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Sample records for blue fluorescent protein

  1. Photophysics of the blue fluorescent protein

    International Nuclear Information System (INIS)

    The blue fluorescent protein (BFP) is a mutant of the green fluorescent protein, where the phenolic ring of the chromophore has been replaced by imidazole cycle of histidine residue. The usability of BFP as a fluorescent marker is hampered by its low fluorescence quantum yield at room temperature. The intensity of fluorescence increases by a factor of 4.5 when the temperature is decreased from 320 K down to 225 K. The fluorescence is also enhanced by hydrostatic pressure. Both effects have been explained by shift of the equilibrium between hydrogen nonbonded and hydrogen-bonded chromophores. Our semi-empirical quantum chemical calculations show that the fluorescence quantum yield of the BFP chromophore is low due to isomerization in the electronically excited state -twisting of the bridging bond by 90 deg. At this twisted geometry the potential energy surfaces of ground and excited states are situated close to each other facilitating efficient nonradiative decay

  2. Changing blue fluorescent protein to green fluorescent protein using chemical RNA editing as a novel strategy in genetic restoration.

    Science.gov (United States)

    Vu, Luyen T; Nguyen, Thanh T K; Alam, Shafiul; Sakamoto, Takashi; Fujimoto, Kenzo; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2015-11-01

    Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site-directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5'-carboxyvinyl-2'-deoxyuridine ((CV) U) were used for reversible photoligation, and single-stranded 100-nt BFP DNA and in vitro-transcribed full-length BFP mRNA were the targets. Photo-cross-linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross-linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence-analysis results revealed that in vitro-translated proteins were synthesized from mRNAs after site-directed RNA editing. We detected substantial amounts of the target-base-substituted fragment using RFLP and observed highly reproducible spectra of the transition-GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C-to-U transition. Thus, we successfully used non-enzymatic site-directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders. PMID:26031895

  3. Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Faaizah; Pickup, John C., E-mail: john.pickup@kcl.ac.uk

    2013-08-30

    Highlights: •We showed that the NIR fluorophore, 651-Blue Oxazine, is solvatochromic (polarity sensitive). •Blue Oxazine was covalently attached to mutants of glucose/galactose-binding protein (GBP). •Fluorescence intensity of GBP-Blue Oxazine increased with addition of glucose. •Fluorescence from bead-immobilised GBP-Blue Oxazine was detectable through skin in vitro. •This shows proof-of-concept for non-invasive glucose sensing using GBP-Blue Oxazine. -- Abstract: Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25 mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.

  4. Imidazole-assisted catalysis of luminescence reaction in blue fluorescent protein from the photoprotein aequorin

    International Nuclear Information System (INIS)

    Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a dissociable complex of Ca2+-bound apoaequorin and coelenteramide, and is identified as a luciferase that catalyzes the oxidation of coelenterazine by molecular oxygen to emit light. Based on the chemical luminescence of coelenterazine oxidation by an acid-base mechanism, we found that the luminescence activity of BFP-aq was stimulated by imidazole at concentrations of 30-300 mM with coelenterazine and its analogues. The kinetic analyses indicate that imidazole has no effect on the binding affinity of coelenterazine to BFP-aq and may act as a catalytic base, accepting a proton from the -NH- group of coelenterazine and stimulating luminescence activity

  5. Crystallization and preliminary X-ray diffraction analysis of a novel wild-type blue fluorescent protein from Vibrio vulnificus CKM-1

    International Nuclear Information System (INIS)

    The wild type BFPvv has been crystallized for the first time in order to obtain its tertiary structure and to further understand how chromophore formation occurs via a different oxygen-independent mechanism. The use of green fluorescent protein (GFP) for non-invasive in vivo imaging is limited to aerobic systems, as chromophore formation requires oxygen. However, a novel NADPH-dependent blue fluorescent protein from Vibrio vulnificus CKM-1 (BFPvv) that emits blue fluorescence in both aerobic and anaerobic systems has recently been discovered. Wild-type BFPvv was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The resulting BFPvv crystals diffracted to a resolution of 1.9 Å and belonged to space group P3, with unit-cell parameters a = b = 96.62, c = 214.511 Å. Assuming the presence of eight molecules in the unit cell, the solvent content was estimated to be ∼56.16%

  6. Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

    Science.gov (United States)

    Sakharova, N Yu; Mezhevikina, L M; Smirnov, A A; Vikhlyantseva, E F

    2014-05-01

    We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos. PMID:24913583

  7. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    Science.gov (United States)

    Giron, Maria D.; Salto, Rafael

    2011-01-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

  8. Why Do Proteins Glow Blue?

    CERN Document Server

    Sarkar, Sohini; Hazra, Partha; Mandal, Pankaj

    2014-01-01

    Recent literatures reported blue-green emission from amyloid fibril as exclusive signature of fibril formation. This unusual visible luminescence is regularly used to monitor fibril growth. Blue-green emission has also been observed in crystalline protein and in solution. However, the origin of this emission is not known exactly. Our spectroscopic study of serum proteins reveals that the blue-green emission is a property of protein monomer. Evidences suggest that semiconductor-like band structure of proteins with the optical band-gap in the visible region is possibly the origin of this phenomenon. We show here that the band structure of proteins is primarily the result of electron delocalization through the peptide chain, rather than through the hydrogen bond network in secondary structure.

  9. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  10. Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

    OpenAIRE

    Niino, Yusuke; Hotta, Kohji; Oka, Kotaro

    2010-01-01

    Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indica...

  11. Diversity and Evolution of Coral Fluorescent Proteins

    OpenAIRE

    Alieva, Naila O.; Konzen, Karen A.; Field, Steven F.; Meleshkevitch, Ella A.; Hunt, Marguerite E.; Victor Beltran-Ramirez; Miller, David J.; Jörg Wiedenmann; Anya Salih; Matz, Mikhail V

    2008-01-01

    GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full ...

  12. Dispensability of the major coat protein of oat blue dwarf virus in genome replication: Substitution of the open reading frame with the enhanced green fluorescent protein gene

    Science.gov (United States)

    Oat blue dwarf virus (OBDV) is a representative marafivirus that infects monocots and a limited number of dicot species and is vectored propagatively by the leafhopper Macrosteles fascifrons.Recently, we reported the generation of clone pOBDV-2r, the first clone of a marafivirus from which infectiou...

  13. Green fluorescent protein: A perspective

    OpenAIRE

    Remington, S James

    2011-01-01

    A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994–2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationship...

  14. Diversity and evolution of coral fluorescent proteins.

    Directory of Open Access Journals (Sweden)

    Naila O Alieva

    Full Text Available GFP-like fluorescent proteins (FPs are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae and pink chromoprotein from Stylophora pistillata (Pocilloporidae, both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria. The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of

  15. Blue fluorescent organic light emitting diodes with multilayered graphene anode

    International Nuclear Information System (INIS)

    As an innovative anode for organic light emitting devices (OLEDs), we have investigated graphene films. Graphene has importance due to its huge potential in flexible OLED applications. In this work, graphene films have been catalytically grown and transferred to the glass substrate for OLED fabrications. We have successfully fabricated 2 mm × 2 mm device area blue fluorescent OLEDs with graphene anodes which showed 2.1% of external quantum efficiency at 1000 cd/m2. This is the highest value reported among fluorescent OLEDs using graphene anodes. Oxygen plasma treatment on graphene has been found to improve hole injections in low voltage regime, which has been interpreted as oxygen plasma induced work function modification. However, plasma treatment also increases the sheet resistance of graphene, limiting the maximum luminance. In summary, our works demonstrate the practical possibility of graphene as an anode material for OLEDs and suggest a processing route which can be applied to various graphene related devices.

  16. Protein requirement for Trichogaster lalius, blue variety

    Directory of Open Access Journals (Sweden)

    Jener Alexandre Sampaio Zuanon

    2013-02-01

    Full Text Available The objective of the present study was to evaluate the protein requirement of juvenile Trichogaster lalius, blue variety. The experimental design was of randomised blocks (B1 = initial weight of 1.04±0.05 g and B2 = 1.36±0.02 g, with two replicates within each block and five treatments (230, 270, 310, 350 and 390 g CP/kg diet. The fish were fed to satiation, three times a day for 90 days. The study evaluated: survival rate, weight gain, final length, feed intake, feed conversion ratio, protein efficiency ratio, specific growth rate and condition factor. There was a linear effect of dietary protein levels for protein efficiency ratio, specific growth rate (positive linear effect and feed conversion ratio (negative linear effect. For weight gain, final length, feed intake and condition factor a quadratic effect of dietary protein levels was observed, with estimated values of 409.8, 366.2, 317.4 and 365.0 g CP/kg diet, to improve their performance parameters. Analysis of growth based on the length of the fish shows that 366.2 g of CP/kg diet meets the protein requirement of juvenile Trichogaster lalius, blue variety.

