A5029609853- bioluminescence and chemiluminescence research
- Cell Image Analysis Techniques
- Photoreceptor and optogenetics research
- Biosensors and Analytical Detection
- 3D Printing in Biomedical Research
- Computational Drug Discovery Methods
- Nicotinic Acetylcholine Receptors Study
- Bacterial biofilms and quorum sensing
- Neurobiology and Insect Physiology Research
- Viral Infectious Diseases and Gene Expression in Insects
- Transgenic Plants and Applications
- Bacillus and Francisella bacterial research
- Insect and Pesticide Research
Connecticut College
2012-2023
University of Bologna
2007
Pfizer (United States)
2004
Uppsala University
2003
Beetle luciferases catalyze a two-step reaction that includes the initial adenylation of luciferin substrate, followed by an oxidative decarboxylation ultimately produces light. Evidence for homologous acyl-CoA synthetases supports domain alternation catalytic mechanism in which these enzymes' C-terminal rotates ~140° to adopt two conformations are used partial reactions. While many structures exist both conformations, date only biochemical evidence with luciferase. We have determined...
Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, emitter of bioluminescence. We have confirmed generally held notions oxidation step is initiated formation a carbanion intermediate and hydroperoxide (anion) involved. Additionally, structural evidence presented accounts for delivery oxygen reaction site. Herein, we report key convincing spectroscopic participation superoxide anion in...
Beetle luciferases (including those of the firefly) use same luciferin substrate to naturally display light ranging in color from green (λmax ∼530 nm) red ∼635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, and Stroh, J. G. (2002) Am. Chem. Soc. 124, 2112−2113) that synthetic adenylate firefly analogue d-5,5-dimethylluciferin was transformed into emitter 5,5-dimethyloxyluciferin bioluminescence reactions catalyzed by...
Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes small animal models. However, due to attenuation visible light by tissue, and limited set near-infrared bioluminescent enzymes, BLI largely restricted monitoring single vivo. Here we show, that combining stabilised colour mutants firefly luciferase (FLuc) with luciferin (LH2) analogue infraluciferin (iLH2), dual can be achieved The X-ray crystal structure FLuc a high-energy...
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence reactions that require Mg-ATP and molecular oxygen. We had previously developed [Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., Zimmer, (1998) Biochemistry 37, 15311−15319] graphics-based working model active site starting with first X-ray structure [Conti, E., Franks, N. P., Brick, P. (1996) Structure 4, 287−298] enzyme without bound...
Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first adenylation substrate luciferin with Mg-ATP followed by multistep oxidation adenylate to form light emitter oxyluciferin an electronically excited state. beetle luciferases are members a large superfamily, mainly comprised nonbioluminescent enzymes that activate carboxylic acid substrates acyl-adenylate intermediates. Recently, crystal structure member this adenylate-forming...
Five novel firefly luciferin analogues in which the benzothiazole ring system of natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, kinetic properties compounds evaluated recombinant Photinus pyralis wild type luciferase. With exception indole, all substrates containing heterocycle substitutions produced readily measurable flashes light Compared to that luciferin, intensities ranged from...
According to the domain alternation mechanism and crystal structure evidence, acyl-CoA synthetases, one of three subgroups a superfamily adenylating enzymes, catalyze adenylate- thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting active sites through an ~140° rotation C-domain. second half-reaction catalyzed another subgroup, beetle luciferases, is mechanistically dissimilar oxidative process that produces bioluminescence. We have...
Effective methods for monitoring eukaryotic gene expression and regulation based on bioluminescence - the emission of light by living organisms are well established. Typically, a interest is reported with high sensitivity over wide dynamic range from variety engineered luciferase genes beetles marine organisms. The reporter expressed downstream target or promoter detected after exogenous addition luciferin substrates. We describe novel method simultaneous two expressing firefly variants that...
Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557 nm) light, is widely believed to be most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. In a previous study designed produce luciferases simultaneously two expression events, we identified very promising blue-shifted emitter (548 that contained mutations Val241Ile, Gly246Ala, and Phe250Ser [Branchini, B. R., Southworth, T. L.,...
The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis wild-type genes, expanded the applicability these reporters from perspective using more proteins emitting at different wavelengths in same cell-based assay. By spectrally resolving light emitted reporter it is fact possible to simultaneously monitor multiple targets. A luciferase isolated Luciola italica has been recently cloned, thermostable red- green-emitting mutants were random site-directed...
The marine annelid Chaetopterus variopedatus produces bioluminescence by an unknown and potentially novel mechanism. We have advanced the study of this fascinating phenomenon, which has not been investigated for nearly 60 years after initial studies were first reported species. Here, we show that luminous slime produced worm exhibits blue fluorescence matches emission. This result suggests oxyluciferin emitter is present. However, while decays over time green increasingly revealed likely...
Abstract Unlike the enchanting yellow‐green flashes of light produced on warm summer evenings by Photinus pyralis , most common firefly species in North America, orange lights scintillans are infrequently observed. These species, and likely all bioluminescent beetles, use same substrates beetle luciferin, ATP oxygen to produce light. It is structure particular luciferase enzyme that key determining color emitted We report here molecular cloning P. luc gene expression characterization...
We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains N-domain recombinant Photinus pyralis luciferase joined to C-domain Luciola italica luciferase. Compared P. enzyme, novel PpyLit chimera exhibited 1.8-fold flash-height specific activity, 2.0-fold integration-based 2.9-fold catalytic efficiency (kcat/Km), and 1.4-fold greater quantum yield. The results this study provide an underlying basis unusual example with are not simply sum contributions two...