Red fluorescent protein variants with incorporated non-natural amino acid analogues

Fluorescent proteins are important tools in biotechnology applications and biosensing. DsRed, a red fluorescent protein, has expanded the colors of fluorescent proteins beyond the more commonly used green fluorescent protein. Many genetic modifications have been performed on DsRed to overcome some of its drawbacks. These primarily focused on overcoming the oligomerization detrimental to DsRed activity, and the parasitic green fluorescence caused by the immature chromophore. One such variant, DsRed-monomer, has minimal green fluorescence and no oligomerization. A few traditional mutagenesis studies have been done with DsRed and its mutants to shift the fluorescence wavelengths creating additions to the pallet of fluorescent protein colors. We have explored incorporation of non-natural amino acid analogues into DsRed-Monomer, obtaining variants with differing emission properties. In this work, two such analogues of tyrosine have been incorporated into DsRed-Monomer: 3-amino-l-tyrosine and 3-fluoro-l-tyrosine. Tyrosine analogues were chosen due to the role of tyrosine in the formation and structure of the protein's chromophore. The variants obtained in our study showed altered emission wavelengths and spectral characteristics. Our study demonstrates that incorporation of non-natural analogues into DsRed-Monomer is a viable approach to alter the spectral characteristics of the protein. We envision that this study will open up the door to non-natural mutagenesis studies with red fluorescent proteins and its mutants.     

Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants

The red fluorescent protein DsRed has been extensively engineered for use as an in vivo research tool. In fast maturing DsRed variants, the chromophore maturation half-time is approximately 40 min, compared to approximately 12 h for wild-type DsRed. Further, DsRed has been converted from a tetramer into a monomer, a task that entailed mutating approximately 20% of the amino acids. These engineered variants of DsRed have proven extremely valuable for biomedical research, but the structural basis for the improved characteristics has not been thoroughly investigated. Here we present a 1.7 A crystal structure of the fast maturing tetrameric variant DsRed.T4. We also present a biochemical characterization and 1.6 A crystal structure of the monomeric variant DsRed.M1, also known as DsRed-Monomer. Analysis of the crystal structures suggests that rearrangements of Ser69 and Glu215 contribute to fast maturation, and that positioning of the Lys70 side chain modulates fluorescence quantum yield. Despite the 45 mutations in DsRed.M1 relative to wild-type DsRed, there is a root-mean-square deviation of only 0.3 A between the two structures. We propose that novel intramolecular interactions in DsRed.M1 partially compensate for the loss of intermolecular interactions found in the tetramer.     

Bright fluorescent dsDNA probes: novel polycationic asymmetric monomethine cyanine dyes based on thiazolopyridine‐quinolinium chromophore

Seven tri‐ and tetracationic monomeric and homodimeric monomethine cyanine dyes based on thiazolo[4,5‐b]pyridinium and quinolinium end groups were synthesised and characterised. The dyes were tested as fluorescent DNA intercalating probes to apply in DNA gel electrophoresis. The DNA samples stained with all dyes from the series demonstrated bright fluorescent signals. DNA fragments were successfully visualised under orange and green filters as well as under standard UV transillumination. Two of the studied dyes revealed higher sensitivity to DNA when compared with the commercial dimeric cyanine dye TOTO‐1. Their sensitivity reached that of the commercial dimeric cyanine dye YOYO‐1, but the emission was shifted to longer wavelengths. These qualities make the dyes suitable to apply in a wide range of medical and scientific analytical methods.     

The creation of a novel fluorescent protein by guided consensus engineering

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.     