  17. Blue, green, and red fluorescence emission signatures of green, etiolated, and white leaves

    International Nuclear Information System (INIS)

    Laser-induced fluorescence emission of green terrestrial vegetation has become of high interest in remote LIDAR techniques in recent years. The remote sensing of blue and red fluorescence signatures appears to be a suitable future tool to determine the state of health of plants. In this contribution we report about major factors which determine the intensities of the UV-laser (337 nm) excited blue fluorescence near 450 nm and the red chlorophyll fluorescence between 650 nm and 800 nm as well as the ratio of the blue to the red fluorescence F450/F690. In contrast to green plants grown in a phytochamber or at low light intensities, plants grown in the field at a high photon flux density (PFD) showed high values for the ratio of the blue to the red fluorescence F450/F690, which was due to a relatively low intensity of the red chlorophyll fluorescence. The strongly increased ratio F450/F690 was primarily caused by a much reduced penetration depth of the exciting UV light into the leaf, which seemed to be due to substances absorbing in the epidermal layers. Etiolated wheat leaves exhibited a stronger blue fluorescence intensity than green leaves and showed also a small maximum around 530 nm (green fluorescence). White leaves of wheat treated with the bleaching herbicide norfluorazone (10−1 M) and leaves from which the photosynthetic pigments had been extracted by acetone showed a fourfold and tenfold increase of the blue fluorescence, respectively. Isolated chloroplasts and thylakoids did not exhibit a blue-green fluorescence. It is concluded that the blue fluorescence is mainly caused by phenolic plant substances located in the cell wall and / or vacuoles of leaves. Partial reabsorption of the emitted blue and red fluorescence by the photosynthetic pigments modifies the shape of the fluorescence spectra. (author)

  18. Protein requirement for Trichogaster lalius, blue variety

    OpenAIRE

    Jener Alexandre Sampaio Zuanon; Antonio Policarpo Souza Carneiro; Lidiane da Silva Nascimento; Donizete Aparecido da Silva; Marcelo Duarte Pontes; Márcio Yoshiyuki Kanashiro; Ana Lúcia Salaro

    2013-01-01

    The objective of the present study was to evaluate the protein requirement of juvenile Trichogaster lalius, blue variety. The experimental design was of randomised blocks (B1 = initial weight of 1.04±0.05 g and B2 = 1.36±0.02 g), with two replicates within each block and five treatments (230, 270, 310, 350 and 390 g CP/kg diet). The fish were fed to satiation, three times a day for 90 days. The study evaluated: survival rate, weight gain, final length, feed intake, feed conversion ratio, prot...

  19. Nitroreductase-triggered activation of a novel caged fluorescent probe obtained from methylene blue.

    Science.gov (United States)

    Bae, Jungeun; McNamara, Louis E; Nael, Manal A; Mahdi, Fakhri; Doerksen, Robert J; Bidwell, Gene L; Hammer, Nathan I; Jo, Seongbong

    2015-08-18

    A near-infrared fluorescent probe based on methylene blue (p-NBMB) was developed for the detection of nitroreductase. Conjugating methylene blue with a p-nitrobenzyl moiety enables it to be activated by nitroreductase-catalyzed 1,6-elimination, resulting in the release of an active methylene blue fluorophore. PMID:26165999

  20. Single-molecule spectroscopy of fluorescent proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the d

  1. Chemiluminescence characteristics of cumarin derivatives as blue fluorescers in peroxyoxalate-hydrogen peroxide system

    Science.gov (United States)

    Chaichi, Mohammad Javad; Karami, Ali Reza; Shockravi, Abbas; Shamsipur, Mojtaba

    2003-04-01

    The chemiluminescence characteristics of seven different cumarin derivatives were studied in detail. The fluorescence and chemiluminescence spectra were compared; all cumarins used were found to act as blue fluorescers. The intensity and kinetic parameters for the chemiluminescent systems were evaluated from computer fitting of the resulting intensity-time plots. Among different cumarin derivatives used, 7-amino-4-trifluoromethylcumarin revealed the most promising characteristics as an efficient blue fluorescent emitter.

  2. Fluorescent Protein Approaches in Alpha Herpesvirus Research

    Science.gov (United States)

    Hogue, Ian B.; Bosse, Jens B.; Engel, Esteban A.; Scherer, Julian; Hu, Jiun-Ruey; del Rio, Tony; Enquist, Lynn W.

    2015-01-01

    In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer. PMID:26610544

  3. Fluorescent Protein Approaches in Alpha Herpesvirus Research.

    Science.gov (United States)

    Hogue, Ian B; Bosse, Jens B; Engel, Esteban A; Scherer, Julian; Hu, Jiun-Ruey; Del Rio, Tony; Enquist, Lynn W

    2015-11-01

    In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer. PMID:26610544

  4. Fluorescent Protein Approaches in Alpha Herpesvirus Research

    Directory of Open Access Journals (Sweden)

    Ian B. Hogue

    2015-11-01

    Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

  5. Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study.

    Science.gov (United States)

    Alieva, Roza R; Tomilin, Felix N; Kuzubov, Alexander A; Ovchinnikov, Sergey G; Kudryasheva, Nadezhda S

    2016-09-01

    Coelenteramide-containing fluorescent proteins are products of bioluminescent reactions of marine coelenterates. They are called 'discharged photoproteins'. Their light-induced fluorescence spectra are variable, depending considerably on external conditions. Current work studies a dependence of light-induced fluorescence spectra of discharged photoproteins obelin, aequorin, and clytin on excitation energy. It was demonstrated that photoexcitation to the upper electron-excited states (260-300nm) of the discharged photoproteins initiates a fluorescence peak in the near UV region, in addition to the blue-green emission. To characterize the UV fluorescence, the light-induced fluorescence spectra of coelenteramide (CLM), fluorophore of the discharged photoproteins, were studied in methanol solution. Similar to photoproteins, the CLM spectra depended on photoexcitation energy; the additional peak (330nm) in the near UV region was observed in CLM fluorescence at higher excitation energy (260-300nm). Quantum chemical calculations by time depending method with B3LYP/cc-pVDZ showed that the conjugated pyrazine-phenolic fragment and benzene moiety of CLM molecule are responsible for the additional UV fluorescence peak. Quantum yields of CLM fluorescence in methanol were 0.028±0.005 at 270-340nm photoexcitation. A conclusion was made that the UV emission of CLM might contribute to the UV fluorescence of the discharged photoproteins. The study develops knowledge on internal energy transfer in biological structures - complexes of proteins with low-weight aromatic molecules. PMID:27400455

  6. Fluorescent sensors based on bacterial fusion proteins

    International Nuclear Information System (INIS)

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

  7. Fluorescent sensors based on bacterial fusion proteins

    Science.gov (United States)

    Prats Mateu, Batirtze; Kainz, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Toca-Herrera, José L.

    2014-06-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

  8. Evolution of a fluorinated green fluorescent protein

    OpenAIRE

    Yoo, Tae Hyeon; Link, A. James; Tirrell, David A.

    2007-01-01

    The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence ...

  9. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    NARCIS (Netherlands)

    Shaner, Nathan C; Campbell, Robert E; Steinbach, Paul A; Giepmans, Ben N G; Palmer, Amy E; Tsien, Roger Y

    2004-01-01

    Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red

  10. Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra.

    Science.gov (United States)

    Goun, Alexei; Bondar, Denys I; Er, Ali O; Quine, Zachary; Rabitz, Herschel A

    2016-01-01

    By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters. PMID:27181496

  11. Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra

    Science.gov (United States)

    Goun, Alexei; Bondar, Denys I.; Er, Ali O.; Quine, Zachary; Rabitz, Herschel A.

    2016-05-01

    By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters.

  12. Metal-enhanced fluorescence of single green fluorescent protein (GFP)

    International Nuclear Information System (INIS)

    The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure

  13. Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions.

    Science.gov (United States)

    Bhadauria, V; Miraz, P; Kennedy, R; Banniza, S; Wei, Y

    2010-04-01

    Understanding the infection biology of fungi is the key step in devising suitable control strategies for plant diseases. Recently, the Arabidopsis-Colletotrichum higginsianum (causal agent of anthracnose) system has emerged as a seminal paradigm for deciphering the infection biology underlying fungus-plant interactions. We describe here three staining methods coupled with confocal microscopy: trypan blue, aniline blue and dual trypan blue-aniline blue fluorescence staining. Trypan blue and aniline blue staining were employed to scan the infection structures of the hemibiotrophic fungus C. higginsianum and host response in A. thaliana leaf tissues. The two techniques then were combined to observe the contrast between in planta fungal infection structures, i.e., infection vesicles, primary hyphae and secondary hyphae, and the host plant defense responses, i.e., papilla formation and hypersensitive response. These staining techniques also were applied to the lentil-C. truncatum pathosystem to demonstrate their applicability for multiple pathosystems. PMID:19669979

  14. Fluorescent S-layer fusion proteins

    International Nuclear Information System (INIS)

