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Eugene S. Vysotski - One of the best experts on this subject based on the ideXlab platform.

  • Unusual shift in the visible absorption spectrum of an active ctenophore Photoprotein elucidated by time-dependent density functional theory
    Photochemical & Photobiological Sciences, 2021
    Co-Authors: Felix N. Tomilin, Ludmila P. Burakova, Anastasia V. Rogova, Olga N. Tchaikovskaya, Pavel V. Avramov, Dmitri G. Fedorov, Eugene S. Vysotski
    Abstract:

    Active hydromedusan and ctenophore Ca^2+-regulated Photoproteins form complexes consisting of apoprotein and strongly non-covalently bound 2-hydroperoxycoelenterazine (an oxygenated intermediate of coelenterazine). Whereas the absorption maximum of hydromedusan Photoproteins is at 460–470 nm, ctenophore Photoproteins absorb at 437 nm. Finding out a physical reason for this blue shift is the main objective of this work, and, to achieve it, the whole structure of the protein–substrate complex was optimized using a linear scaling quantum–mechanical method. Electronic excitations pertinent to the spectra of the 2-hydroperoxy adduct of coelenterazine were simulated with time-dependent density functional theory. The dihedral angle of 60° of the 6-( p -hydroxy)-phenyl group relative to the imidazopyrazinone core of 2-hydroperoxycoelenterazine molecule was found to be the key factor determining the absorption of ctenophore Photoproteins at 437 nm. The residues relevant to binding of the substrate and its adopting the particular rotation were also identified.

  • Luminescence Activity Decreases When v-coelenterazine Replaces Coelenterazine in Calcium-Regulated Photoprotein-A Theoretical and Experimental Study.
    Photochemistry and photobiology, 2020
    Co-Authors: Bo-wen Ding, Elena V. Eremeeva, Eugene S. Vysotski, Ya-jun Liu
    Abstract:

    Calcium-regulated Photoproteins are found in at least five phyla of organisms. The light emitted by those Photoproteins can be tuned by mutating the Photoprotein and/or by modifying the substrate coelenterazine (CTZ). Thirty years ago, Shimomura observed that the luminescence activity of aequorin was dramatically reduced when the substrate CTZ was replaced by its analog v-CTZ. The latter is formed by adding a phenyl ring to the π-conjugated moiety of CTZ. The decrease in luminescence activity has not been understood until now. In this paper, through combined quantum mechanics and molecular mechanics calculations as well as molecular dynamics simulations, we discovered the reason for this observation. Modification of the substrate changes the conformation of nearby aromatic residues and enhances the π-π stacking interactions between the conjugated moiety of v-CTZ and the residues, which weakens the charge transfer to form light emitter and leads to a lower luminescence activity. The microenvironments of CTZ in obelin and in aequorin are very similar, so we predicted that the luminescence activity of obelin will also dramatically decrease when CTZ is replaced by v-CTZ. This prediction has received strong evidence from currently theoretical calculations and has been verified by experiments.

  • The interaction of C-terminal Tyr208 and Tyr13 of the first α-helix ensures a closed conformation of ctenophore Photoprotein berovin
    Photochemical & Photobiological Sciences, 2020
    Co-Authors: Ludmila P. Burakova, Elena V. Eremeeva, Eugene S. Vysotski
    Abstract:

    Light-sensitive Ca^2+-regulated Photoprotein berovin is responsible for the bioluminescence of the cteno-phore Beroe abyssicola. It shares many properties of hydromedusan Photoproteins although the degree of identity of its amino acid sequence with those of Photoproteins is low. There is a hydrogen bond between C-terminal Pro and Arg situated in the N-terminal α-helix of hydromedusan Photoproteins that supports a closed conformation of the internal cavity of the Photoprotein molecule with bound 2-hydro-peroxycoelenterazine. The C- and N-terminal hydrogen bond network is necessary to properly isolate the Photoprotein active site from the solvent and consequently to provide a high quantum yield of the bioluminescence reaction. In order to find out which berovin residues perform the same function we modified the N- and C-termini of the protein by replacing or deleting various amino acid residues. The studies on berovin mutants showed that the interaction between C-terminal Tyr208 and Tyr13 localized in the first α-helix of the Photoprotein is important for the stabilization and proper orientation of the oxygenated coelenterazine adduct within the internal cavity as well as for supporting the closed Photoprotein conformation. We also suggest that the interplay between Tyr residues in ctenophore Photoproteins occurs rather through the π–π interaction of their phenyl rings than through hydrogen bonds as in hydro-medusan Photoproteins.