A New Multifunctional Triazine?Carbazole Compound with High Triplet Energy for High-Performance Blue Fluorescence,Green and Red Phosphorescent Host,and Hybrid White Organic Light-Emitting Diodes

A blue fluorescent compound, 9-[4-(4,6-diphenoxy-1,3,5-triazin-2-yl)phenyl]-9H-carbazole (POTC), the triplet energy level of which reaches 2.76 eV, has been designed and synthesized. POTC is an excellent blue emitter as well as host for green and red phosphors, and therefore, matches the requirements of the host for single-emitting-layer fluorescence and phosphorescence hybrid white organic light-emitting diodes (OLEDs). The blue, green, red, and white devices based on POTC show maximum external quantum efficiencies (EQEs) of 2.4, 22.4, 13.0, and 8.1 %, respectively. Even at a high brightness of 1000 cd m⁻², these values maintain EQEs of 2.3, 22.1, 11.1, and 7.0 %, respectively, indicating less than 15 % roll-offs from the maxima.     

Phosphor‐in‐fluorescent‐glasses for high color rendering white light emitting diodes

Fluorescent glass frits were prepared and used to synthesize phosphor‐in‐fluorescent glass composites (PiFGs) to realize stable white light emitting diodes with high color‐rendering properties. Commercial red, green, and blue phosphors were co‐sintered and red phosphors were partially replaced by Eu³⁺ in glass frits. Phosphor‐in‐glass composites were placed on UV‐light emitting diodes (UV‐LEDs) to generate white light. Pure white light with a luminous efficacy=58.4 lm/W, general color rendering index R_a=87 and special color rendering index for strong red R₉=73 was realized with glass frits containing 7 mol% Eu₂O₃ and RGB ratio of 35:20:15. Luminous efficacy, R_a and R₉ increased as red phosphors were replaced by red‐fluorescent glass frits.     

A Genetically Encodable System for Sequence‐Specific Detection of RNAs in Two Colors

Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non‐fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths.     

Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment – typically at a single site or at a few sites per protein – of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l -phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production – using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain – of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.     

Synthesis and characterization of a novel fluorene‐alt‐carbazole polymer to host green,blue, and red phosphorescence

A novel fluorene‐alt‐carbazole polymer host Poly(9,9‐dioctyl‐9H‐fluorene‐2,7‐diyl‐alt‐N‐tetrahydropyran‐3,6‐carbazole) (PFCz), composed of N‐tetrahydropyran‐3,6‐carbazole and 9,9‐dioctyl‐2,7‐fluorene in the polymer backbone, was synthesized by Suzuki coupling. The PFCz possesses good thermal stability and proper lowest unoccupied molecular orbital (LUMO)/highest occupied molecular orbital (HOMO) energy levels to facilitate the injection and transport of electrons and holes. Upon doping with blue, green, and red phosphors, red ‐ green ‐ blue (R‐G‐B) phosphorescent devices hosted by PFCz have been fabricated and investigated. In contrast to those of blue and green devices, the red devices give better performances with a maximum luminous efficiency of 4.88 cd/A and a maximum power efficiency of 1.85% at 149.84 cd/m², due to favorable triplet energy level (E_T) of PFCz for red phosphor, bis(2‐methyldibenzo[f,h]quinoxaline)(acetylacetonate)iridium(III) [Ir(MDQ)₂(acac)]. Additionally, with different doped concentrations of Ir(MDQ)₂(acac), the PFCz‐related red devices emit nearly pure red light with Commission Internationale de L'Eclairage (CIE) coordinates of (0.57, 0.38), (0.60, 0.38), (0.61, 0.38), and (0.62, 0.38), which were very close to the standard red (0.66, 0.34) by the National Television System Committee. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43234.     

