A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.     

BIOLUMINESCENCE OF BRAZILIAN FIREFLIES (COLEOPTERA: LAMPYRIDAE): SPECTRAL DISTRIBUTION and pH EFFECT ON LUCIFERASE-ELICITED COLORS. COMPARISON WITH ELATERID and PHENGODID LUCIFERASES

Twenty-five Brazilian species (nine genera: Phorinus, Photinoides, Macrolampis, Aspisoma, Cratomorphus, Amydetes, Photuris, Bicellonychia, Pyrogaster) of adult fireflies were found to emit light in vivo in the green-yellow range (Λ_max=548–573 nm) of the spectrum, more frequently near the green region, in contrast with North-American species, which predominantly emit yellow light. Distinct ecological contexts where these species evolved, such as the habitat (open field vs forests) and the duration of twilight, are discussed as possible factors responsible for these differences. Except for Photuris and Bicellonychia spp., the in vivo and in vitro bioluminescence spectra for various species of a given genus agree within ±5 nm. Lowering the pH caused the typical red shift in the in vitro bioluminescence spectrum from lampyrid luciferases (six species), which has been interpreted as due to the presence of a basic residue in the enzyme active site catalyzing fast enolization of the initially formed excited keto-oxyluciferin (red emitter) to the excited enol form (yellow-green emitter). The in vitro bioluminescence colors obtained from larval or adult elaterid (five species) and phengodid (three species) luciferases studied here, spanning the green-red region, do not respond to pH changes. This could indicate either the absence of the neighboring basic center (in red-emitting luciferases) or the presence of a non-pH affected proximal basic residue in the active site of the luciferase (in yellow-green-emitting luciferases).     

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.     

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.     

The influence of Ala243 (Gly247), Arg215 and Thr226 (Asn230) on the bioluminescence spectra and pH-sensitivity of railroad worm,click beetle and firefly luciferases

Among beetle luciferases, the pH-sensitive firefly luciferases have been studied extensively. Much less is known about pH-insensitive luciferases, which include click beetle and railroad worm luciferases. Previously, we found that the residues R215 and T226 (N230) are important for green light emission. Here we show that the conserved residue A243 in pH-insensitive luciferases and the corresponding G247 in pH-sensitive luciferases affect the emission spectrum and influence pH-sensitivity. In contrast to railroad worm green light-emitting (PxvGR) and firefly luciferases, the substitution of R215 in Pyrearinus termitilluminans click beetle luciferase (Pte) had no effect on the spectrum, showing that R215 is not essential for green light emission in all beetle luciferases. A homology-based model of Pte luciferase shows that R215 and T226 are close enough to interact. To investigate if there was an interaction between these conserved residues, double mutants were constructed. The double substitution R215S/T226N in Pte luciferase abolished the activity. In PxvGR luciferase the same double mutant resulted in a redshift (lambda(max) = 595 nm), whose magnitude was lower than the value expected for an additive effect. These results suggest that the effects of R215S and T226N are partially interdependent. The double substitution T226N/A243G had an additive redshift effect on the spectrum of PxvGR luciferase, whereas it had a smaller effect on the spectrum of Pte luciferase. Altogether, these results suggest that the above substitutions have different effects on the active site of click beetle and railroad worm luciferases.     

Yellow-green and red firefly bioluminescence from 5,5-dimethyloxyluciferin

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) similar 530 nm) to red (lambda(max) similar 635 nm). The original mechanism of bioluminescence color determination advanced by White and co-workers was based on the concept that the keto and enol tautomers of the emitter oxyluciferin produce red and green light, respectively. Alternatively, McCapra proposed that color variation is associated with conformations of the keto form of excited-state oxyluciferin. We have prepared the adenylate of D-5,5-dimethylluciferin and shown that it is transformed into the putative emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the green-emitting click beetle. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, may be taken as the first experimental support for McCapra's mechanism of firefly bioluminescence color or any other proposal that requires only a single keto form of oxyluciferin.     

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.     

