The role of protein globule in firefly luciferase catalysis.

The important role of the dynamic structure of firefly luciferase in enzyme functioning is a subject of this literature review. Due to the domain alternation, the optimal configuration of the active site is created for each stage of the luciferin oxidation. The diversity of bioluminescence spectra is explained by the combined emission of several coexisting forms of electronically excited oxyluciferin. The superposition of two or three emitter forms recorded in the bioluminescence spectra indicates that different luciferase conformers coexist in the reaction medium in dynamic equilibrium. The relationship between the thermal stability of the protein globule and the bioluminescence spectra is also discussed.

Luciola mingrelica firefly luciferase as a marker in bioluminescent immunoassays.

Chemical modification of the enzymes with biospecific macromolecules is used in various fields of biotechnology to impart new functions or improve their properties and is a fast and convenient way to get the final products. The preparation of highly active, stable, and functionally active conjugates of the thermostable luciferase through the NH2-groups or free SH-groups of the enzyme with target molecules of different molecular weight (albumin, avidin from chicken eggs, antibodies, and progesterone) is described. The obtained conjugates were successfully tested as a reporter in bioluminescent immunoassay for the detection of the molecules and pathogens. Thus, the luc-albumin (Luc-Alb) and luc-insulin (Luc-Ins) conjugates were used in competitive ELISA for the detection of an analyte (albumin or insulin) in the samples. Luc-progesterone (Luc-Pg) was used in the rapid homogeneous immunoassay of progesterone by the BRET technique with the detection limit of 0.5 ng/ml. Luciferase conjugates with avidin (Luc-Avi) and secondary and primary antibodies (Luc-RAM and Luc-Sal) were used for enzyme immunoassay detection of Salmonella paratyphi A cells with the cell detection limit of 5 × 104 CFU/ml. To reduce the detection limit of Salmonella cells, we developed a pseudo-homogeneous bioluminescent enzyme immunoassay of cells using a new matrix for the analyte capture-polystyrene microparticles coated with Pluronic F108, covalently labeled with Sal antibodies. This allowed to achieve efficient trapping of cells from solution, significantly reduced nonspecific sorption and decreased the cell detection limit to 2.7 × 103 CFU/ml without prior concentration of the sample. The methodology that was developed in this study can be applied for the development of novel bioanalytical systems based on firefly luciferases.

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.

Application of Bioluminescent Methods to Study the Effect of the Membrane-active Antibiotic Colistin on Bacterial Cells.

For the first time, recombinant Escherichia coli cells expressing thermostable Luciola mingrelica firefly luciferase were used to study the effect of the membrane-active antibiotic colistin on live cells. Simple, fast, and highly sensitive bioluminescent methods were developed for measurement of luciferase activity and ATP concentration inside and outside E. coli cells incubated in a nutrient medium, or in saline. Luciferase proved to be an informative protein marker for detecting the irreversible changes in cell membrane permeability. The study of kinetics of intra- and extracellular ATP concentration at different concentrations of colistin showed that the rate of decrease in intracellular ATP concentration significantly exceeded the rate of accumulation of extracellular ATP concentration. This fact could not be explained only by the release of ATP from the cell with an increase in the permeability of the outer cell membrane under the action of colistin. The loss of a significant part of intracellular ATP in presence of the colistin is probably due to a decrease in the activity of the respiratory chain enzymes and ATP synthase which operate in the cytoplasmic cell membrane, which leads to a decrease in the rate of ATP synthesis or even to its halt.

Mechanisms of increased mitochondria-dependent necrosis in Wiskott-Aldrich syndrome platelets.

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs 3.9% in controls; P<0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P<0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia.

A Novel Streptavidin-luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA.

A streptavidin-luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin-luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin-binding activities. It was shown that fusion has the same Km values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10-18 -10-13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin-streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin-luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.

Firefly Luciferase-based Fusion Proteins and their Applications in Bioanalysis.

Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin-binding domain and streptavidin, with proteins A and G, antibodies, with DNA- and RNA-binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase-based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET-based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein-protein interactions, assaying of metabolites involved in cell communication and cell signaling.

Color-shifting mutations in the C-domain of L. mingrelica firefly luciferase provide new information about the domain alternation mechanism.

We identified three color-shifting mutations-Phe467Ser, Glu490Val, and Glu490Lys-in the C-domain of the wild-type recombinant L. mingrelica luciferase. These mutations had moderate effect on the specific activity and thermal stability of the enzyme but changed the pH-dependence of its bioluminescence spectra. We constructed the model structures of the enzyme in three known conformations (open, adenylation, and oxidation conformation). The structural analysis and experimental data provided no evidences that these residues participate in structure-forming interactions in the open or oxidation conformation or that their mutations alter the overall structure of the enzyme. Given that the bioluminescence spectra reflect the microenvironment of the emitter (oxyluciferin in an electronically excited state), we concluded that the mutated residues affect the active site during the emission of light via short-range interactions. We found that it is only in the adenylation conformation that the residues Phe467 and Glu490 approach the N-domain, whereas the domain rotation associated with the oxidation conformation completely removes them from the active site. Therefore, the emission most likely occurs from the adenylation conformation.

A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine.

We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1ml saline and incubated for 15min at room temperature and then for 1h at 37°C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К=0.36±0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone.

The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) .

Bioanalytical systems based on bioluminescence resonance energy transfer using firefly luciferase.

