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1.
Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore‐binding GAF domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C‐terminally located histidine kinase. In this work, we fused this third GAF domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue‐light control from a LOV domain. A series of RGS‐AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC‐deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red‐light‐converted form of RGSΔ14‐Δ4AC (in vitro) was about three times more active than the green‐light‐converted form. Expression of the fusion protein RGSΔ14‐Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.  相似文献   

2.
Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.  相似文献   

3.
Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm−1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.  相似文献   

4.
Feruloylated soy glycerides (FSG) are a natural‐based, ultraviolet (UV) absorbing, antioxidant vegetable oil synthesized from the lipase‐catalyzed transesterification of ethyl ferulate and soybean oil. Commercial broad spectrum UV absorbing formulations contain multiple UV absorbing compounds that absorb UV radiation in specific regions. The most commonly used compounds are avobenzone (AVO, λmax 356 nm) and octinoxate (ONX, λmax 310 nm), which absorb primarily ultraviolet A and ultraviolet B radiation, respectively. The FSG chromophore is chemically similar to ONX but has a λmax of 328 nm, approximately the median λmax of AVO and ONX. Equimolar mixtures of AVO–ONX and AVO–FSG, 50 μM:50 μM, solutions in ethanol were compared to determine whether FSG was fungible for ONX in total absorbance capacity, photostability when exposed to UV radiation, and broad spectrum absorbance coverage before and after exposure to UV radiation. While it was determined that AVO–FSG mixtures possessed statistically indistinguishable total absorbance capacity compared to AVO–ONX solutions, AVO–FSG possessed slightly better photostability after 4 hours of UV exposure based on 95% confidence interval comparisons from weighted regression equations. Substituting FSG for half of the ONX (e.g., 50 μM:25 μM:25 μM AVO–ONX–FSG) resulted in the best mixture with total absorbance capacity and photostability statistically equal to the AVO–ONX mixtures but with statistically superior broad spectrum UV absorbance compared to AVO–ONX and AVO–FSG mixtures. The natural, vegetable oil‐based FSG can be substituted on an equimolar bases for ONX in mixtures with AVO to produce formulations with similar to superior efficacy.  相似文献   

5.
Poly(amide‐imide)s (PAI) bearing azobenzene chromophore groups were prepared by allowing a hydroxyl‐containing azobenzene dye (Disperse Red 1) to react with and reactive‐terminated PAI with weight–average molecular weights ranging from ~ 1.2 to 2.0 × 104 g/mol. Such PAI were prepared by the condensation of trimellitic anhydride (TMA) and 4,4′‐methylene diphenyl diisocyanate (MDI). The final polymers presented a deep red color, with an absorption maxima in N,N‐dimethylformamide (DMF) solution at 490 nm, close to the azobenzene reactant used (Disperse Red 1) and molecular weights slightly higher than the pristine polymer, showing that the azo chromophore incorporation reaction does not lead to side reactions. The azofunctionalized polymer presented a high Tg value (170°C) that could be increased by a thermal curing process to 240°C. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 841–847, 2007  相似文献   

6.
The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato—a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green–red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live‐cell imaging applications.  相似文献   

7.
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 (ET) 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.  相似文献   

8.
Were it not for an algorithm patch, the color‐appearance model CIECAM02 would sometimes be forced to evaluate fractional powers of negative numbers. The artifact arises because the red and green primaries of the initial CAT02 chromatic adaptation lie outside the positive gamut of the Hunt–Pointer–Estévez (HPE) primaries that subsequently convey the color signal in the model. Relocating the chromaticities of the HPE red and green primaries so as to lie on the CAT02 red–green line alleviates the problem, but adds a bit of X and Y to the revised HPE blue. An (x,y) diagram aids in visualizing the CIECAM02 gamut overlaps, with an extension that accommodates the HPE RGB triangle's enclosure of the point at infinity. © 2006 Wiley Periodicals, Inc. Col Res Appl, 31, 142–145, 2006  相似文献   

9.
Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C‐terminus of guanylate‐kinase‐associated protein (GKAP). Here, small‐molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high‐affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein–protein interaction. Chemical synthesis via a hetero‐Diels–Alder strategy is employed for hit optimization, and structure–activity relationship studies are performed. Best hits possess Ki values in the 10 μM range, and binding to the PDZ domain is confirmed by 1H,15N HSQC NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD‐95, syntrophin, and DVL3 was studied using 1H,15N HSQC NMR spectroscopy.  相似文献   