THE CHEMICAL MECHANISM and EVOLUTIONARY DEVELOPMENT OF BEETLE BIOLUMINESCENCE

Abstract— Bioluminescence, as a phenotype, has many evolutionary origins, and thus is an example of natural reinvention many times over. Although peculiar, it arises from the same biochemical principles and evolutionary mechanisms as other biochemical reactions. Of these many different bioluminescent systems, that of the luminous beetles is one of the best understood, having been extensively studied for over 50 years. The luminescence ensues from oxidation of a molecule unique to luminous beetles, beetle luciferin, through a catalytic mechanism evolved from ancestral coenzyme A synthetases. Thus, the character of this bioluminescent reaction is in part a consequence of that evolutionary history. Beetle bioluminescence is furthermore unusual in having a range of luminescent colors found among different beetle species and sometimes even within individual beetles. Structural features of the luciferases are responsible for these color differences, although the underlying mechanism is not yet clear.     

Hetero‐metallic Lanthanide CPs Involving Sodium: Synthesis,Crystal Structure,and Luminescence Properties

[TbNa(4‐msal)₄(phen)₂]_n ( 1 ) (4‐msal = 4‐methyl salicylic acid), a new hetero‐metallic lanthanide coordination polymer (CP) involving sodium was synthesized. It crystallizes in the monoclinic space group P2₁/n, with a = 20.4809(9) Å, b = 9.8183(2) Å, c = 26.1987(11) Å, α = 90.00°, β = 112.922(5)°, γ = 90.00°, V = 4852.2(3) ų, and Z = 4. The complex was characterized by single crystal and powder X‐ray diffraction, elemental analysis (EA), and Fourier transform infrared (FT‐IR) and luminescence spectroscopy. The luminescence properties of a powder sample of 1 were studied at room temperature and the luminescence lifetime and total quantum yield (QY) were determined.     

Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Five new firefly luciferin ( 1 ) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains ( 2 – 4 ) and heterocyclic rings derived from amino acids ( 5 and 6 ) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin ( 6 ), possessing a pyrroline‐substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP‐carboluciferin (AMP=adenosine monophosphate) and AMP‐firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.     

Synthetic Routes to Coelenterazine and Other Imidazo[1,2‐a]pyrazin‐3‐one Luciferins: Essential Tools for Bioluminescence‐Based Investigations

In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2‐a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence (“glow” systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2‐a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.     

A Novel Catalytic Function of Synthetic IgG‐Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine

The synthetic IgG‐binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine‐utilizing luciferase. The Z domain derivatives (ZZ‐gCys, Z‐gCys and Z‐domain) were purified and the luminescence properties were characterized by comparing with coelenterazine‐utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ‐gCys (100%) > Z‐gCys (36.8%) > Z‐domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ‐gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z‐gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG‐binding region of the Z domain.     

BIOLUMINESCENCE: BIOCHEMICAL AND PHYSIOLOGICAL ADVANCES

Bioluminescent organisms are to be found among the bacteria, fungi, unicellular algae, and most of the major animal phyla, some of which contain hundreds of luminescent species. Early biochemical studies reflected this taxonomic diversity and often a primary focus of attention was the uniqueness of each newly isolated system. Relatively few in vitro cross-reactions were discovered among unrelated species and five of the first six luciferins to be characterized proved to be structurally unrelated compounds. The functions of bioluminescence in many organisms were unknown and, while several theories arose, these theories seldom addressed the part that light production might play in the ecology of an organism or a population. Major advances on each of these fronts have been made during the past decade. Biochemical research has centered on the chemical mechanism(s) of luminescence and a single type of chemical species, a dioxetanone, has emerged as a common intermediate in several (but not all) bioluminescence and chemiluminescence systems. Likewise, in the last 10 years, numerous interphyletic cross-reactions have been discovered and the ecological functions of bioluminescence for a large number of species have been established. Intensive studies are continuing on reaction mechanisms, especially those involving the bioluminescent bacteria, and much remains to be learned about the protein biochemistry of all luminescence systems. Amino acid sequence determinations and X-ray crystallographic studies have been initiated in several laboratories and, in others, attempts are being made to clone the genes that code for bioluminescence proteins. Sensitized bioluminescence has been implicated in representatives of both prokaryotic and eukaryotic organisms. Low level biological chemiluminescence has been investigated in a variety of systems including liver microsomes and phagocytic leucocytes using sensitive photon counting devices. Perhaps the area of greatest growth, however, has been the application of bioluminescence and chemiluminescence techniques as tools of clinical research. The need for safe, sensitive and specific assay methods has, for example, stimulated the development of immobilized luciferases and luciferase-linked enzyme systems. In addition, luminescence immunoassay has emerged as a reasonable alternative to the commonly used, but more troublesome, method of radioimmunoassay. This trend toward applications development has shifted the emphasis of some of the university laboratories and in general has improved the lines of communication between basic and applied research groups working in the area of bioluminescence.     