  • Recombinant Ca^2+-regulated Photoproteins of ctenophores: current knowledge and application prospects
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Lyudmila P. Burakova, Eugene S. Vysotski
    Abstract:

    Bright bioluminescence of ctenophores is conditioned by Ca^2+-regulated Photoproteins. Although they share many properties characteristic of hydromedusan Ca^2+-regulated Photoproteins responsible for light emission of marine animals belonging to phylum Cnidaria , a substantial distinction still exists. The ctenophore Photoproteins appeared to be extremely sensitive to light—they lose the ability for bioluminescence on exposure to light over the entire absorption spectrum. Inactivation is irreversible because keeping the inactivated Photoprotein in the dark does not recover its activity. The capability to emit light can be restored only by incubation of inactivated Photoprotein with coelenterazine in the dark at alkaline pH in the presence of oxygen. Although these Photoproteins were discovered many years ago, only the cloning of cDNAs encoding these unique bioluminescent proteins in the early 2000s has provided a new impetus for their studies. To date, cDNAs encoding Ca^2+-regulated Photoproteins from four different species of luminous ctenophores have been cloned. The amino acid sequences of ctenophore Photoproteins turned out to completely differ from those of hydromedusan Photoproteins (identity less than 29%) though also similar to them having three EF-hand Ca^2+-binding sites. At the same time, these Photoproteins reveal the same two-domain scaffold characteristic of hydromedusan Photoproteins. This review is an attempt to systemize and critically evaluate the data scattered through various articles regarding the structural features of recombinant light-sensitive Ca^2+-regulated Photoproteins of ctenophores and their bioluminescent and physicochemical properties as well as to compare them with those of hydromedusan Photoproteins. In addition, we also discuss the prospects of their biotechnology applications.

  • Recombinant Ca2+-regulated Photoproteins of ctenophores: current knowledge and application prospects.
    Applied microbiology and biotechnology, 2019
    Co-Authors: Lyudmila P. Burakova, Eugene S. Vysotski
    Abstract:

    Bright bioluminescence of ctenophores is conditioned by Ca2+-regulated Photoproteins. Although they share many properties characteristic of hydromedusan Ca2+-regulated Photoproteins responsible for light emission of marine animals belonging to phylum Cnidaria, a substantial distinction still exists. The ctenophore Photoproteins appeared to be extremely sensitive to light—they lose the ability for bioluminescence on exposure to light over the entire absorption spectrum. Inactivation is irreversible because keeping the inactivated Photoprotein in the dark does not recover its activity. The capability to emit light can be restored only by incubation of inactivated Photoprotein with coelenterazine in the dark at alkaline pH in the presence of oxygen. Although these Photoproteins were discovered many years ago, only the cloning of cDNAs encoding these unique bioluminescent proteins in the early 2000s has provided a new impetus for their studies. To date, cDNAs encoding Ca2+-regulated Photoproteins from four different species of luminous ctenophores have been cloned. The amino acid sequences of ctenophore Photoproteins turned out to completely differ from those of hydromedusan Photoproteins (identity less than 29%) though also similar to them having three EF-hand Ca2+-binding sites. At the same time, these Photoproteins reveal the same two-domain scaffold characteristic of hydromedusan Photoproteins. This review is an attempt to systemize and critically evaluate the data scattered through various articles regarding the structural features of recombinant light-sensitive Ca2+-regulated Photoproteins of ctenophores and their bioluminescent and physicochemical properties as well as to compare them with those of hydromedusan Photoproteins. In addition, we also discuss the prospects of their biotechnology applications.

Sylvia Daunert - One of the best experts on this subject based on the ideXlab platform.

  • red shifted aequorin variants incorporating non canonical amino acids applications in in vivo imaging
    PLOS ONE, 2016
    Co-Authors: Kristen Marie Grinstead, Laura Rowe, Emre Dikici, Charles Mark Ensor, Smita Joel, Pirouz Daftarian, Jean Marc Zingg, Sylvia Daunert
    Abstract:

    The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic Photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these Photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

  • Spectral tuning of Photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs
    Protein engineering design & selection : PEDS, 2008
    Co-Authors: Laura Rowe, Charles Mark Ensor, Anna Rothert, C. Logue, Sapna K. Deo, Sylvia Daunert
    Abstract:

    Aequorin and obelin are Photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these Photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic Photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic Photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic Photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

  • Photoproteins in Bioanalysis: DAUNERT: PhotoproteinS IN BIOANALYSIS O-BK - Photoproteins in Bioanalysis
    2006
    Co-Authors: Sylvia Daunert
    Abstract:

    Preface. List of Contributors. 1 The Photoproteins (Osamu Shimomura). 1.1 Discovery of Photoprotein. 1.2 Various Types of Photoproteins Presently Known. 1.2.1 Radiolarian (Protozoa) Photoproteins. 1.2.2 Coelenterate Photoproteins. 1.2.3 Ctenophore Photoproteins. 1.2.4 Pholasin (Pholas Luciferin). 1.2.5 Chaetopterus Photoprotein. 1.2.6 Polynoidin. 1.2.7 Symplectin. 1.2.8 Luminodesmus Photoprotein. 1.2.9 Ophiopsila Photoprotein. 1.3 Basic Strategy of Extracting and Purifying Photoproteins. 1.4 The Photoprotein Aequorin. 1.4.1 Extraction and Purifi cation of Aequorin. 1.4.1.1 Hydrophobic Interaction Chromatography. 1.4.2 Properties of Aequorin. 1.4.2.1 Stability. 1.4.2.2 Freeze-drying. 1.4.3 Specifi city to Ca 2+ . 1.4.4 Luminescence of Aequorin by Substances Other Than Divalent Cations. 1.4.5 Mechanism of Aequorin Luminescence and Regeneration of Aequorin. 1.4.5.1 Structure of Aequorin. 1.4.5.2 Luminescence Reaction. 1.4.5.3 Regeneration. 1.4.6 Inhibitors of Aequorin Luminescence. 1.4.7 Recombinant Aequorin. 1.4.8 Semi-synthetic Aequorins. 1.4.8.1 e-Aequorins. References. 2 Luminous Marine Organisms (Steven H.D. Haddock). 2.1 Introduction. 2.1.1 Non-luminous Taxa. 2.1.2 Luminous Taxa. 2.2 Taxonomic Distribution of Bioluminescence. 2.2.1 Bacterial Luminescence. 2.2.2 Dinofl agellate Luciferin. 2.2.3 Cypridina (Vargula) Luciferin. 2.2.4 Coelenterazine. 2.2.5 Other Luciferins: Known and Unknown. 2.3 Functions. 2.3.1 Startle or Distract. 2.3.2 Burglar Alarm. 2.3.3 Counterillumination. 2.3.4 Mating Displays. 2.3.5 Prey Attraction. References. 3 Beetle Luciferases: Colorful Lights on Biological Processes and Diseases (Vadim R. Viviani and Yoshihiro Ohmiya). 3.1 Introduction. 3.2 Beetle Luciferases. 3.3 Bioanalytical Assays of ATP. 3.3.1 Biomass Estimation and Microbiological Contamination. 3.3.2 Cytotoxicity and Cell Viability Tests. 3.3.3 Enzymatic Assays. 3.4 Luciferases as Reporter Genes. 3.4.1 Dual and Multiple Reporter Assays. 3.5 Biophotonic Imaging in Animals: A Living Light on Diseases. 3.5.1 Pathogen Infection in Living Models. 3.5.2 Drug Screening. 3.5.3 Tumor Proliferation and Regression Studies. 3.5.4 Gene Delivery and Gene Therapy. 3.5.5 Luciferase as Biomarkers for Cell Traffi cking Studies. 3.5.6 Immunoassays. 3.6 Biophotonic Imaging in Plants. 3.7 Biosensors: Sensing the Environment. 3.8 Novel Luciferases: Different Colors for Different Occasions. References. 4 Split Luciferase Systems for Detecting Protein-Protein Interactions in Mammalian Cells Based on Protein Splicing and Protein Complementation (Yoshio Umezawa). 4.1 Introduction. 4.2 Protein Splicing-based Split Firefl y Luciferase System [23]. 4.2.1 Split Luciferase Works as a Probe for Protein Interaction. 4.3 Split Renilla Luciferase Complementation System [33]. 4.3.1 Time Course of the Interaction Between Y941 and SH2n. 4.3.2 Location of the Interaction Between Y941 and SH2n. References. 5 Photoproteins in Nucleic Acid Analysis (Theodore K. Christopoulos, Penelope C. Ioannou, and Monique Verhaegen). 5.1 Hybridization Assays. 5.2 Quantitative Polymerase Chain Reaction. 5.3 Genotyping of Single-nucleotide Polymorphisms. 5.4 Conjugation of Aequorin to Oligodeoxynucleotide Probes. 5.5 Development of New Recombinant Bioluminescent Reporters. 5.6 Signal Amplifi cation by in Vitro Expression of DNA Reporters Encoding Bioluminescent Proteins. 5.7 Conclusions. References. 6 Bioluminescence Resonance Energy Transfer in Bioanalysis (Suresh Shrestha and Sapna K. Deo). 6.1 Introduction. 6.2 BRET Principle, Effi ciency, and Instrumentation. 6.3 Comparison of BRET and FRET. 6.4 Examples of BRET Donor-Acceptor Pairs. 