    This work describes the construction and characterisation of fluorescent S-layer fusion proteins used as building blocks for the fabrication of nanostructured monomolecular biocoatings on silica particles with defined fluorescence properties. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the pH-dependant cyan, green and yellow variant of the green fluorescent protein (GFP) and the red fluorescent protein mRFP1. These fluorescent S-layer fusion proteins, acting as scaffold and optical sensing element simultaneously, were able to reassemble in solution and on silica particles forming 2D nanostructures with p2 lattice symmetry (a=11 ±0.5 nm, b=14 ±0.4 nm, g=80 ±1o). The pH-dependant fluorescence behaviour was studied with fluorimetry, confocal microscopy and flow cytometry. These fluorescent S-layer fusion proteins can be used as pH-sensor. 50% of the fluorescence intensity decreases at their calculated pKa values (pH6 - pH5). The fluorescence intensity of the GFP variants vanished completely between pH4 and pH3 whereas the chromophore of the red protein mRFP1 was only slightly affected in acidic conditions. At the isoelectric point of the S-layer coated silica particles (pH4.6 ±0.2) an increase in particle aggregation was detected by flow cytometry. The cyan and yellow fluorescent proteins were chosen to create a bi-fluorescent S-layer tandem fusion protein with the possibility for resonance energy transfer (FRET). A transfer efficiency of 20% and a molecular distance between the donor (ECFP) and acceptor (YFP) chromophores of around 6.2 nm could be shown. This bi-fluorescent ECFP-SgsE-YFP tandem fusion protein was able to reassemble on solid surfaces. The remarkable combination of fluorescence and self-assembly and the design of bi-functional S-layer tandem fusion protein matrices makes them to a promising tool in nanobiotechnology. (author)

  15. A high performance fluorescence switching system triggered electrochemically by Prussian blue with upconversion nanoparticles

    Science.gov (United States)

    Zhai, Yiwen; Zhang, Hui; Zhang, Lingling; Dong, Shaojun

    2016-05-01

    A high performance fluorescence switching system triggered electrochemically by Prussian blue with upconversion nanoparticles was proposed. We synthesized a kind of hexagonal monodisperse β-NaYF4:Yb3+,Er3+,Tm3+ upconversion nanoparticle and manipulated the intensity ratio of red emission (at 653 nm) and green emission at (523 and 541 nm) around 2 : 1, in order to match well with the absorption spectrum of Prussian blue. Based on the efficient fluorescence resonance energy transfer and inner-filter effect of the as-synthesized upconversion nanoparticles and Prussian blue, the present fluorescence switching system shows obvious behavior with high fluorescence contrast and good stability. To further extend the application of this system in analysis, sulfite, a kind of important anion in environmental and physiological systems, which could also reduce Prussian blue to Prussian white nanoparticles leading to a decrease of the absorption spectrum, was chosen as the target. And we were able to determine the concentration of sulfite in aqueous solution with a low detection limit and a broad linear relationship.A high performance fluorescence switching system triggered electrochemically by Prussian blue with upconversion nanoparticles was proposed. We synthesized a kind of hexagonal monodisperse β-NaYF4:Yb3+,Er3+,Tm3+ upconversion nanoparticle and manipulated the intensity ratio of red emission (at 653 nm) and green emission at (523 and 541 nm) around 2 : 1, in order to match well with the absorption spectrum of Prussian blue. Based on the efficient fluorescence resonance energy transfer and inner-filter effect of the as-synthesized upconversion nanoparticles and Prussian blue, the present fluorescence switching system shows obvious behavior with high fluorescence contrast and good stability. To further extend the application of this system in analysis, sulfite, a kind of important anion in environmental and physiological systems, which could also reduce Prussian blue to

  16. Fluorescence Studies of Protein Crystal Nucleation

    Science.gov (United States)

    Pusey, Marc; Sumida, John

    2000-01-01

    We have postulated that, in the case of tetragonal chicken egg white lysozyme, crystal growth occurs by the addition of pre-critical nuclei sized n-mers that form in the bulk solution, and that the n-mer growth units were multiples of the tetrameric 4(sub 3) helical structure. These have the strongest intermolecular bonds in the crystal and are therefore likely to be the first species formed. High resolution AFM studies provide strong supporting evidence for this model, but the data also suggest that the actual species in solution may not be identical in structure to that found in the crystal. We are using fluorescence resonance energy transfer (FRET) to study the initial solution phase self-assembly process, using covalent fluorescent derivatives which crystallize in the characteristic P4(sub 3)2(sub 1)2(sub 1) space group. FRET studies are being carried out between the cascade blue (CB-lys, donor, Ex(sub max) 366 nm, Em 420 nm) and lucifer yellow (LY-lys, acceptor, Ex(sub max) 430 nm, Em 528 nm) asp101 derivatives. The estimated R(sub 0) for this probe pair, the distance where 50% of the donor energy is transferred to the acceptor, is approx. 1.2 nm, compared to 2.2 nm between the side chain carboxyls of adjacent asp101's in the crystalline 4(sub 3) helix. The short donor lifetime of 2.80 ns (chi(sup 2) = 0.644), coupled with the large average distances between the molecules (greater than or equal to 50 nm) in solution, ensure that any energy transfer observed is not due to random diffusive interactions. Lifetime data show that CB-lys has a single lifetime when it is the only species in solution. Similarly, LY-lys also exhibits a single lifetime of 4.63 ns (chi(sup 2) = 0.42) when alone in solution. Addition of LY-lys to CB-lys results in the appearance of a third lifetime component of 0.348ns for the CB-lys. The fractional intensities of the different species present can be used to estimate the distribution of monomer and n-mers in solution. The self

  17. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  18. Proteins expressed in blue-green sharpshooter leafhoppers

    Science.gov (United States)

    We used a metagenomics approach to identify proteins from the blue-green sharpshooter, Graphocephala atropunctata (Hemiptera: Cicadellidae) which is an important vector of Pierce’s disease of grapes. The 44 proteins are being used as markers to monitor and identify current and exotic introductions o...

  19. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  20. Fluorescence of reduced charge montmorillonite complexes with methylene blue: Experiments and molecular modeling

    Czech Academy of Sciences Publication Activity Database

    Klika, Z.; Pustková, P.; Praus, P.; Kovář, P.; Pospíšil, M.; Malý, P.; Grygar, Tomáš; Kulhánková, L.; Čapková, P.

    2009-01-01

    Roč. 339, č. 2 (2009), s. 416-423. ISSN 0021-9797 Institutional research plan: CEZ:AV0Z40320502 Keywords : methylene blue * reduced charge montmorillonite s * fluorescence Subject RIV: DB - Geology ; Mineralogy Impact factor: 3.019, year: 2009

  1. Efficient fluorescent red, green, and blue organic light-emitting devices with a blue host of spirobifluorene derivative

    Energy Technology Data Exchange (ETDEWEB)

    Lee, R.-H. [Department of Chemical and Material Engineering, National Yunlin University of Science and Technology, Yunlin 640, Taiwan (China)], E-mail: lerongho@yuntech.edu.tw; Huang, Y.-W.; Wang, Y.-Y. [Department of Chemical and Material Engineering, National Yunlin University of Science and Technology, Yunlin 640, Taiwan (China); Chang, H.-Y. [EChem Hightech CO., LTD, Hsin-Chu Industrial Park, Hu-Kou, Hsin-Chu, Taiwan (China)

    2008-06-02

    Efficient fluorescent blue, green, and red (RGB) organic light-emitting devices (OLEDs) were fabricated using a blue host material of pyrimidine-containing spirobifluorene derivative 2,7-bis[2-(4-tert-butylphenyl)pyrimidine-5-yl]-9,9'-spirobifluorene (TBPSF) doped with blue dye perylene, green dye 10-(2-benzothiazolyl)-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H,5H, 11H-benzo[l] pyrano[6,7,8-ij] quinolizin-11-one (C545T), and red dye 4-(dicyanomethylene)-2-t-butyl-6-(1,1,7,7-tetramethyljulolidyl-9-enyl) -4H-pyran (DCJTB), respectively. The brightness and current efficiency of the perylene doped blue device reached 10117 cd/m{sup 2} and 2.97 cd/A. Green emission of the C545T doped device reached 8500 cd/m{sup 2} and 13.0 cd/A. Red emission of the DCJTB doped device can be as high as 9000 cd/m{sup 2} and 2.0 cd/A, respectively. High color purity of the blue (Commission Internationale de L'Eclairage (CIE{sub x,y}) coordinates (CIE, x = 0.27, y = 0.24)), green (CIE, x = 0.19, y = 0.63) and red (CIE, x = 0.62, y = 0.37) emissions were achieved for RGB dyes doped TBPSF OLEDs. High brightness, large current efficiency, and good color purity of TBPSF-based RGB OLEDs were obtained by the configuration optimization device, such as inserting the hole and electron-injection materials, and suitable dopant content and light emitting layer thickness.

  2. Trace fluorescent labeling for protein crystallization

    Energy Technology Data Exchange (ETDEWEB)

    Pusey, Marc, E-mail: marc.pusey@ixpressgenes.com; Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph [iXpressGenes Inc., 601 Genome Way, Huntsville, AL 35810 (United States)

    2015-06-27

    The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.

