. Christopher J. Chang, Andreas Stahl, and coworkers at the University of California, Berkeley, have now developed a bioluminescent molecular probe for detecting copper in specific organs. The team used the probe to reveal a copper deficiency associated with fatty liver disease, a condition in which fat accumulates in liver cells, leading to inflammation and potentially cirrhosis (Proc. Natl. Acad. Sci. USA 2016, DOI: 10.1073/pnas.1613628113). The probe is a caged luciferin molecule that is held in a nonluminescent state by a tris[(2-pyridyl)methyl]amine ligand. When copper is present, oxidative cleavage of the ligand releases luciferin, which is then free to be a substrate for the enzyme luciferase and produce a bioluminescent signal that is proportional to the copper concentration. The researchers achieved organ-specific copper detection by using

Thoughts on the diversity of convergent evolution of bioluminescence on earth

AbstractThe widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms.Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms.Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

Molecular enigma of multicolor bioluminescence of firefly luciferase.

Firefly luciferase-catalyzed reaction proceeds via the initial formation of an enzyme-bound luciferyl adenylate intermediate. The chemical origin of the color modulation in firefly bioluminescence has not been understood until recently. The presence of the same luciferin molecule, in combination with various mutated forms of luciferase, can emit light at slightly different wavelengths, ranging from red to yellow to green. A historical perspective of development in understanding of color emission mechanism is presented. To explain the variation in the color of the bioluminescence, different factors have been discussed and five hypotheses proposed for firefly bioluminescence color. On the basis of recent results, light-color modulation mechanism of firefly luciferase propose that the light emitter is the excited singlet state of OL(-) [(1)(OL(-))*], and light emission from (1)(OL(-))* is modulated by the polarity of the active-site environment at the phenol/phenolate terminal of the benzothiazole fragment in oxyluciferin.

In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter

Living organisms produce hydrogen peroxide (H(2)O(2)) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. The effects of H(2)O(2) on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of H(2)O(2) within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with H(2)O(2) to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for H(2)O(2), combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of H(2)O(2) generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in H(2)O(2) levels by directly imaging elevations in H(2)O(2) within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor H(2)O(2) fluxes in real time in living animals offers opportunities to dissect H(2)O(2)'s disparate contributions to health, aging, and disease.

Complexation study of luciferin with metal ions in acetonitrile employing theoretical and experimental methods

Luciferin complexation with some metal ions in acetonitrile has been investigated by fluorescence spectroscopy, UV–Vis absorbance, and conductometry. The complexation study was carried out based on some preliminary theoretical investigations. Formation constant values (K f) were calculated for complex formation between luciferin and metal ions. The experimental results revealed that these cations have the ability to quench the intrinsic fluorescence of luciferin through a quenching procedure. The experimental results showed that Cu 2+ ion has the best interaction with luciferin molecule. Furthermore, the pair wise interaction energy between luciferin and Cu 2+ ion were also calculated employing DFT method.

Fluorescent properties of firefly luciferases and their complexes with luciferin.

Fluorescence of luciferases from Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417, Trp-426) was studied. Analysis of quenching of tryptophan fluorescence showed that the tryptophan residue conserved in all luciferases is not accessible for charged quenchers, which is explained by the presence of positively and negatively charged amino acid residues in the close vicinity to it. An effective energy transfer from tryptophan to luciferin was observed during quenching of tryptophan fluorescence of both luciferases with luciferin. From the data on the energy transfer, the distance between the luciferin molecule and Trp-417 (419) in the luciferin luciferase complex was calculated: 11-15 A for P. pyralis and 12-17 A for L. mingrelica luciferases. The role of the conserved Trp residue in the catalysis is discussed.

Picosecond time-resolved absorption spectroscopy of luciferin

We present the results of picosecond time-resolved absorption spectroscopy of luciferin, substrate of the bioluminescence reaction. Both absorption and emission from the excited states contribute to the detected signal. Time evolution of the differential optical density at different wavelengths is analyzed. Characteristic rate constants of the relaxation processes are determined. The possible relationship between the behavior of the luciferin molecule in the excited state and the properties of the luciferin–luciferase complex is discussed.

Weak chemiluminescence of bilirubin and its stimulation by aldehydes.