6.5 Applications of BRET in Bioanalysis. 6.5.1 Homogeneous Assays. 6.5.2 Protein-Protein Interactions and High-throughput Screening. 6.6 Conclusions. References. 7 Photoproteins as in Vivo Indicators of Biological Function (Rajesh Shinde, Hui Zhao, and Christopher H. Contag). 7.1 Overview. 7.2 Probes Used for in Vivo Bioluminescence Imaging. 7.3 Probes Used for in Vivo Fluorescence Imaging. 7.4 Detection Technologies. 7.5 Current Applications. 7.5.1 Oncology. 7.5.2 Infectious Disease. 7.5.3 Bacterial Infections. 7.5.4 Viral Infections. 7.5.5 Viral-mediated Gene Transfer. 7.5.6 Cell Biology. 7.5.7 Stem Cell Biology. 7.6 Protease Sensors. 7.7 Conclusions. References. 8 Photoproteins as Reporters in Whole-cell Sensing (Jessika Feliciano, Patrizia Pasini, Sapna K. Deo, and Sylvia Daunert). 8.1 Introduction. 8.1.1 Biosensors Using Intact Cells. 8.1.2 Reporter Genes in Genetically Engineered Whole-cell Sensors. 8.2 The Luciferases. 8.2.1 Bacterial Luciferases. 8.2.1.1 luxAB Bioreporters. 8.2.1.2 luxCDABE Bioreporters. 8.2.1.3 Naturally Luminescent Bioreporters. 8.2.2 Eukaryotic Luciferases. 8.2.2.1 Firefl y Luciferase. 8.2.2.2 Sea Pansy Luciferase. 8.3 Aequorin. 8.4 Fluorescent Proteins. 8.4.1 Green Fluorescent Protein. 8.4.2 Red Fluorescent Protein. 8.5 Multiplexing. 8.6 Applications. 8.6.1 Stress Factors and Genotoxicants. 8.6.2 Environmental Pollutants. 8.6.3 Quorum-sensing Signaling Molecules. 8.6.4 Antibiotics. 8.7 Technological Advances. References. 9 Luminescent Proteins in Binding Assays (Aldo Roda, Massimo Guardigli, Elisa Michelini, Mara Mirasoli, and Patrizia Pasini). 9.1 Introduction. 9.2 Protein-Protein and Protein-Ligand Interaction Assays. 9.2.1 FRET and BRET Techniques. 9.2.2 FRET and BRET Applications. 9.2.3 Other Detection Principles. 9.3 Antibody-based Binding Assays. 9.3.1 Chemical Conjugation. 9.3.2 Gene Fusion. 9.3.3 Dual-analyte Assays. 9.3.4 Expression Immunoassays. 9.3.5 BRET-based Immunoassays. 9.4 Biotin-Avidin Binding Assays. 9.5 Nucleic Acid Hybridization Assays. 9.6 Other Binding Assays. 9.7 Concluding Remarks. References. 10 Luminescent Proteins: Applications in Microfl uidics and Miniaturized Analytical Systems (Emre Dikici, Laura Rowe, Elizabeth A. Moschou, Anna Rothert, Sapna K. Deo, and Sylvia Daunert). 10.1 Miniaturization and Microfl uidics. 10.2 Photoproteins and Applications in Miniaturized Detection Systems. 10.2.1 Green Fluorescent Protein. 10.2.1.1 GFP in Miniaturized Microfl uidic-based Assays. 10.2.2 Luciferase. 10.2.2.1 Luciferase in Miniaturized Microfl uidic-based Assays. 10.2.3 Aequorin. 10.2.3.1 Aequorin in Miniaturized Microfl uidic-based Assays. 10.3 Future Perspectives. References. 11 Advances in Instrumentation for Detecting Low-level Bioluminescence and Fluorescence (Eric Karplus). 11.1 Introduction. 11.2 Low Light Levels. 11.3 Methods of Coupling the Signal to the Detector. 11.3.1 Proximity Focusing. 11.3.2 Microscope Objectives. 11.3.3 Macro Lenses. 11.3.4 Fiber Optics. 11.4 Evaluating the Performance of an Optical System. 11.4.1 Numerical Aperture. 11.4.2 Transmission Effi ciency. 11.4.3 Magnifi cation. 11.5 Detector Technologies. 11.6 Selecting the Right Detector. 11.7 Detector Sensitivity. 11.8 Detector Noise. 11.9 Statistics of Photon Counting. 11.10 Summary. References. 12 Photoproteins and Instrumentation: Their Availability and Applications in Bioanalysis (Leslie Doleman, Stephanie Bachas-Daunert, Logan Davies, Sapna K. Deo, and Sylvia Daunert). Aequorin. Obelin. Luciferases. Aequorea and Anthozoa Fluorescent Proteins. Coelenteraziness. Luminometers. Fluorometers. Portable Luminometers. Disclaimer. Subject Index.