  3. Blue-native PAGE in plants: a tool in analysis of protein-protein interactions

    OpenAIRE

    Millar A Harvey; Braun Hans-Peter; Eubel Holger

    2005-01-01

    Abstract Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE). BN-PAGE has become the method of choice for the investigation of the respiratory pr...

  4. Effects of special blue fluorescent light on hepatic mixed-function oxidase activity in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Davis, D.R.; Yeary, R.A.; Randall, G.

    1981-01-01

    Phototherapy has been widely used in the treatment of neonatal hyperbilirubinemia. Recent reports, however, have indicated that fluorescent light may be toxic and mutagenic to mammalian cells. these findings suggest possible long-term side effects with the use of phototherapy. This study was undertaken to determine the effects of phototherapy on hepatic microsomal enzyme activity. The exposure of Sprague-Dawley and Gunn rats to special blue fluorescent light at an average irradiance of 1,200 microW/cm2 resulted in no significant changes in liver microsomal enzyme activity for aniline hydroxylase, p-nitroanisole-O-demethylase, ethylmorphine-N-demethylase, cytochrome c reductase or the quantity of cytochrome P-450. A significant decrease in aniline hydroxylase and p-nitroanisole-O-demethylase activity was observed when liver microsomes were exposed in vitro to special blue fluorescent light. Photoactivated bilirubin did not effect the activity of the mixed-function oxidase enzymes measured under the conditions of this study.

  5. Miniature fluorescence detection system for protein chips

    Science.gov (United States)

    Kim, Hoseong; Choi, Jaeho; Lee, Kook-Nyung; Kim, Yongkwon

    2005-01-01

    We report the development of miniature fluorescence detection systems that employ miniature prism, mirrors and low cost CCD camera to detect the fluorescence emitted from 40 fluorescently-labeled protein patterns without scanner. This kind of miniature fluorescence detection systems can be used in point of care. We introduce two systems, one uses prism + mirror block and the other uses prism and two mirrors. A large NA microscope eyepiece and low cost CCD camera are used. We fabricated protein chip containing multi-pattern BSA labeled with Cy5, using MEMS technology and modified the surface chemically to clean and to immobilize proteins. The measurements show that the combination of prism and mirrors can homogenize elliptical excitation light over the sample with higher optical efficiency, and increase the separation between excitation and fluorescence light at the CCD to give higher signal intensity and higher signal to noise ratio. The measurements also show that protein concentrations ranging from 10 ng/ml to 1000 ng/ml can be assayed with very small error. We believe that the proposed fluorescence detection system can be refined to build a commercially valuable hand-held or miniature detection device.

  6. Sentinel lymph nodes detection with an imaging system using Patent Blue V dye as fluorescent tracer

    Science.gov (United States)

    Tellier, F.; Steibel, J.; Chabrier, R.; Rodier, J. F.; Pourroy, G.; Poulet, P.

    2013-03-01

    Sentinel lymph node biopsy is the gold standard to detect metastatic invasion from primary breast cancer. This method can help patients avoid full axillary chain dissection, thereby decreasing the risk of morbidity. We propose an alternative to the traditional isotopic method, to detect and map the sentinel lymph nodes. Indeed, Patent Blue V is the most widely used dye in clinical routine for the visual detection of sentinel lymph nodes. A Recent study has shown the possibility of increasing the fluorescence quantum yield of Patent Blue V, when it is bound to human serum albumin. In this study we present a preclinical fluorescence imaging system to detect sentinel lymph nodes labeled with this fluorescent tracer. The setup is composed of a black and white CCD camera and two laser sources. One excitation source with a laser emitting at 635 nm and a second laser at 785 nm to illuminate the region of interest. The prototype is operated via a laptop. Preliminary experiments permitted to determine the device sensitivity in the μmol.L-1 range as regards the detection of PBV fluorescence signals. We also present a preclinical evaluation performed on Lewis rats, during which the fluorescence imaging setup detected the accumulation and fixation of the fluorescent dye on different nodes through the skin.

  7. Synthesis and spectral characterization of a new blue fluorescent tripod for detecting metal cations and protons

    Energy Technology Data Exchange (ETDEWEB)

    Staneva, Desislava [University of Chemical Technology and Metallurgy, 1756 Sofia (Bulgaria); Makki, Mohammad S.I.; Sobahi, Tariq R. [Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589 (Saudi Arabia); Bosch, Paula [Institute of Science and Technology of Polymers, CSIC, Juan de la Cierva 3, 28006, Madrid (Spain); Abdel-Rahman, Reda M. [Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589 (Saudi Arabia); Asiri, Abdullah [Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589 (Saudi Arabia); Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589 (Saudi Arabia); Grabchev, Ivo, E-mail: i.grabchev@chem.uni-sofia.bg [Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589 (Saudi Arabia); Sofia University “St. Kliment Ohridski”, Faculty of Medicine, 1407 Sofia (Bulgaria)

    2015-06-15

    A new symmetrical blue fluorescent tripod containing three 1,8-naphtalimide fragments has been synthesized. Its spectral properties have been investigated in organic solvents of different polarity. Its basic photophysical characteristics depend strongly on the polarity of the organic solvents. In nonpolar solvents (diethyl ether, chloroform and toluene) the new tripod emits eximer fluorescence. The new tripod has been investigated as a ligand for detection of metal ions (Ag{sup +}, Co{sup 2+}, Pb{sup 2+}, Zn{sup 2+}, Ni{sup 2+}, and Cu{sup 2+}) in acetonitrile and acetonitrile/H{sub 2}O (1:4, v/v). The influence of protons in an ethanol–water (1:4, v/v) solution and in toluene and diethyl ether solvents on the tripod fluorescence intensity has been investigated and it has been shown that the protons destroy the excimer formations recovering the monomer fluorescence. - Highlights: • New blue fluorescent tripod containing three 1,8-naphtalimide fragments was described. • In nonpolar solvents the new tripod emits eximer fluorescence. • The new tripod has been investigated as a ligand for metal ions and protons.

  8. Transgenic rats with green, red, and blue fluorescence: powerful tools for bioimaging, cell trafficking, and differentiation

    Science.gov (United States)

    Murakami, Takashi; Kobayashi, Eiji

    2005-04-01

    The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats" system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.

  9. Visible light communication using a blue GaN mu LED and fluorescent polymer color converter

    OpenAIRE

    Chun, Hyunchae; Manousiadis, Pavlos; Rajbhandari, Sujan; Vithanage, D.A.; Faulkner, G.; Tsonev, Dobroslav; Mckendry, Jonathan J.D.; Videv, Stefan; Xie, Enyuan; Gu, Erdan; Martin D. Dawson; Haas, Harald; Turnbull, Graham; Samuel, Ifor D. W.; O'Brien, Dominic

    2014-01-01

    This letter presents a novel technique to achieve high-speed visible light communication (VLC) using white light generated by a blue GaN mu LED and a yellow fluorescent copolymer. We generated white light suitable for room illumination by optimizing the ratio between the blue electroluminescence of the mu LED and yellow photoluminescence of the copolymer color converter. Taking advantage of the components' high bandwidth, we demonstrated 1.68 Gb/s at a distance of 3 cm (at 240 lx illumination...

  10. Fluorescent Magnesium Nanocomplex in Protein Scaffold for Cell Nuclei Imaging Application

    Energy Technology Data Exchange (ETDEWEB)

    Pandya, Alok [Ahmedabad Univ. (India); Tripathi, Apritam [Ahmedabad Univ. (India); Purohit, Rahul [Ahmedabad Univ. (India); Singh, Sanjay [Ahmedabad Univ. (India); Nandasiri, Manjula I. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Karakoti, Ajay S. [Ahmedabad Univ. (India); Singh, Surinder P. [National Physical Lab., New Delhi (India); Shanker, Rishi [Ahmedabad Univ. (India)

    2015-10-27

    Here in, we report a facile strategy for the synthesis of water-soluble ultra-fine blue emitting fluorescent Magnesium nanoparticles-protein complex (MgNC). This MgNC is demonstrated to exhibit excellent photo stability and biocompatibility. It was also observed that MgNC stain cell nuclei with high specifcity.

  11. Versatile protein tagging in cells with split fluorescent protein

    OpenAIRE

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Jonathan S Weissman; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respec...

  12. Fluorescent deep-blue and hybrid white emitting devices based on a naphthalene-benzofuran compound

    KAUST Repository

    Yang, Xiaohui

    2013-08-01

    We report the synthesis, photophysics and electrochemical properties of naphthalene-benzofuran compound 1 and its application in organic light emitting devices. Fluorescent deep-blue emitting devices employing 1 as the emitting dopant embedded in 4-4′-bis(9-carbazolyl)-2,2′-biphenyl (CBP) host show the peak external quantum efficiency of 4.5% and Commission Internationale d\\'Énclairage (CIE) coordinates of (0.15, 0.07). Hybrid white devices using fluorescent blue emitting layer with 1 and a phosphorescent orange emitting layer based on an iridium-complex show the peak external quantum efficiency above 10% and CIE coordinates of (0.31, 0.37). © 2013 Published by Elsevier B.V.