Bioanalytical systems based on the Bioluminescence Resonance Energy Transfer (BRET) are widely used in fundamental biochemical studies, as well as for screening and analysis of biologically active compounds. The Renilla luciferase is the most often used energy donor in this system despite the fact that it has low bioluminescence quantum yield and demonstrates not so stable luminescence in time as the firefly luciferase. Moreover, the bioluminescence λmax is observed in the green region of the spectrum, which complicates signal recording in tissues during in vivo experiments. The firefly luciferases do not have such drawbacks and show great promise for applications in BRET systems. Different versions of BRET systems based on firefly luciferases and the methods for increasing their efficiency are considered in this review; examples of the use of BRET systems based on the firefly luciferases for highly sensitive determination of proteases and for homogeneous immunoassays are presented.

Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.

Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.

Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence.

Bioluminescence spectra of firefly luciferases demonstrate highly pH-sensitive spectra changing the color from green to red light when pH is lowered from alkaline to acidic. This reflects a change of ratio of the green and red emitters in the bimodal spectra of bioluminescence. We show that the mutations strongly stabilizing green (Y35N) or red (H433Y) emission compensate each other leading to the WT color of firefly luciferase. We further used this compensating ability of Y35N to search for strong red-shifting mutations in the C-domain of firefly luciferase by random mutagenesis. The discovered mutation E457K substantially increased the contribution of the red emitter and caused a 12 nm red shift of the green emitter as well. E457 is highly conservative not only in beetle luciferases but also in a whole ANL superfamily of adenylating enzymes and forms a conservative structural hydrogen bond with V471. Our results suggest that the removal of this hydrogen bond only mildly affects luciferase properties and that most of the effect of E457K is caused by the introduction of positive charge. E457 forms a salt bridge with R534 in most ANL enzymes including pH-insensitive luciferases which is absent in pH-sensitive firefly luciferases. The mutant A534R shows that this salt bridge is not important for pH-sensitivity but considerably improves in vivo thermostability. Although E457 is located far from the oxyluciferin-binding site, the properties of the mutant E457K suggest that it affects color by influencing the AMP binding.

Approaches to engineer stability of beetle luciferases.

Luciferase enzymes from fireflies and other beetles have many important applications in molecular biology, biotechnology, analytical chemistry and several other areas. Many novel beetle luciferases with promising properties have been reported in the recent years. However, actual and potential applications of wild-type beetle luciferases are often limited by insufficient stability or decrease in activity of the enzyme at the conditions of a particular assay. Various examples of genetic engineering of the enhanced beetle luciferases have been reported that successfully solve or alleviate many of these limitations. This mini-review summarizes the recent advances in development of mutant luciferases with improved stability and activity characteristics. It discusses the common limitations of wild-type luciferases in different applications and presents the efficient approaches that can be used to address these problems.

Thermostabilization of firefly luciferase by in vivo directed evolution.

Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.

Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelica firefly luciferase.

Insufficient thermal stability of firefly luciferases often limits their application in a wide range of fields. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases. However, for Hotaria parvula firefly luciferase, that shares 98% degree of homology with Luciola mingrelica luciferase, the A217L mutation is known to dramatically decrease catalytic activity. We analyzed the environment of A217 in the 3D-structure of L. mingrelica luciferase with the purpose of identifying possible additional mutations that would allow retention of the high thermal stability of the A217L mutant while preserving the high level of activity. The G216N/A217L double mutant of L. mingrelica luciferase demonstrated significantly improved stability at 42 and 45 °C but retained only 10% of activity; the loss in activity was accompanied by a large red shift of bioluminescence emission maximum from 566 to 611 nm compared with the wild-type enzyme. The triple mutant G216N/A217L/S398M exhibited high thermal stability of the double mutant together with the high activity and bioluminescence spectra close to that of the wild-type luciferase.

Improved bioluminescent assay of somatic cell counts in raw milk.

Somatic cell count (SCC) in milk is considered to be a valuable indicator of cow mastitis. For assessment of SCC in milk, the bioluminescent assay based on determination of ATP from somatic cells ([ATPsom]) in milk was proposed earlier. However, this assay is still not widely used in practice owing to lower reliability compared with conventional methods such as direct microscopy and flow cytometry. We revised the bioluminescent SCC assay and developed a simple protocol based on determination of the total non-bacterial ATP concentration in milk. It was shown that the novel ATP-releasing agent Neonol-10 (oxy-ethylated iso-nonyl phenol) has superior performance providing 100% lysis of somatic cells while not disrupting bacterial cells of milk at a concentration of 1.5% w/w. There was high correlation (R2=0.99) between measured bioluminescence and SCC as measured by direct microscopy. The observed detection limit of the bioluminescent milk SCC assay was as low as 900 cell/ml, time of analysis was 2-3 min per sample. The proposed method has high potential for on-site mastitis diagnostics.

Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods.

The results of the author's laboratory on the interaction of Luciola mingrelica firefly luciferase with substrates and their analogs using both steady-state and time resolved fluorescence are reviewed. The contribution of fluorescence of Trp and Tyr residues of the protein to its intrinsic fluorescence spectrum was estimated. Studies of quenching of Trp and Tyr fluorescence by luciferin and ATP allowed one to determine binding constants of the luciferase with substrates and to show that the binding of one substrate to the luciferase decreases the affinity of the enzyme for the other one. Fluorescence of oxyluciferin and its analogs (dimethyl- and monomethyloxyluciferins) was shown to be a good model of native firefly bioluminescence. A comparison of the fluorescence spectra of oxyluciferin and its analogs in aqueous solutions and in the presence of the luciferase revealed specific and nonspecific effects of the microenvironment on the equilibrium between different ionic forms of oxyluciferin. An approach based on photo-physical concepts of the correlation between luminescence spectra and structure of the emitter and its microenvironment was proposed and this approach was used to analyze bioluminescence spectra of wild-type and mutant luciferases.