  • Photoproteins in Bioanalysis - Luminescent Proteins: Applications in Microfluidics and Miniaturized Analytical Systems
    Photoproteins in Bioanalysis, 2006
    Co-Authors: Emre Dikici, Laura Rowe, Anna Rothert, Sapna K. Deo, Elizabeth A. Moschou, Sylvia Daunert
    Abstract:

    The use of light-emitting proteins for the detection of biomolecules provides fast and sensitive methods which overcome the disadvantages of radioactive labels and the high cost of fluorescent dyes. This reference work summarizes modern advanced techniques and their applications and includes practical examples of assays based on Photoproteins. The book presents contemporary key topics like luminescent marine organisms, DNA probes, reporter gene assays and Photoproteins, ratiometric sensing, use of Photoproteins for in vivo functional imaging and luminescent proteins in binding assays, to name just a few, and is complemented by recent advances in instrumentation

  • Protein Science Encyclopedia - Photoproteins as Reporters in Whole‐cell Sensing
    Photoproteins in Bioanalysis, 2006
    Co-Authors: Jessika Feliciano, Sapna K. Deo, Patrizia Pasini, Sylvia Daunert
    Abstract:

    Originally published in: Photoproteins in Bioanalysis. Edited by Sylvia Daunert and Sapna K. Deo. Copyright © 2006 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-31016-6 The sections in this article are Introduction Biosensors Using Intact Cells Reporter Genes in Genetically Engineered Whole-cell Sensors The Luciferases Bacterial Luciferases luxAB Bioreporters luxCDABE Bioreporters Naturally Luminescent Bioreporters Eukaryotic Luciferases Firefly Luciferase Sea Pansy Luciferase Aequorin Fluorescent Proteins Green Fluorescent Protein Red Fluorescent Protein Multiplexing Applications Stress Factors and Genotoxicants Environmental Pollutants Quorum-sensing Signaling Molecules Antibiotics Technological Advances Acknowledgments Keywords: Photoproteins in bioanalysis; Photoproteins as reporters in whole-cell sensing; luciferases; aequorin; fluorescent proteins; multiplexing; applications; technological advances

Rosario Rizzuto - One of the best experts on this subject based on the ideXlab platform.

  • using targeted variants of aequorin to measure ca2 levels in intracellular organelles
    CSH Protocols, 2014
    Co-Authors: Veronica Granatiero, Maria Patron, Anna Tosatto, Giulia Merli, Rosario Rizzuto
    Abstract:

    Aequorin is a Ca(2+)-sensitive Photoprotein isolated from the jellyfish Aequorea victoria. It is an ideal probe for measuring Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because it can be modified to include specific targeting sequences. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group coelenterazine is released and a photon is emitted. This protocol presents procedures for expressing, targeting, and reconstituting aequorin in intact and permeabilized mammalian cells and describes how to use this Photoprotein to measure intracellular [Ca(2+)] in various subcellular compartments.

  • The Use of Aequorin and Its Variants for Ca2+ Measurements
    Cold Spring Harbor protocols, 2014
    Co-Authors: Veronica Granatiero, Maria Patron, Anna Tosatto, Giulia Merli, Rosario Rizzuto
    Abstract:

    Ca(2+)-sensitive Photoproteins are ideal agents for measuring the Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because they can be modified to include specific targeting sequences. Aequorin was the first Ca(2+)-sensitive Photoprotein probe used to measure the [Ca(2+)] inside specific intracellular organelles in intact cells. Aequorin is a 22-kDa protein produced by the jellyfish Aequorea victoria. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group is released and a photon is emitted. Aequorin has become widely used for intracellular Ca(2+) measurements because it offers many advantages: For example, it can be targeted with precision, functions over a wide range of [Ca(2+)], and shows low buffering capacity. In this article we describe the main characteristics of the aequorin probe and review the reasons why it is widely used to measure intracellular [Ca(2+)].

  • subcellular calcium measurements in mammalian cells using jellyfish Photoprotein aequorin based probes
    Nature Protocols, 2013
    Co-Authors: Massimo Bonora, Rosario Rizzuto, Carlotta Giorgi, Angela Bononi, Saverio Marchi, Simone Patergnani, Alessandro Rimessi, Paolo Pinton
    Abstract:

    Subcellular calcium measurements in mammalian cells using jellyfish Photoprotein aequorin-based probes

  • recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling
    Biochemical Journal, 2001
    Co-Authors: A Chiesa, E Rapizzi, Valeria Tosello, Paolo Pinton, M De Virgilio, K E Fogarty, Rosario Rizzuto
    Abstract:

    Luminous proteins include primary light producers, such as aequorin, and secondary Photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene expression, and so on. The Ca(2+)-sensitive Photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reticulum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions, such as the bulk cytosol and the subplasmalemmal rim), and was used to analyse Ca(2+) homoeostasis at the subcellular level. We will discuss this application, reviewing its advantages and disadvantages and the experimental procedure. The applications of green fluorescent protein (GFP) are even broader. Indeed, the ability to molecularly engineer and recombinantly express a strongly fluorescent probe has provided a powerful tool for investigating a wide variety of biological events in live cells (e.g. tracking of endogenous proteins, labelling of intracellular structures, analysing promoter activity etc.). More recently, the demonstration that, using appropriate mutants and/or fusion proteins, GFP fluorescence can become sensitive to physiological parameters or activities (ion concentration, protease activity, etc.) has further expanded its applications and made GFP the favourite probe of cell biologists. We will here present two applications in the field of cell signalling, i.e. the use of GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases.

  • transfected aequorin in the measurement of cytosolic ca2 concentration ca2 c a critical evaluation
    Journal of Biological Chemistry, 1995
    Co-Authors: Marisa Brini, Tullio Pozzan, Robert Marsault, Carlo Bastianutto, Javier Alvarez, Rosario Rizzuto
    Abstract:

    Abstract Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., [Medline] Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant Photoproteins for measuring, in mammalian cells, the [Ca2+] in the cytoplasm, a compartment for which effective Ca2+probes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca2+] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 μM) are associated with a significant buffering of the [Ca2+]ctransients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the Photoprotein also allows an accurate estimate of the large [Ca2+]cincreases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca2+]cin cotransfection experiments. In particular, we show that when cotransfected with an α1-adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the untransfected cells.

Zhi-jie Liu - One of the best experts on this subject based on the ideXlab platform.

  • Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type Photoprotein.
    Journal of photochemistry and photobiology. B Biology, 2016
    Co-Authors: Ludmila P. Burakova, Elena V. Eremeeva, Svetlana V Markova, Pavel V. Natashin, Natalia P. Malikova, Zhi-jie Liu, Chongyun Cheng, Eugene S. Vysotski
    Abstract:

    Abstract The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated Photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 A resolution which expectedly revealed a high similarity with the structures of other hydromedusan Photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan Photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active Photoprotein, in contrast to C-terminal Pro of other Photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan Photoproteins with different bioluminescence properties.

  • all ca 2 binding loops of light sensitive ctenophore Photoprotein berovin bind magnesium ions the spatial structure of mg 2 loaded apo berovin
    Journal of Photochemistry and Photobiology B-biology, 2015
    Co-Authors: Ludmila Burakova, Eugene S. Vysotski, Pavel V. Natashin, Natalia P. Malikova, Fengfeng Niu, Zhi-jie Liu
    Abstract:

    Light-sensitive Photoprotein berovin accounts for a bright bioluminescence of ctenophore Beroe abyssicola. Berovin is functionally identical to the well-studied Ca(2+)-regulated Photoproteins of jellyfish, however in contrast to those it is extremely sensitive to the visible light. Berovin contains three EF-hand Ca(2+)-binding sites and consequently belongs to a large family of the EF-hand Ca(2+)-binding proteins. Here we report the spatial structure of apo-berovin with bound Mg(2+) determined at 1.75A. The magnesium ion is found in each functional EF-hand loop of a Photoprotein and coordinated by oxygen atoms donated by the side-chain groups of aspartate, carbonyl groups of the peptide backbone, or hydroxyl group of serine with characteristic oxygen-Mg(2+) distances. As oxygen supplied by the side-chain of the twelfth residue of all Ca(2+)-binding loops participates in the magnesium ion coordination, it was suggested that Ca(2+)-binding loops of berovin belong to the mixed Ca(2+)/Mg(2+) rather than Ca(2+)-specific type. In addition, we report an effect of physiological concentration of Mg(2+) on bioluminescence of berovin (sensitivity to Ca(2+), rapid-mixed kinetics, light-sensitivity, thermostability, and apo-berovin conversion into active protein). The different impact of physiological concentration of Mg(2+) on berovin bioluminescence as compared to hydromedusan Photoproteins was attributed to different affinities of the Ca(2+)-binding sites of these Photoproteins to Mg(2+).