  13. Functionally specified protein signatures distinctive for each of the different blue copper proteins

    Directory of Open Access Journals (Sweden)

    Anishetty Sharmila

    2004-09-01

    Full Text Available Abstract Background Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. Results Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids

  14. Engineered fluorescent proteins illuminate the bacterial periplasm

    Directory of Open Access Journals (Sweden)

    Thorben Dammeyer

    2012-10-01

    Full Text Available The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP, remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat pathway, but actively fold in the periplasm following general secretory pathway (Sec and signal recognition particle (SRP mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  15. Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy.

    Science.gov (United States)

    Duwé, Sam; De Zitter, Elke; Gielen, Vincent; Moeyaert, Benjamien; Vandenberg, Wim; Grotjohann, Tim; Clays, Koen; Jakobs, Stefan; Van Meervelt, Luc; Dedecker, Peter

    2015-10-27

    "Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins. PMID:26308583

  16. Green fluorescent protein is lighting up fungal biology

    Science.gov (United States)

    Lorang, J.M.; Tuori, R.P; Martinez, J.P; Sawyer, T.L.; Redman, R.S.; Rollins, J. A.; Wolpert, T.J.; Johnson, K.B.; Rodriguez, R.J.; Dickman, M. B.; Ciuffetti, L.M.

    2001-01-01

    Prasher (42) cloned a cDNA for the green fluorescent protein (GFP) gene from the jellyfishAequorea victoria in 1992. Shortly thereafter, to the amazement of many investigators, this gene or derivatives thereof were successfully expressed and conferred fluorescence to bacteria andCaenorhabditis elegans cells in culture (10,31), followed by yeast (24, 39), mammals (40), Drosophila (66),Dictyostelium(23, 30), plants (28,49), and filamentous fungi (54). The tremendous success of GFP as a reporter can be attributed to unique qualities of this 238-amino-acid, 27-kDa protein which absorbs light at maxima of 395 and 475 nm and emits light at a maximum of 508 nm. The fluorescence of GFP requires only UV or blue light and oxygen, and therefore, unlike the case with other reporters (β-glucuronidase, β-galacturonidase, chloramphenicol acetyltransferase, and firefly luciferase) that rely on cofactors or substrates for activity, in vivo observation ofgfp expression is possible with individual cells, with cell populations, or in whole organisms interacting with symbionts or environments in real time. Complications caused by destructive sampling, cell permeablization for substrates, or leakage of products do not occur. Furthermore, the GFP protein is extremely stable in vivo and has been fused to the C or N terminus of many cellular and extracellular proteins without a loss of activity, thereby permitting the tagging of proteins for gene regulation analysis, protein localization, or specific organelle labeling. The mature protein resists many proteases and is stable up to 65°C and at pH 5 to 11, in 1% sodium dodecyl sulfate or 6 M guanidinium chloride (reviewed in references 17and 67), and in tissue fixed with formaldehyde, methanol, or glutaraldehyde. However, GFP loses fluorescence in methanol-acetic acid (3:1) and can be masked by autofluorescent aldehyde groups in tissue fixed with glutaraldehyde. Fluorescence is optimal at pH 7.2 to 8.0 (67).

  17. Changes of the laser-induced blue, green and red fluorescence signatures during greening of etiolated leaves of wheat

    International Nuclear Information System (INIS)

    The UV-laser-induced blue, green and red fluorescence-emission spectra were used to characterize the pigment status of etiolated leaves of wheat (Triticum aestivum L.) during a 48 h greening period under white light conditions. Upon UV-light excitation (337 nm) leaves not only show a fluorescence emission in the red spectral region between 650 and 800nm (chlorophyll fluorescence with maxima near 690nm and 735 nm), but also in the blue and green regions between 400 to 570 nm with maxima or shoulders near 450 nm (blue) and 530 nm (green). During greening of etiolated leaves the chlorophyll-fluorescence ratio F690/F735 strongly correlated with the total chlorophyll content and the ratio of the chlorophylls to the carotenoids (a+b/x+c). The ratio of the blue to the green fluorescence F450/F530 was also correlated with the total chlorophyll content and the ratio of chlorophylls to total carotenoids (a+b/x+c). Consequently, there also existed a correlation between the chlorophyll-fluorescence ratio F690/F735 and the ratio of the blue to green fluorescence F450/F530. In contrast, the ratios of the blue to red fluorescences F450/F690 and F450/F735 did not show clear relations to the pigment content of the investigated plants. The particular shape of the UV-laser-induced-fluorescence emission spectra of wheat leaves as well as the dependencies of the fluorescence ratios on the pigment content are due to a partial and differential reabsorption of the emitted fluorescences by the photosynthetic pigments

  18. Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2015-10-01

    Full Text Available The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

  19. Fluorescence of Alexa Fluor dye tracks protein folding

    OpenAIRE

    Simon Lindhoud; Westphal, Adrie H.; Visser, Antonie J.W.G.; Jan Willem Borst; van Mierlo, Carlo P. M.

    2012-01-01

    Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluoresc...

  20. Blue-native PAGE in plants: a tool in analysis of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Millar A Harvey

    2005-11-01

    Full Text Available Abstract Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE. BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms, including bacteria, yeasts, animals and plants. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science.

  1. Identification and expression of a gfpK79R mutant of green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A mutant strain of E.coli showing strong green fluorescence is obtained during the study of cloning and expression of blue fluorescent protein gene bfp. A recombinant plasmid pHN122 has been isolated. It is demonstrated by enzyme digestion, subcloning and sequence analysis that it contains both of bfp and gfpK79R mutant genes. The results of spectrometric analysis show that GFPK79R expressed by pHN122 is the same as the wild type GFP, but its fluorescent intensity is increased 1.25~2.44-fold.

  2. Comparative Study of Lettuce and Radish Grown Under Red and Blue LEDs and White Fluorescent Lamps

    Science.gov (United States)

    Mickens, Matthew A.; Massa, Gioia; Newsham, Gerard; Wheeler, Raymond; Birmele, Michele

    2016-01-01

    Growing vegetable crops in space will be an essential part of sustaining astronauts during long-range missions. To drive photosynthesis, red and blue light-emitting diodes (LEDs) have attracted attention because of their efficiency, longevity, small size, and safety. In efforts to optimize crop yield, there is also recent interest in analyzing the subtle effects of additional wavelengths on plant growth. For instance, since plants often look purplish gray under red and blue LEDs, the addition of green light allows easy recognition of disease and the assessment of plant health status. However, it is important to know if wavelengths outside the traditional red and blue wavebands have a direct effect on enhancing or hindering the mechanisms involved in plant growth. In this experiment, a comparative study was performed on two short cycle crops of red romaine lettuce (Lactuca sativa cv. "Outredgeous") and radish (Raphanus sativa cv. 'Cherry Bomb'), which were grown under two light treatments. The first treatment being red (630 nm) and blue (450 nm) LEDs alone, while the second treatment consisted of daylight tri-phosphor fluorescent lamps (CCT approximately 5000 K) at equal photosynthetic photon flux (PPF). The treatment effects were evaluated by measuring the fresh biomass produced, plant morphology and leaf dimensions, leaf chlorophyll content, and adenosine triphosphate (ATP) within plant leaf/storage root tissues.

  3. Endogenous green fluorescent protein (GFP) in Amphioxus

    OpenAIRE

    Deheyn, D. D.; K. Kubokawa; McCarthy, J. K.; Murakami, A.; Porrachia, M.; Rouse, G.W.; Holland, N.D.

    2007-01-01

    Green fluorescent proteins (GFPs) are well known for their intensive use in cellular and molecular biology in applications that take advantage of the GFPs self-folding and built-in fluorophore characteristics as biomarker. Occurrence and function of GFPs in nature is less known. For a long time GFPs were described only from some cnidarians, and it is only recently that they were also found in copepod crustaceans. Here we describe the occurrence of a GFP from three species of amphioxus, namely...

  4. Synthesis and intrinsic blue fluorescence study of hyperbranched poly(ester-amide-ether)

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A series of hyperbranched poly(ester-amide-ether)s (H-PEAEs) were synthesized via the A2+CB3 approach by the self-transesterification of ethyl ester-amide-ethers end-capped with three hydroxyl groups and ethyl ester group at two terminals.The molecular structures were characterized with 1H NMR and FT-IR spectroscopy.The number average molecular weights were estimated by GPC analysis to possess bimodal wide distribution from 1.57 to 2.09.The strong inherent blue fluorescence was observed at 330 nm for excitation and 390 nm for emission.Moreover,the emission intensity and fluorescence quantum yield increased along with the incorporated ether chain length,as well as almost linearly with the H-PEAE concentration in an aqueous solution.For comparing the fluorescence performance,the linear poly(ester-amide-ether) (L-PEAE) and hyperbranched poly(ester-amide) (H-PEA) were synthesized.The results showed that the coexistence of ether bond and carboxyl group in the molecular chain was essential for generating the strong fluorescence.However,the compact backbone of H-PEAE would be propitious to the enhancement of fluorescence properties.