Integrated synthesis and ripening of AgInS2 QDs in droplet microreactors: An update fluorescence regulating via suitable temperature combination

Aqueous phase synthesized ternary I–III–VI₂ Quantum dots (QDs) are getting more and more attention in biology researches, for their good biocompatibility and easy-to-adjust fluorescence properties. However, the quantum yield (QY) of these aqueous phase synthesized QDs are often pretty low, which seriously hindered their further applications in this field. In general, the ripening of the QDs helps to enhance their QY, closely related to the ripening temperature. But it is still hard to precisely control the fluorescence performance of the QDs products, due to the difficulties in precise temperature control and cumbersome temperature adjusting operations in batch reactors. Here we proposed an integrated droplet microfluidic chip for the automated and successive AgInS₂ QDs synthesis and ripening, with both temperatures controlled independently, precisely but easily. Taking advantage of the space-time transformation of the droplet microfluidic chips, the suitable temperature combination for AgInS₂ QDs synthesis and ripening was studied, and the high-performance AgInS₂ QDs were obtained. In addition, the reason for the decrease of QY of AgInS₂ QDs at higher ripening temperature was also explored.     

Optical properties of a tetradentate bis(beta-diketonate) europium(III) complex

Eu₂(BPOPB)₃H₂O, an europium complex chelated with bis(β-diketone), was synthesized. Its properties have been investigated by absorption spectrum, emission spectrum and luminescence lifetime measurement. The complex displays strong red luminescence upon irradiation at the ligand band around 355 nm, which indicates that the bis-β-diketonate ligand BPOPB is an efficient sensitizer. The Judd–Ofelt parameters obtained from the emission spectrum of Eu₂(BPOPB)₃H₂O have been used to calculate the total spontaneous emission probabilities (A), the radiative lifetime (τ_rad), the fluorescence branching ratio (β) and the stimulated emission cross-sections (σ). The luminescence lifetimes are determined to be 402 and 169 μs for Eu₂(BPOPB)₃H₂O and Eu(DBM)₃(H₂O)₂, respectively. The relationship between the structures of rare-earth complexes and luminescence lifetimes was analyzed. The radiative properties reveal that Eu₂(BPOPB)₃H₂O is potential to be an efficient luminescent material.     

Luminescence spectrum and kinetics of laser-excited HgNH3 complex

When an Hg/NH₃ gas mixture was irradiated by laser light around 260 nm, luminescence appeared in the range of 290–360 nm. The spectrum of the luminescence was almost the same as that induced by the Hg photosensitized reaction of NH₃, and thus, the laser-induced luminescence was assigned to emission of an excited HgNH₃ complex. The luminescence spectrum as well as the excitation spectrum could be reproduced by the Franck-Condon factors calculated from the bound-free transition between the Hg-NH₃ repulsive ground state and the excited bound state. Time-evolution measurements on the luminescence induced by the pulsed irradiation of the laser indicated that initially formed HgNH₃ excimer had to make a collisional transition to a state which could emit the luminescence around 340 nm.     

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH₂ assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.