10.
Polo‐like kinase 1 (PLK1) plays crucial functions in multiple stages of mitosis and is considered to be a potential drug target for cancer therapy. The functions of PLK1 are mediated by its N‐terminal kinase domain and C‐terminal polo‐box domain (PBD). Most inhibitors targeting the kinase domain of PLK1 have a selectivity issue because of a high degree of structural conservation within kinase domains of all protein kinases. Here, we combined virtual and experimental screenings to identify green tea catechins as potent inhibitors of the PLK1 PBD. Initially, (?)‐epigallocatechin, one of the main components of green tea polyphenols, was found to significantly block the binding of fluorescein‐labeled phosphopeptide to the PBD at a concentration of 10 μm. Next, additional catechins were evaluated for their dose‐dependent inhibition of the PBD and preliminary structure–activity relationships were derived. Cellular analysis further showed that catechins interfere with the proper subcellular localization of PLK1, lead to cell‐cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types, such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa). Our data provides new insight into understanding the anticancer activities of green tea catechins.  相似文献   

11.
Composite hydrogel films are prepared by photocrosslinking of partially polymerized poly(2‐hydroxyethyl methacrylate‐co‐methyl methacrylate) prepolymer solution dispersed with aragonite precipitated calcium carbonate (PCC) particles. The effects of PCC aspect ratio (AR, 4.2–6.6) and volume fraction (?PCC) are studied with respect to the prepolymer viscosity, the maximum packing fraction (?m), and optical properties. ?m obtained from Kreigher–Dougherty fitting is found to decrease with increasing AR of PCC particles, from ?m = 0.33 (AR = 4.2) to ?m = 0.21 (AR = 6.6). Scanning electron microscopy images revealed that the particles are uniformly dispersed and randomly orientated in the polymer network when ?PCC < ?m. Optically transparent neat hydrogel films become opaque with increasing particle concentration and AR because of random light scattering from the PCC particles. A whiteness up to 80 is obtained from 100 μm thick composite hydrogel film in the wet state with PCC AR = 6.6 and ?PCC = 0.19 (near ?m). POLYM. ENG. SCI., 2012. © 2012 Society of Plastics Engineers  相似文献   

12.
More than 50 new inhibitors of the oncogenic Stat3 protein were identified through a structure–activity relationship (SAR) study based on the previously identified inhibitor S3I‐201 (IC50=86 μM , Ki>300 μM ). A key structural feature of these inhibitors is a salicylic acid moiety, which, by acting as a phosphotyrosine mimetic, is believed to facilitate binding to the Stat3 SH2 domain. Several of the analogues exhibit higher potency than the lead compound in inhibiting Stat3 DNA binding activity, with an in vitro IC50 range of 18.7–51.9 μM , and disruption of Stat3–pTyr peptide interactions with Ki values in the 15.5–41 μM range. One agent in particular exhibited potent inhibition of Stat3 phosphorylation in both breast and multiple myeloma tumor cells, suppressed the expression of Stat3 target genes, and induced antitumor effects in tumor cells harboring activated Stat3 protein.  相似文献   

13.
Fluorescent proteins (FPs) are a powerful tool for examining tissues, cells, and subcellular components in vivo and in vitro. FusionRed is a particular FP variant mutated from mKate2 that, in addition to lower cytotoxicity and aggregation rates, has shown potential for acting as a tunable photoswitch. This was posited to stem partially from the presence of a bulky side chain at position 158 and a further stabilizing residue at position 157. In this work, we apply computational techniques including classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics simulations (QM/MM) to explore the effect of mutagenesis at these locations in FusionRed on the chromophore structure, the excited-state surface, and relative positional stability of the chromophore in the protein pocket. We find specific connections between the statistical sampling of the underlying protein structure and the nonradiative decay mechanisms from excited-state dynamics. A single mutation (C158I) that restricts the motion of the chromophore through a favorable hydrophobic interaction corresponds to an increase in fluorescence quantum yield (FQY), while a second rescue mutation (C158I-A157N) partially restores the flexibility of the chromophore and photoswitchability with favorable water interactions on the surface of the protein that counteracts the original interaction. We suggest that applying this understanding of structural features that inhibit or favor rotation on the excited state can be applied for rational design of new, tunable and red photoswitches.  相似文献   

14.
Galectin‐8 is a β‐galactoside‐recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3‐O‐[1‐carboxyethyl]‐β‐d ‐galactopyranoside (compound 6 ) as a ligand to target the N‐terminal domain of galectin‐8 (galectin‐8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3′‐O‐sialylated lactose (3′‐SiaLac), with galectin‐8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin‐8N for 6 was 32.8 μm , whereas no significant affinity was detected for the C‐terminal domain of galectin‐8 (galectin‐8C). The crystal structure of the galectin‐8N– 6 complex validated the predicted binding conformation and revealed the exact protein–ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin‐8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide‐based scaffold as a binder of galectin‐8.  相似文献   