  • All Ca2 +-binding loops of light-sensitive ctenophore Photoprotein berovin bind magnesium ions: The spatial structure of Mg2 +-loaded apo-berovin
    Journal of photochemistry and photobiology. B Biology, 2015
    Co-Authors: Ludmila P. Burakova, Eugene S. Vysotski, Pavel V. Natashin, Natalia P. Malikova, Fengfeng Niu, Zhi-jie Liu
    Abstract:

    Abstract Light-sensitive Photoprotein berovin accounts for a bright bioluminescence of ctenophore Beroe abyssicola. Berovin is functionally identical to the well-studied Ca2 +-regulated Photoproteins of jellyfish, however in contrast to those it is extremely sensitive to the visible light. Berovin contains three EF-hand Ca2 +-binding sites and consequently belongs to a large family of the EF-hand Ca2 +-binding proteins. Here we report the spatial structure of apo-berovin with bound Mg2 + determined at 1.75 A. The magnesium ion is found in each functional EF-hand loop of a Photoprotein and coordinated by oxygen atoms donated by the side-chain groups of aspartate, carbonyl groups of the peptide backbone, or hydroxyl group of serine with characteristic oxygen-Mg2 + distances. As oxygen supplied by the side-chain of the twelfth residue of all Ca2 +-binding loops participates in the magnesium ion coordination, it was suggested that Ca2 +-binding loops of berovin belong to the mixed Ca2 +/Mg2 + rather than Ca2 +-specific type. In addition, we report an effect of physiological concentration of Mg2 + on bioluminescence of berovin (sensitivity to Ca2 +, rapid-mixed kinetics, light-sensitivity, thermostability, and apo-berovin conversion into active protein). The different impact of physiological concentration of Mg2 + on berovin bioluminescence as compared to hydromedusan Photoproteins was attributed to different affinities of the Ca2 +-binding sites of these Photoproteins to Mg2 +.

  • Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan Photoproteins
    The FEBS journal, 2014
    Co-Authors: Pavel V. Natashin, Eugene S. Vysotski, Svetlana V Markova, John Lee, Zhi-jie Liu
    Abstract:

    Ca2+-regulated Photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan Photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant Photoproteins vary in the range 462–495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these Photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008–1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca2+ discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan Photoproteins.

  • Crystal structure of a Ca2+-discharged Photoprotein: implications for mechanisms of the calcium trigger and bioluminescence.
    The Journal of biological chemistry, 2004
    Co-Authors: Lu Deng, Eugene S. Vysotski, Svetlana V Markova, Zhi-jie Liu, John Lee, John P. Rose, Bi-cheng Wang
    Abstract:

    Ca2+-regulated Photoproteins are members of the EF-hand calcium-binding protein family. The addition of Ca2+ produces a blue bioluminescence by triggering a decarboxylation reaction of protein-bound hydroperoxycoelenterazine to form the product, coelenteramide, in an excited state. Based on the spatial structures of aequorin and several obelins, we have postulated mechanisms for the Ca2+ trigger and for generation of the different excited states that are the origin of the different colors of bioluminescence. Here we report the crystal structure of the Ca2+-discharged Photoprotein obelin at 1.96-A resolution. The results lend support to the proposed mechanisms and provide new structural insight into details of these processes. Global conformational changes caused by Ca2+ association are typical of the class of calcium signal modulators within the EF-hand protein superfamily. Accommodation of the Ca2+ ions into the loops of the EF-hands is seen to propagate into the active site of the protein now occupied by the coelenteramide where there is a significant repositioning and flipping of the His-175 imidazole ring as crucially required in the trigger hypothesis. Also the H-bonding between His-22 and the coelenterazine found in the active Photoprotein is preserved at the equivalent position of coelenteramide, confirming the proposed rapid excited state proton transfer that would lead to the excited state of the phenolate ion pair, which is responsible for the blue emission of bioluminescence.

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  • Unusual shift in the visible absorption spectrum of an active ctenophore Photoprotein elucidated by time-dependent density functional theory
    Photochemical & Photobiological Sciences, 2021
    Co-Authors: Felix N. Tomilin, Ludmila P. Burakova, Anastasia V. Rogova, Olga N. Tchaikovskaya, Pavel V. Avramov, Dmitri G. Fedorov, Eugene S. Vysotski
    Abstract:

    Active hydromedusan and ctenophore Ca^2+-regulated Photoproteins form complexes consisting of apoprotein and strongly non-covalently bound 2-hydroperoxycoelenterazine (an oxygenated intermediate of coelenterazine). Whereas the absorption maximum of hydromedusan Photoproteins is at 460–470 nm, ctenophore Photoproteins absorb at 437 nm. Finding out a physical reason for this blue shift is the main objective of this work, and, to achieve it, the whole structure of the protein–substrate complex was optimized using a linear scaling quantum–mechanical method. Electronic excitations pertinent to the spectra of the 2-hydroperoxy adduct of coelenterazine were simulated with time-dependent density functional theory. The dihedral angle of 60° of the 6-( p -hydroxy)-phenyl group relative to the imidazopyrazinone core of 2-hydroperoxycoelenterazine molecule was found to be the key factor determining the absorption of ctenophore Photoproteins at 437 nm. The residues relevant to binding of the substrate and its adopting the particular rotation were also identified.