Preparation of photoluminescence films containing rare earth complexes by UV photograft polymerization

To avoid the fluorescence quenching resulting from the uneven dispersion of fluorescent rare earth complexes in photoluminescence films, which were prepared by blending until recently, photoluminescence films were prepared in which the chemical bond combination occurs between the fluorescent rare earth complexes and the macromolecular material. Acrylic acid (AA) was grafted onto polyethylene (PE) film through liquid‐phase UV photograft polymerization. Then the grafted films (PAA‐g‐PE) were reacted with a solution of Eu³⁺ and thenoyltrifluoroacetone (TTA) in alcohol–water and with a solution of Tb³⁺ and acetylacetone (AcAc) in chloroform–water, respectively. Thus, red (Eu³⁺–TTA–PAA‐g‐PE) and green (Tb³⁺–ACAC–PAA‐g‐PE) photoluminescence films were obtained. The fluorescence and infrared spectra of the photoluminescence films were recorded. Compared with their corresponding solid fluorescent complexes, both the excitation and emission wavelengths of the photoluminescence films prepared in this way had been changed remarkably, indicating that the fluorescent rare earth complexes had been chemically bonded onto the PE film. Moreover, the effects of the conditions (including pH value, reaction time, and temperature) of the reaction of the grafted film with the solution containing Eu³⁺ and TTA on the fluorescence intensity of the red photoluminescence film were investigated. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 662–667, 2003     

A versatile radiochromic dosimeter for low‐medium gamma radiation and its application to food irradiation

This article presents a novel radiochromic film for selective detection of low‐medium (0–10 kGy) gamma radiation (⁶⁰Co) doses. This dosimeter is based on a printed fluorescent multilayer structure comprising a paper substrate having layers of copper phthalocyanine (DY220) (a green emitter material) on the bottom, and layers of poly[2‐methoxy‐5(2′‐ethylhexyloxy)‐p‐phenylenevinylene] (MEH‐PPV) (a green‐light absorber, red emitter, and radiation sensitive polymer) on the top. The effect of gamma radiation on the optical properties of DY220/MEH‐PPV was described: it was observed as a strong correlation between radiation dose and fluorescent, color coordinates for CIE (1931) chromatic diagram, and Pantone color reference of the dosimeter. The rate of these changes can be altered by manipulation of top–bottom layers to represent easily the radiation dose to be determined in a wide range. This versatile dosimeter has many uses in the field of food radiation for monitoring, quality assurance, and control of the gamma radiation process. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45729.     

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.     

Evaluation of Reaction Efficiency of Thioacidolysis for Cleavage of β-O-4 Interunitary Linkages by Using β-O-4 Type Artificial Lignin Polymer

Abstract

Dimeric lignin model compounds with non-phenolic and phenolic moieties and a β -O-4 type artificial lignin polymer were subjected to thioacidolysis to evaluate the reaction efficiency of thioacidolysis for cleavage of β -O-4 interunitary linkage. The obtained yields of thioethylated monomeric products from the dimeric lignin model compounds reached nearly 100% under the conventional condition, whereas that from the artificial lignin polymer was as low as 74%. Neither prolonged reaction time nor increased concentration of ethanethiol (the thioacidolysis reagent) enhanced the resulting monomer yields from the polymer (69–79%). Thioacidolysis of the lignin model compounds followed by HPSEC analysis also showed the dimeric model compounds were degraded almost quantitatively, but that the artificial lignin polymer was not. Thioacidolysis followed by desulfurization gave at least one dimeric product resulting from incomplete β -O-4 cleavage at significant yield. These results suggested the conventional thioacidolysis could not achieve quantitative cleavage of β -O-4 linkages in lignin macromolecules.     

Cation Transport Through Liquid Membranes Mediated by Photoreactive Crown Ethers. Effects of Alkali Metal Cations on Their Photoreactivities and Transporting Properties

Photoreactivities and cation transporting properties of monomeric, dimeric, and polymeric crown ethers which contain cinnamoyl residues were investigated. Photochemical cycloaddition reaction of the cinnamoyl moieties was accelerated by NaCl or KCl for the dimeric and polymeric derivatives but not for the monomeric one. The dimeric and polymeric crown ethers after irradiation in the presence of KCl showed a significant enhancement in the transport rates for Rb⁺ cation.     

