Genetically encodable bioluminescent system from fungi Kotlobay, Alexey A.; Sarkisyan, Karen S.; Mokrushina, Yuliana A. ...
Proceedings of the National Academy of Sciences - PNAS,
12/2018, Letnik:
115, Številka:
50
Journal Article
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Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, ...molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.
Integral membrane proteins are important components of a cell. Their structural and functional studies require production of milligram amounts of proteins, which nowadays is not a routine process. ...Cell-free protein synthesis is a prospective approach to resolve this task. However, there are few known membrane mimetics that can be used to synthesize active membrane proteins in high amounts. Here, we present the application of commercially available “Facade” detergents for the production of active rhodopsin. We show that the yield of active protein in lipid bicelles containing Facade-EM, Facade-TEM, and Facade-EPC is several times higher than in the case of conventional bicelles with CHAPS and DHPC and is comparable to the yield in the presence of lipid-protein nanodiscs. Moreover, the effects of the lipid-to-detergent ratio, concentration of detergent in the feeding mixture, and lipid composition of the bicelles on the total, soluble, and active protein yields are discussed. We show that Facade-based bicelles represent a prospective membrane mimetic, available for the production of membrane proteins in a cell-free system.
The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in ...heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results ...provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.
Let there be light: The oxyluciferin from the bioluminescent worm Fridericia heliota was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.
Green fluorescent protein (GFP) is widely used as an excellent reporter module of the fusion proteins. The unique structure of GFP allows isolation of the active fluorescent protein directly from the ...crude cellular sources by extraction with organic solvents. We demonstrated the stable expression of four short polypeptides fused to GFP in
Escherichia coli cells, including antimicrobial cationic peptides, which normally kill bacteria. EGFP module protected fusion partners from the intracellular degradation and allowed the purification of the chimerical proteins by organic extraction. The nature of the polypeptide fused to GFP, as opposed to the order of GFP and the polypeptide modules in the fusion protein, influenced the efficiency of the described purification technique.
The purple chromoprotein (asFP595) from Anemonia sulcata belongs to the family of green fluorescent protein (GFP). Absorption and emission spectra of asFP595 are similar to those of a number of ...recently cloned GFP-like red proteins of the DsRed subfamily. The earlier proposed asFP595 chromophore structure Martynov, V. I.; et al. (2001) J. Biol. Chem. 276, 21012−21016 was postulated to result from an “alternative cyclization” giving rise to a pyrazine-type six-membered heterocycle. Here we report that the asFP595 chromophore is actually very close in chemical structure to that of zFP538, a yellow fluorescent protein Zagranichny, V. E.; et al. (2004) Biochemistry 43, 4764−4772. NMR spectroscopic studies of four chromophore-containing peptides (chromopeptides) isolated under mild conditions from enzymatic digests of asFP595 and one chromopeptide obtained from DsRed revealed that all of them contain a p-hydroxybenzylideneimidazolinone moiety formed by Met-65/Gln-66, Tyr-66/67, and Gly-67/68 of asFP595/DsRed, respectively. Two asFP595 chromopeptides are proteolysis products of an isolated full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other asFP595 chromopeptides were isolated as proteolysis products of the purified chromophore-containing C-terminal fragment. One of these has an oxo group at Met-65 Cα and is a hydrolysis product of another one, with the imino group at Met-65 Cα. The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain cleavage at a very unexpected site, the former peptide bond between Cys-64 C‘ and Met-65 Nα. Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins.
Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with ...streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in
Escherichia coli cells. Association of SAV–aequorin and BCCP–EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510
nm and decreasing 470
nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP–EGFP for binding to SAV–aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.
The yellow fluorescent protein (zFP538) from coral Zoanthus sp. belongs to a family of green fluorescent protein (GFP). Absorption and emission spectra of zFP538 show an intermediate bathochromic ...shift as compared with a number of recently cloned GFP-like red fluorescent and nonfluorescent chromoproteins of the DsRed subfamily. Here we report that the zFP538 chromophore is very close, if not identical, in chemical structure to that of DsRed. To gain insight into the mechanism of zFP538 fluorescence and chromophore structure and chemistry, we studied three chromophore-containing peptides isolated from enzymatic digests of zFP538. Like GFP and DsRed chromophores, these contain a p-hydroxybenzylideneimidazolinone moiety formed by Lys-66, Tyr-67, and Gly-68 of zFP538. One of the peptides studied, the hexapeptide FKYGDR derivative, is a proteolysis product of the zFP538 full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other peptides are the derivatives of the pentapeptide KYGDR resulted from the protein in which the chromophore maturation process had been completed. One of these has an oxogroup at Lys-66 Cα and is a hydrolysis product of another one, with the imino group at Lys-66 Cα. The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain fragmentation at a very unexpected site, the former peptide bond between Phe-65 C‘ and Lys-66 Nα. Also observed in the entire protein under mild denaturing conditions, this fragmentation is likely the feature of native zFP538 chromophore that distinguishes it chemically from the DsRed chromophore.
Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active ...obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity ofin vitrotranslation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.
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