Bilirubin in an alkaline solution exhibits a weak chemiluminescence (CL) under aerobic conditions. This spontaneous CL was markedly enhanced by the addition of various aldehydes. The fluorescent emission spectrum of bilirubin, excited by weak intensity light at 350 nm, coincided with its CL emission spectrum (peak at 670 nm). CL emission from bilirubin was not quenched by active oxygen scavengers. This suggests that triplet oxygen reacts with bilirubin, and forms an oxygenated intermediate (hydroperoxide) as a primary emitter (oxidative scission of tetrapyrrole bonds in bilirubin is not involved in this CL). The Ehrlich reaction (test for monopyrroles) and hydrolsulphite reaction (test for dipyrroles) on the CL reaction mixture and unreacted bilirubin showed no differences. When the CL was initiated by singlet oxygen, rather than superoxide anion, monopyrrole, was detected in the reaction products by gel chromatography. The inhibitory effect of a scavenger of singlet oxygen on CL was eliminated in the presence of formaldehyde. Therefore, triplet carbonyl, formed by singlet oxygen through the dioxetane structure in bilirubin, is not an emitter. The reaction mechanism of bilirubin CL and the formation of a hydroperoxide intermediate is discussed in relation to the chemical structure of luciferin molecules from bioluminescent organisms.

Dinoflagellate luminescence: Purification of a NAD(P)H-dependent reductase and of its substrate

The soluble enzymatic luminescent system of the dinoflagellate Pyrocystis lunula (luciferase-luciferin) is coupled with an enzymatic NAD(P)H-dependent reaction. The enzyme is a soluble reductase ( M r 47,000) which catalyzes, in the presence of NAD(P)H, the reduction of a molecule called P630. Reduced P630 has the same spectral characteristics as the purified luciferin. The luciferase can oxidize this reduced molecule with a light emission at 480 nm. These observations suggest that reduced P630 is a luciferin molecule. The oxidized form seems, in these conditions, to be the precursor of luciferin.

Substrate and substrate analogue binding properties of Renilla luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis catalyzes the oxidative decarboxylation of luciferin consuming 1 mol of O2 per mol of luciferin oxidized and producing 1 mol of CO2, 1 mol of oxyluciferin, and light (lambdaB, 480 nm) with a 5.5% quantum yield. In this work we have examined the binding characteristics of luciferin, luciferin analogues, and competitive inhibitors of the luciferin-luciferase reaction. The results show that luciferin binding and orientation in the single luciferin binding site of luciferase are highly specific for and dependent upon the three group substituents of the luciferin molecule while the imidazolone-pyrazine nucleus of luciferin is not directly involved in binding. Anaerobic luciferin binding promotes a rapid concentration-dependent aggregation of luciferase which results in irreversible inactivation of the enzyme. This aggregation phenomenon is not observed upon binding of oxyluciferin, luciferyl sulfate, or luciferin analogues in which the substituent at the 2 position of the imidazolone-pyrazine ring has been substantially altered.

Substrate-binding properties of firefly luciferase: I. Luciferin-binding site

Various techniques were used for characterization of the luciferin-binding site of the firefly luciferase. Both equilibrium-dialysis and fluorescence-titration techniques revealed the existence of two identical, noninteracting binding sites for dehydroluciferin, which is a potent competitive inhibitor. Kinetically obtained dissociation constants of various luciferin analogues and other competitive inhibitors revealed that most of the binding energies of these compounds reside in the backbone ring structure and that various substituents did not influence the binding to any significant extent. Exceptions to this are the methyl group at the 6-position of the benzothiazole ring and the carboxylic acid group. The binding energy of the luciferin backbone structure to the luciferin-binding site is 7.5 kcal/mole. Two parts of the luciferin molecule, the benzothiazole ring portion and the thiazoline ring portion, contribute 6.0 kcal/mole and 1.5 kcal/mole respectively to the binding energy. Comparison of the binding energies of several heterocyclic compounds structurally related to benzothiazole suggests that the nitrogen atom of the benzothiazole ring may be important in orienting the compound to a fixed position in the luciferinbinding site. The binding studies with N-ethylmaleimide-inactivated luciferase gave further evidence that the two sulfhydryl groups essential for the luciferase activity are located at the luciferin binding sites, most likely one at each binding site. Excitation spectra of luciferase-bound dehydroluciferin indicated that the phenolic group of dehydroluciferin is in unionized form on the enzyme even in a medium of high pH. This effect and a large enhancement of the blue fluorescence peak at 440 mμ suggest the environment of luciferin-binding site to be quite hydrophobic in agreement with earlier observations.