  5. Fixation-resistant photoactivatable fluorescent proteins for CLEM.

    Science.gov (United States)

    Paez-Segala, Maria G; Sun, Mei G; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A; Macklin, John J; Patel, Ronak; Allen, John R; Howe, Elizabeth S; Piszczek, Grzegorz; Hess, Harald F; Davidson, Michael W; Wang, Yalin; Looger, Loren L

    2015-03-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. PMID:25581799

  6. Efficient fluorescent deep-blue and hybrid white emitting devices based on carbazole/benzimidazole compound

    KAUST Repository

    Yang, Xiaohui

    2011-07-28

    We report the synthesis, photophysics, and electrochemical characterization of carbazole/benzimidazole-based compound (Cz-2pbb) and efficient fluorescent deep-blue light emitting devices based on Cz-2pbb with the peak external quantum efficiency of 4.1% and Commission Internationale dÉnclairage coordinates of (0.16, 0.05). Efficient deep-blue emission as well as high triplet state energy of Cz-2pbb enables fabrication of hybrid white organic light emitting diodes with a single emissive layer. Hybrid white emitting devices based on Cz-2pbb show the peak external quantum efficiency exceeding 10% and power efficiency of 14.8 lm/W at a luminance of 500 cd/m2. © 2011 American Chemical Society.

  7. Detection of single fluorescent proteins inside eukaryotic cells using two-photon fluorescence

    OpenAIRE

    Hou, Ximiao; Cheng, Wei

    2012-01-01

    Imaging single fluorescent proteins in a live cell is a challenging task because of the strong cellular autofluorescence. Autofluorescence can be minimized by reducing fluorescence excitation volume. Total internal reflection fluorescence (TIRF) microscopy has been routinely used to reduce excitation volume and detect single protein molecules in or close to cell membrane. However, the limited penetration depth of evanescent field excludes imaging of single fluorescent proteins that reside dee...

  8. OSL response bleaching of BeO samples, using fluorescent light and blue LEDs

    Science.gov (United States)

    Groppo, D. P.; Caldas, L. V. E.

    2016-07-01

    The optically stimulated luminescence (OSL) is widely used as a dosimetric technique for many applications. In this work, the OSL response bleaching of BeO samples was studied. The samples were irradiated using a beta radiation source (90Sr+90Y); the bleaching treatments (fluorescent light and blue LEDs) were performed, and the results were compared. Various optical treatment time intervals were tested until reaching the complete bleaching of the OSL response. The best combination of the time interval and bleaching type was analyzed.

  9. Small quinolinium-based enzymatic probes via blue-to-red ratiometric fluorescence.

    Science.gov (United States)

    Wang, Pan; Du, Jiajun; Liu, Huijing; Bi, Guoqiang; Zhang, Guoqing

    2016-02-01

    A small fluorescence ratiometric probe consisting of a single dye species, N-methyl-6-hydroxyquinolinium (MHQ), and coupled enzymatic substrates, exhibits a dramatic colour change (deep blue to red) and possesses a huge response ratio (over 2000 fold) upon specific recognition of target enzymes. Such dramatic responses are attributed to the excited-state proton transfer processes of MHQ molecules in water. Here the detection of β-galactosidase and porcine pancreatic lipase is successfully demonstrated and this class of molecules has the potential to be developed as a "naked-eye" probe in vitro. PMID:26788553

  10. Response of epidermal blue-green fluorescence emission from Barley leaves to uv radiation stress

    Czech Academy of Sciences Publication Activity Database

    Karlický, Václav; Nezval, J.; Štroch, Michal; Špunda, Vladimír

    Brno: Global change research centre, Academy of Sciences of the Czech Republic, v. v. i, 2013 - (Stojanov, R.; Žalud, Z.; Cudlín, P.; Farda, A.; Urban , O.; Trnka, M.), s. 232-236 ISBN 978-80-904351-8-6. [Global Change and Resilience. Brno (CZ), 22.05.2013-24.05.2013] R&D Projects: GA MŠk(CZ) ED1.1.00/02.0073; GA ČR GA522/09/0468 Institutional support: RVO:67179843 Keywords : blue-green fluorescence * UV-B and UV-A radiation * barley * plant stress Subject RIV: EH - Ecology, Behaviour

  11. Green fluorescent protein as a reporter of prion protein folding

    Directory of Open Access Journals (Sweden)

    Dalton Kevin

    2006-08-01

    Full Text Available Abstract Background The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP, as a method to examine the refolding of the amino terminal domain of mouse prion protein. Results Fusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence. Conclusion Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

  12. Fluorescence Studies of Protein Crystallization Interactions

    Science.gov (United States)

    Pusey, Marc L.; Smith, Lori; Forsythe, Elizabeth

    1999-01-01

    We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

  13. Versatile protein tagging in cells with split fluorescent protein.

    Science.gov (United States)

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A; Ishikawa, Hiroaki; Leonetti, Manuel D; Marshall, Wallace F; Weissman, Jonathan S; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  14. Evidence for covalent binding of epicocconone with proteins from synchronous fluorescence spectra and fluorescence lifetimes

    Indian Academy of Sciences (India)

    Debashis Panda; Anindya Datta

    2007-03-01

    Synchronous fluorescence and time-resolved fluorescence spectroscopic studies that reveal the interaction of epicocconone with human serum albumin is significantly different from its interaction with surfactant assemblies. This observation, along with steady-state fluorescence data, indicates groundstate interaction between the fluorophore epicocconone and the protein. Similarity in fluorescence properties with the adduct of the fluorophore with -butylamine indicates that bonding occurs at the Nterminus of the protein.

  15. Heat generation and light scattering of green fluorescent protein-like pigments in coral tissue

    Science.gov (United States)

    Lyndby, Niclas H.; Kühl, Michael; Wangpraseurt, Daniel

    2016-05-01

    Green fluorescent protein (GFP)-like pigments have been proposed to have beneficial effects on coral photobiology. Here, we investigated the relationships between green fluorescence, coral heating and tissue optics for the massive coral Dipsastraea sp. (previously Favia sp.). We used microsensors to measure tissue scalar irradiance and temperature along with hyperspectral imaging and combined imaging of variable chlorophyll fluorescence and green fluorescence. Green fluorescence correlated positively with coral heating and scalar irradiance enhancement at the tissue surface. Coral tissue heating saturated for maximal levels of green fluorescence. The action spectrum of coral surface heating revealed that heating was highest under red (peaking at 680 nm) irradiance. Scalar irradiance enhancement in coral tissue was highest when illuminated with blue light, but up to 62% (for the case of highest green fluorescence) of this photon enhancement was due to green fluorescence emission. We suggest that GFP-like pigments scatter the incident radiation, which enhances light absorption and heating of the coral. However, heating saturates, because intense light scattering reduces the vertical penetration depth through the tissue eventually leading to reduced light absorption at high fluorescent pigment density. We conclude that fluorescent pigments can have a central role in modulating coral light absorption and heating.

  16. A new approach to dual-color two-photon microscopy with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Rebane Aleks

    2010-02-01

    Full Text Available Abstract Background Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA efficiency. Results Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. Conclusion Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

  17. Phycobiliprotein fusion proteins: versatile intensely fluorescent constructs

    Science.gov (United States)

    Glazer, Alexander N.; Cai, Yuping A.; Tooley, Aaron J.

    2004-06-01

    Since 1982, phycobiliproteins have served as fluorescent labels in a wide variety of cell and molecule analyses. The exceptional spectroscopic properties of these labels include very high absorbance coefficients and quantum yields, and large Stokes shifts. The spectroscopic diversity of these reagents is restricted to a subset of naturally occurring phycobiliproteins with stable assembly states in vitro, whose target specificity is generated by chemical conjugation to proteins or small molecules. The latter step generates heterogeneity. These limitations have been overcome by expressing various recombinant phycobiliprotein constructs in the cyanobacterium Anabaena sp. PCC7120. Modular recombinant phycobiliprotein-based labels were constructed with some or all of the following features (a) an affinity purification tag; (b) a stable oligomerization domain (to maintain stable higher order assemblies of the phycobiliprotein monomers at very low protein concentration); (c) a biospecific recognition domain. Such phycobiliprotein constructs are readily purified from crude cell extracts by affinity chromatography and used directly as fluorescent labels. To generate constructs for intracellular in vivo labeling, the entire pathways for the biosynthesis of the His-tagged holo- α (phycocyanobilin-bearing) subunit of phycocyanin (emission max. 641 nm) and of the His-tagged holo-α (phycobiliviolin-bearing) subunit of phycoerythrocyanin (emission max. 582 nm) were reconstituted in Escherichia coli.

  18. Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

    Science.gov (United States)

    Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

    1998-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

  19. Fluorescent protein engineering by in vivo site-directed mutagenesis

    OpenAIRE

    Ceballos, Melvys Valledor; Hu, Qinghua; Schiller, Paul,; Myers, Richard S.

    2012-01-01

    In vivo site-directed mutagenesis by ssDNA recombineering is a facile method to change the color of fluorescent proteins without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the E. coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the fluorescent protein. E...