  • The interaction of C-terminal Tyr208 and Tyr13 of the first α-helix ensures a closed conformation of ctenophore Photoprotein berovin
    Photochemical & Photobiological Sciences, 2020
    Co-Authors: Ludmila P. Burakova, Elena V. Eremeeva, Eugene S. Vysotski
    Abstract:

    Light-sensitive Ca^2+-regulated Photoprotein berovin is responsible for the bioluminescence of the cteno-phore Beroe abyssicola. It shares many properties of hydromedusan Photoproteins although the degree of identity of its amino acid sequence with those of Photoproteins is low. There is a hydrogen bond between C-terminal Pro and Arg situated in the N-terminal α-helix of hydromedusan Photoproteins that supports a closed conformation of the internal cavity of the Photoprotein molecule with bound 2-hydro-peroxycoelenterazine. The C- and N-terminal hydrogen bond network is necessary to properly isolate the Photoprotein active site from the solvent and consequently to provide a high quantum yield of the bioluminescence reaction. In order to find out which berovin residues perform the same function we modified the N- and C-termini of the protein by replacing or deleting various amino acid residues. The studies on berovin mutants showed that the interaction between C-terminal Tyr208 and Tyr13 localized in the first α-helix of the Photoprotein is important for the stabilization and proper orientation of the oxygenated coelenterazine adduct within the internal cavity as well as for supporting the closed Photoprotein conformation. We also suggest that the interplay between Tyr residues in ctenophore Photoproteins occurs rather through the π–π interaction of their phenyl rings than through hydrogen bonds as in hydro-medusan Photoproteins.

  • Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type Photoprotein.
    Journal of photochemistry and photobiology. B Biology, 2016
    Co-Authors: Ludmila P. Burakova, Elena V. Eremeeva, Svetlana V Markova, Pavel V. Natashin, Natalia P. Malikova, Zhi-jie Liu, Chongyun Cheng, Eugene S. Vysotski
    Abstract:

    Abstract The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated Photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 A resolution which expectedly revealed a high similarity with the structures of other hydromedusan Photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan Photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active Photoprotein, in contrast to C-terminal Pro of other Photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan Photoproteins with different bioluminescence properties.

  • Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca2+-regulated Photoprotein berovin
    Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2016
    Co-Authors: Ludmila P. Burakova, Elena V. Eremeeva, Galina A Stepanyuk, Eugene S. Vysotski
    Abstract:

    Bright bioluminescence of ctenophores is caused by Ca2+-regulated Photoproteins. Although these Photoproteins are functionally identical to and share many properties of cnidarian Photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian Photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian Photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the Photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca2+-regulated Photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand–protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore Photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge–charge interaction.

  • All Ca2 +-binding loops of light-sensitive ctenophore Photoprotein berovin bind magnesium ions: The spatial structure of Mg2 +-loaded apo-berovin
    Journal of photochemistry and photobiology. B Biology, 2015
    Co-Authors: Ludmila P. Burakova, Eugene S. Vysotski, Pavel V. Natashin, Natalia P. Malikova, Fengfeng Niu, Zhi-jie Liu
    Abstract:

    Abstract Light-sensitive Photoprotein berovin accounts for a bright bioluminescence of ctenophore Beroe abyssicola. Berovin is functionally identical to the well-studied Ca2 +-regulated Photoproteins of jellyfish, however in contrast to those it is extremely sensitive to the visible light. Berovin contains three EF-hand Ca2 +-binding sites and consequently belongs to a large family of the EF-hand Ca2 +-binding proteins. Here we report the spatial structure of apo-berovin with bound Mg2 + determined at 1.75 A. The magnesium ion is found in each functional EF-hand loop of a Photoprotein and coordinated by oxygen atoms donated by the side-chain groups of aspartate, carbonyl groups of the peptide backbone, or hydroxyl group of serine with characteristic oxygen-Mg2 + distances. As oxygen supplied by the side-chain of the twelfth residue of all Ca2 +-binding loops participates in the magnesium ion coordination, it was suggested that Ca2 +-binding loops of berovin belong to the mixed Ca2 +/Mg2 + rather than Ca2 +-specific type. In addition, we report an effect of physiological concentration of Mg2 + on bioluminescence of berovin (sensitivity to Ca2 +, rapid-mixed kinetics, light-sensitivity, thermostability, and apo-berovin conversion into active protein). The different impact of physiological concentration of Mg2 + on berovin bioluminescence as compared to hydromedusan Photoproteins was attributed to different affinities of the Ca2 +-binding sites of these Photoproteins to Mg2 +.