15.
In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand‐ and structure‐based pharmacophore modeling was used to identify structurally diverse small‐molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE ). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)‐5‐(5‐(2‐(1H‐benzo[d]imidazol‐2‐yl)‐2‐cyanovinyl)furan‐2‐yl)isophthalic acid; IC50=4.6±0.1 μm ], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)‐3‐(5‐(2‐cyano‐2‐(5‐methyl‐1H‐benzo[d]imidazol‐2‐yl)vinyl)thiophen‐2‐yl)benzoic acid; IC50=0.3±0.2 μm ], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H37Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.  相似文献   

16.
Mutants of cellular retinoic acid‐binding protein II (CRABPII), engineered to bind all‐trans‐retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis‐imine geometry, which has a high pKa, leading to protonation of the imine and subsequent “turn‐on” of color. Yellow light irradiation yields the trans‐imine isomer, which has a depressed pKa, leading to loss of color because the imine is not protonated. The protein‐bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance.  相似文献   

17.
In vivo expression of colored proteins without post‐translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell‐permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red‐shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2‐, 4‐, 5‐, 6‐, and 7‐azaindole) 4‐azaindole has exhibited the largest Stokes shift (~130 nm) in steady‐state fluorescence measurements. It is also highly biocompatible and as 4‐azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono‐ and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4‐azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole‐containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo‐ or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady‐state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.  相似文献   

18.
This research examined the male and female novice designers toward color associations for the concepts used for ‘warnings’, ‘action required’, and ‘signs and equipment status’ through a questionnaire‐based study. A total of 178 Hong Kong Chinese final year undergraduate design students (89 males and 89 females) participated in the study. The test used required the participants to indicate their choice of one of nine colors to associations with each of 38 concepts in a color‐concept table, so that any one color could be associated with any one of the concepts. For both male and female groups of novice designers, chi‐square tests revealed a strong color association for each concept tested in this study (< .05). The results showed males and females agreed on some color‐concept association stereotypes which were therefore gender neutral. The male and female novice designers had the same color associations and similar levels of stereotype strengths for 21 concepts. The nine strongest and therefore most useful color‐concept association stereotypes for both male and female novice designers were: red‐danger, red‐fire, red‐hot, red‐stop, red‐emergency, red‐error, blue‐cold, blue‐male, and green‐exit. However, the male and female novice designers had different color association stereotypes for the standby (green vs. yellow), emergency exit (green vs. red), and toxic (purple vs. black) concepts, and the strengths of the 14 remaining associations for both groups were not at equivalent levels. Overall, it is anticipated that the findings of this study will act as a useful reference for novice designers and other design practitioners to optimize color coding in the design of ‘warnings’, ‘action required’, and ‘signs and equipment status’ messages.  相似文献   

19.
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 Ra=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, Ra and R9 increased as red phosphors were replaced by red‐fluorescent glass frits.  相似文献   

20.
A novel speedy and super‐water‐absorbing non‐woven cloth with hierarchical three‐dimensional network (3D‐SS‐PET) was fabricated through the induction of UV copolymerization on polyethylene terephthalate (PET) fibers followed by a volume phase transition. The macroscopic three‐dimensional network implied that the PET non‐woven substrates are complicated three‐dimensional fibrous materials including oriented fibers in preferential or random directions. The microscopic three‐dimensional network is poly(acrylic acid‐co‐acrylamide) (poly(AA‐co‐AM)) crosslinked copolymer layers on the fiber surface. The rapid volume phase transition was achieved by immersing the swelled non‐woven poly(AA‐co‐AM) modified PET (PET‐g‐AA‐co‐AM) in ethanol. The above process was an essential step to prepare the copolymer chain; after that the fiber surface was extended to form abundant capillary channels and plenty of space between fibers. The water contact angle decreased remarkably from 130° to 0°, while the absorbing capacity of the saturated water and the average water‐absorbing rate experienced an increasing trend, rising from 300 to 324.6 g g?1 in 24 h and 18.6 and 222 g (g min)?1 in 40 s, respectively. It was concluded that surface hydrophilicity and capillaries of the hydrophilic modified macroscopic fibrous structure enhanced the water‐absorbing rate and the swelling process contributed to the higher water absorption capacity. This speedy and super‐water‐absorbing material exhibits great potentiality in diapers, sanitary napkins, wound dressings, surgical pads, and hygroscopic and sweat‐free underwear in extremely cold areas. © 2018 Society of Chemical Industry  相似文献   

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