  20. Resonantly Enhanced Multi-Photon Ionization Spectrum of the Neutral Green Fluorescent Protein Chromophore

    OpenAIRE

    greenwood, jason; Miles, Jordan; De Camillis, Simone; Mulholland, Peter; Zhang, Lijuan; Parkes, Michael A.; Hailes, Helen C.; Fielding, Helen H.

    2014-01-01

    The photophysics of the green fluorescent protein is governed by the electronic structure of the chromophore at the heart of its β-barrel protein structure. We present the first two-color, resonance-enhanced, multiphoton ionization spectrum of the isolated neutral chromophore in vacuo with supporting electronic structure calculations. We find the absorption maximum to be 3.65 ± 0.05 eV (340 ± 5 nm), which is blue-shifted by 0.5 eV (55 nm) from the absorption maximum of the protein in its neut...

  1. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    International Nuclear Information System (INIS)

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46–103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 °C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit

  2. A green fluorescent protein with photoswitchable emission from the deep sea.

    Directory of Open Access Journals (Sweden)

    Alexander Vogt

    Full Text Available A colorful variety of fluorescent proteins (FPs from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

  3. Highly efficient non-doped deep blue fluorescent emitters with horizontal emitting dipoles using interconnecting units between chromophores.

    Science.gov (United States)

    Kim, Kwon-Hyeon; Baek, Jang Yeol; Cheon, Chan Woo; Moon, Chang-Ki; Sim, Bomi; Choi, Myeong Yong; Kim, Jang-Joo; Kim, Yun-Hi

    2016-09-21

    New deep blue fluorescent emitters composed of anthracene as an electron rich unit, a diphenyltriazine as a strong electron acceptor unit, and phenyl or xylene as interconnecting units were synthesised. The interconnecting unit between chromophores increased the singlet transition energy and the ratio of horizontal emitting dipoles. As a result, a non-doped blue fluorescent organic light-emitting diode (OLED) using a new emitter was demonstrated, with an external quantum efficiency (EQE) of 6.6% and Commision Internationale de l'Eclairage (CIE) colour coordinates of (0.145, 0.068). This device performance has been the highest EQE observed in deep blue non-doped OLEDs with CIE coordinates less than (0.145, 0.068) to date. PMID:27524501

  4. A photoactivatable green-fluorescent protein from the phylum Ctenophora

    OpenAIRE

    Haddock, Steven H. D.; Mastroianni, Nadia; Christianson, Lynne M.

    2015-01-01

    Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the xYG chromophore motif of all other characterized GFP-type proteins. These pro...

  5. Controllable synthesis of green and blue fluorescent carbon nanodots for pH and Cu(2+) sensing in living cells.

    Science.gov (United States)

    Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Zhao, Bo; Wen, Xiangping; Zhang, Guomei; Dong, Chuan; Shuang, Shaomin

    2016-03-15

    We report a controllable strategy for fabrication of green and blue fluorescent carbon nanodots (CDs), and demonstrate their applications for pH and Cu(2+) sensing in living cells. Green and blue fluorescent CDs have been synthesized by hydrothermal method and pyrolysis of leeks, respectively, providing an easy way for the production of CDs without the request of tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. Green fluorescent CDs (G-CDs) exhibit high tolerance to pH values and external cations. Blue fluorescent CDs (B-CDs) can be applied to pH and Cu(2+) sensing. The linear range of Cu(2+) detection is 0.01-10.00 μM and the detection limit is 0.05 μM. For pH detection, there is a good linearity in the pH range of 3.5-10.0. The linear and rapid response of B-CDs to Cu(2+) and pH is valuable for Cu(2+) and pH sensing in living cells. Confocal fluorescent imaging of human cervical carcinoma cells indicates that B-CDs could visualize Cu(2+) and pH fluctuations in living cells with negligible autofluorescence. PMID:26485174

  6. Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

    Directory of Open Access Journals (Sweden)

    Kuang Lin-Yun

    2008-10-01

    Full Text Available Abstract Background The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed. Results We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP, and prey proteins are tagged with the N-terminal portions of either Venus (nVenus or Cerulean (nCerulean. Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin α-1 in the cytoplasm, whereas interaction of VirE2 with a different importin α isoform, importin α-4, occurs predominantly in the nucleus. Conclusion Multi

  7. In vivo cellular imaging using fluorescent proteins - Methods and Protocols

    Directory of Open Access Journals (Sweden)

    M. Monti

    2012-12-01

    Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

  8. Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

    Science.gov (United States)

    Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

    2016-02-01

    Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice. PMID:26549191

  9. Determination of the Electron Self-Exchange Rates of Blue Copper Proteins by Super-WEFT NMR Spectroscopy

    DEFF Research Database (Denmark)

    Ma, Lixin; Philipp, Else Astrid; Led, Jens J.

    Anabaena variabilis plastocyanin, blue copper proteins, electron self-exchange rates, electron transfer, super-WEFT NMR......Anabaena variabilis plastocyanin, blue copper proteins, electron self-exchange rates, electron transfer, super-WEFT NMR...

  10. Fluorescence plate reader for quantum dot-protein bioconjugation analysis.

    Science.gov (United States)

    Carvalho, Kilmara H G; Brasil, Aluizio G; Cabral Filho, Paulo E; Tenório, Denise P L A; de Siqueira, Ana C A; Leite, Elisa S; Fontes, Adriana; Santos, Beate S

    2014-05-01

    We present here a new and alternative method that uses a Fluorescence Plate Reader in a different approach, not to study protein-protein interactions, but to evaluate the efficiency of the protein bioconjugation to quantum dots (QDs). The method is based on the QDs' native fluorescence and was successfully tested by employing two different QDs-proteins conjugation methodologies, one by promoting covalent binding and other by inducing adsorption processes. For testing, we used bioconjugates between carboxyl coated CdTe QDs and bovine serum albumin, concanavalin A lectin and anti-A antibody. Flow cytometry and fluorescence spectroscopy studies corroborated the results found by the Fluorescence Plate Reader assay. This kind of analysis is important because poor bioconjugation efficiency leads to unsuccessful applications of the fluorescent bioconjugates. We believe that our method presents the possibility of performing semi-quantitative and simultaneous analysis of different samples with accuracy taking the advantage of the high sensitivity of optical based measurements. PMID:24734547

  11. Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.

    Science.gov (United States)

    Landete, José M; Langa, Susana; Revilla, Concepción; Margolles, Abelardo; Medina, Margarita; Arqués, Juan L

    2015-08-01

    Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production. PMID:26129953

  12. Fluorescence of Alexa fluor dye tracks protein folding.

    Directory of Open Access Journals (Sweden)

    Simon Lindhoud

    Full Text Available Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488, which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

  13. Light-induced conformational changes and energy transfer in red fluorescent protein

    International Nuclear Information System (INIS)

    Reversible conformational changes have been photo-induced in the red fluorescent protein DsRed at low temperature by wavelength-selective laser irradiation. We have found two new fluorescent forms: a shifted-red (SR-) and a new green (G'-) form that absorb and emit, respectively, ∼14 nm to the red and ∼80 nm to the blue of the 'mature' red (R-) form present in an un-illuminated sample of DsRed. Further, we have identified the 0-0 transitions of the various forms by spectral hole burning and estimated their ground-state energy differences and barrier heights by means of temperature-dependent excitation and fluorescence spectroscopy between 1.6 and 295 K. We have also proven that 'downhill' energy transfer takes place between these forms within the tetrameric structure of DsRed

  14. Fluorescence Quantum Yield Measurements of Fluorescent Proteins: A Laboratory Experiment for a Biochemistry or Molecular Biophysics Laboratory Course

    Science.gov (United States)

    Wall, Kathryn P.; Dillon, Rebecca; Knowles, Michelle K.

    2015-01-01

    Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts…

  15. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  16. Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it

    Science.gov (United States)

    Morikawa, Takamitsu J.; Fujita, Hideaki; Kitamura, Akira; Horio, Takashi; Yamamoto, Johtaro; Kinjo, Masataka; Sasaki, Akira; Machiyama, Hiroaki; Yoshizawa, Keiko; Ichimura, Taro; Imada, Katsumi; Nagai, Takeharu; Watanabe, Tomonobu M.

    2016-01-01

    Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Förster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells. PMID:26956628

  17. LucY: A Versatile New Fluorescent Reporter Protein.

    Directory of Open Access Journals (Sweden)

    Michele E Auldridge

    Full Text Available We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.

  18. Constitutive and Inducible Green Fluorescent Protein Expression in Bartonella henselae

    OpenAIRE

    Lee, Anthea K.; Falkow, Stanley

    1998-01-01

    The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselae chromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37°C was isolated.

  19. Common fluorescent proteins for single-molecule localization microscopy

    Science.gov (United States)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  20. Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

    OpenAIRE

    Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

    2011-01-01

    Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturat...

  1. Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Stepanenko, Olesya V; Baloban, Mikhail; Bublikov, Grigory S; Shcherbakova, Daria M; Stepanenko, Olga V; Turoverov, Konstantin K; Kuznetsova, Irina M; Verkhusha, Vladislav V

    2016-01-01

    Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs. PMID:26725513

  2. Painting Proteins Blue: β-(1-Azulenyl)-L-Alanine as a Probe for Studying Protein-Protein Interactions

    Science.gov (United States)

    Moroz, Yurii S.; Binder, Wolfgang; Nygren, Patrik; Caputo, Gregory A.; Korendovych, Ivan V.