Comparison of the structural and functional effects of monomeric and dimeric human apolipoprotein A-II in high density lipoprotein particles

High density lipoprotein (HDL) is throught to play a significant role in the process of reverse cholesterol transport. It has become clear that the apolipoprotein (apo) composition of HDL is important in determining the metabolic fate of this particle. The major proteins of human HDL are apoAI and APOAII; the latter protein is a disulfide-linked dimer in humans and higher primates but monomeric in the other species. The consequences of the apo Cys6-Cys6 disulfide bridge in apoAII for human HDL structure and function are not known. To address this issue, the influence of the Cys6-Cys6 disulfide bridge on the interaction of human apoAII with palmitoyl-oleoyl phosphatidylcholine has been studied. The size and valence of a series of homogeneous discoidal complexes containing either monomeric (reduced and carboxymethylated) or dimeric apoAII have been determined, and their ability to remove cholesterol from rat Fu5AH hepatoma cells grown in culture has been compared. The apoAII dimer and monomer form discoidal complexes of similar size, with twice as many of the latter molecule required per disc. Removal of the disulfide bond influences the stability of the helical segments around the edge of the disc as seen by a decrease in α-helix content of the monomeric protein. The discoidal particles containing the monomeric form of apoAII are somewhat more effective than particles containing either dimeric apoAII or apoAI in removing cellular cholesterol. Overall, reduction of the disulfide bridge of apoAII probably does not have a major effect in the determination of HDL particle sizein vivo. It follows that the evolution of the Cys6-Cys6 disulfide bond in higher primates probably has not had a major effect on the function of the apoAII molecule.     

Tunable solid‐state fluorescence emission and red upconversion luminescence of novel anthracene‐based fluorophores

Novel, tunable solid‐state emitters based on anthracene groups were synthesised and characterised by spectroscopy and elemental analysis. Their solid‐state photoluminescence properties were studied. These fluorophores display interesting solid‐state emission properties with an emission at wavelengths ranging from 550 to 650 nm when excited by a 325 nm helium–cadmium laser at room temperature. In particular, among them, 1,6‐di(9‐anthryl)hexa‐1,5‐diene‐3,4‐dione, 2‐[4‐(2‐benzoxazolyl)phenyl]‐4,5‐bis[2‐(9‐anthryl)vinyl]‐1H‐imidazole and 2,3‐bis[2‐(9‐anthryl)vinyl]quinoxaline show red, yellow and green emission, respectively, at 650, 584 and 550 nm. The results demonstrated that the luminescent colours can be tuned from red to yellow and green by simply varying molecular structure. Besides, 1,6‐di(9‐anthryl)hexa‐1,5‐diene‐3,4‐dione also exhibited an upconverted red fluorescent emission peak at around 675 nm under femtosecond excitation at 800 nm.     

Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.     

Removal of aldehydes by amino acids and their salts in ambient air

The removal of gaseous aldehydes by amino acids and by their sodium salts and hydrochlorides was studied in ambient air with the relative humidity of 30% at 25°C. Amino acid sodium salts, diamino acids, sodium p-aminobenzoate (PABANa), and o-aminobenzoate (OABANa), and p-aminobenzoic acid (PABA) on sepiolite, both having a carboxylato functionality (? COO^?) together with an amino (? NH₂) group, were highly reactive with aldehydes. In contrast, PABA which has free carboxylic acid functionality (? COOH: dimeric) was not so reactive with aldehydes. Normal amino acids and their hydrochlorides having ammonio (? NH₂⁺) and ? COO^? or ? COOH (dimeric) groups were less reactive with aldehydes. The reactivity was closely related to the degree of dissociation of carboxylate anion; as the degree of dissociation increases, the compound becomes more reactive. p-Aminobenzoic acid hydrochloride (PABA · HCl), having ? NH₃⁺ and ? COOH (monomeric) groups, was the most reactive (with ethanal) of all the amino acids and their salts examined. Amino acid sodium salts, diamino acid, PABANa, OABANa, and PABA on sepiolite are proved to be excellent removers of aldehydes in ambient air. Among them, PABA · HCl is particularly good for ethanal.