    2013-01-01

    We demonstrated that β-(1-Azulenyl)-L-Alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them. PMID:23207368

  3. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

    Felske, A; Vandieken, V; Pauling, B V;

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...

  4. Mechanism of chromophore assisted laser inactivation employing fluorescent proteins.

    Science.gov (United States)

    McLean, Mark A; Rajfur, Zenon; Chen, Zaozao; Humphrey, David; Yang, Bing; Sligar, Stephen G; Jacobson, Ken

    2009-03-01

    Chromophore assisted laser inactivation (CALI) is a technique that uses irradiation of chromophores proximate to a target protein to inactivate function. Previously, enhanced green fluorescent protein (EGFP) mediated CALI has been used to inactivate EGFP-fusion proteins in a spatio-temporally defined manner within cells, but the mechanism of inactivation is unknown. To help elucidate the mechanism of protein inactivation mediated by fluorescent protein CALI ([FP]-CALI), the activities of purified glutathione-S-transferase-FP (GST-EXFP) fusions were measured after laser irradiation in vitro. Singlet oxygen and free radical quenchers as well as the removal of oxygen inhibited CALI, indicating the involvement of a reactive oxygen species (ROS). At higher concentrations of protein, turbidity after CALI increased significantly indicating cross-linking of proximate fusion proteins suggesting that damage of residues on the surface of the protein, distant from the active site, results in inactivation. Control experiments removed sample heating as a possible cause of these effects. Different FP mutants fused to GST vary in their CALI efficiency in the order enhanced green fluorescent protein (EGFP) > enhanced yellow fluorescent protein (EYFP) > enhanced cyan fluorescent protein (ECFP), while a GST construct that binds fluorescein-based arsenical hairpin binder (FlAsH) results in significantly higher CALI efficiency than any of the fluorescent proteins (XFPs) tested. It is likely that the hierarchy of XFP effectiveness reflects the balance between ROS that are trapped within the XFP structure and cause fluorophore and chromophore bleaching and those that escape to effect CALI of proximate proteins. PMID:19199572

  5. Rapid synthesis of blue emitting ZnO nanoparticles for fluorescent applications

    Science.gov (United States)

    Jule, Leta T.; Dejene, Francis B.; Roro, Kittessa T.; Urgessa, Zelalem N.; Botha, Johannes R.

    2016-09-01

    ZnO nanoparticles (NPs), with size ∼16-20 nm were produced using simple, cost effective and rapid synthesis method. In this method zinc salt (typically zinc acetate dehydrate) is directly annealed in air at a temperature from 200 ° C to 500 ° C for 2 h to form ZnO (NPs). This synthesis method, only requires zinc precursor to produce NPs that can emit visible emission without external doping. X-ray diffraction (XRD) patterns confirm the prepared ZnO NPs is polycrystalline structure with wurtzite phase. The observed variation in scanning electron microscopy (SEM) images showed spherical shape of the ZnO NPs. It was found that the NPs exhibited the estimated direct bandgap (Eg) of 3.28 eV, 3.29 eV, 3.33 eV and 3.39 eV for a decomposition temperature of 500, 400, 300 and 200 ° C . Energy dispersive X-ray (EDX) analysis showed that carbon is the only impurity at lower temperature which was most likely originated from the acetate group. The photoluminescence (PL) spectra of ZnO NPs showed the appearance of a blue emission, attributed to Zn interstitials, whose intensity reduces with increase in decomposition temperature and the underlying mechanism are discussed. For the samples prepared at 200 ° C and 300 °C a temperature dependent PL was studied and found out that there are about three transition lines at ∼3.01 eV, ∼3.21 eV and ∼3.33 eV, which are ascribed to zinc vacancy (Vzn), donor-acceptor pairs(DAP) and excitons bound to structural defects respectively. It is hoped that ZnO NPs produced using this method would be ideal for blue light emitting fluorescent application as it is catalyst free growth, uses simple equipment, less hazardous and easy to control particle size and morphologies by scalable temperature.

  6. Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, Heather G.; Roberto, Francisco F.

    2006-02-07

    The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  7. Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, Heather G.; Roberto, Francisco F.

    2006-01-17

    The present invention comprises a Mytilus edulis cDNA sequenc having a nucleotide sequence that encodes for the Mytilus edulis foot protein-1 (Mefp-1), an example of a mollusk foot protein. Mefp-1 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-1 gene will allow researchers to produce Mefp-1 protein using genetic engineering techniques. The discovery of Mefp-1 gene sequence will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  8. Engineering and Characterization of a Superfolder Green Fluorescent Protein

    International Nuclear Information System (INIS)

    Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP

  9. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    OpenAIRE

    Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    International audience In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent prote...

  10. Fluorescence labeling of carbon nanotubes and visualization of a nanotube-protein hybrid under fluorescence microscope.

    Science.gov (United States)

    Yoshimura, Shige H; Khan, Shahbaz; Maruyama, Hiroyuki; Nakayama, Yoshikazu; Takeyasu, Kunio

    2011-04-11

    Biological applications of carbon nanotubes have been hampered by the inability to visualize them using conventional optical microscope, which is the most common tool for the observation and measurement of biological processes. Recently, a number of fluorescence labeling methods for biomolecules and various fluorescence probes have been developed and widely utilized in biological fields. Therefore, labeling carbon nanotubes with such fluorophores under physiological conditions will be highly useful in their biological applications. In this Article, we present a method to fluorescently label nanotubes by combining a detergent and a fluorophore commonly used in biological experiments. Fluorophores carrying an amino group (Texas Red hydrazide or BODIPY FL-hydrazide) were covalently attached to the hydroxyl groups of Tween 20 using carbonyldiimidazole. Fluorescence microscopy demonstrated that nanotubes were efficiently solubilized and labeled by this fluorescently labeled detergent. By using this technique, we also demonstrated multicolor fluorescence imaging of a nanotube-protein hybrid. PMID:21395219

  11. Highly fluorescence-stable blue CdZnS/ZnS quantum dots against degradable environmental conditions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ki-Heon; Lee, Jeong-Hoon; Kang, Hee-Don; Han, Chang-Yeol [Department of Materials Science and Engineering, Hongik University, Seoul 121-791 (Korea, Republic of); Bae, Seung Muk [Kunsan National University Center For Research Facilities, Kunsan National University, Gunsan, Jeonbuk 573-701 (Korea, Republic of); Lee, Yangjin; Hwang, Jun Yeon [Institute of Advanced Composite Materials, Korea Institute of Science and Technology, Jeonbuk 565-905 (Korea, Republic of); Yang, Heesun, E-mail: hyang@hongik.ac.kr [Department of Materials Science and Engineering, Hongik University, Seoul 121-791 (Korea, Republic of)

    2014-10-15

    Graphical abstract: Blue-emitting CdZnS/ZnS alloy QDs exhibited the excellent fluorescent stability against a thorough purification cycle, long-term UV illumination, and hydrophobic-to-hydrophilic ligand exchange. - Highlights: • Highly efficient, color-pure blue CdZnS/ZnS QDs are synthesized. • Fluorescent properties of the QDs remain unchanged against various degradable conditions. • Closely-packed solid QD film is compared with QD solution in PL QY and emission decay dynamics. - Abstract: We report on synthesis of highly efficient, color-pure blue CdZnS/ZnS quantum dots (QDs) and their excellent fluorescent stability against degradable environmental conditions. The CdZnS/ZnS QDs finely emission-tunable in the range of 440–461 nm exhibit high photoluminescence (PL) quantum yields (QYs) of 81–92% along with an exceptionally narrow spectral bandwidth of 19 nm. These QDs are subjected to various post-treatments of a thorough purification cycle, long-term UV illumination, and hydrophobic-to-hydrophilic ligand exchange, each of which is usually detrimental to QD fluorescence. As a result, no noticeable deterioration in the fluorescent properties is observed, indicative of the insensitiveness of our CdZnS/ZnS QDs to the above degradable processes. Furthermore, PL properties of a solid film of multiple QD layers are assessed and compared with those of a diluted QD solution, showing an unprecedentedly small QY reduction of solid QD film versus QD solution. The above promising results are justified mainly by emphasizing the role of a thick ZnS shell present in CdZnS/ZnS QDs.

  12. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    Directory of Open Access Journals (Sweden)

    Mudrik Nikolay N

    2002-04-01

    Full Text Available Abstract Background Within the family of green fluorescent protein (GFP homologs, one can mark two main groups, specifically, fluorescent proteins (FPs and non-fluorescent or chromoproteins (CPs. Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595 from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield ( Conclusions We located a novel point in asCP sequence (position 165 mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

  13. Latest methods of fluorescence-based protein crystal identification

    International Nuclear Information System (INIS)

    Fluorescence, whether intrinsic or by using trace fluorescent labeling, can be a powerful aid in macromolecule crystallization. Its use in screening for crystals is discussed here. Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation (<0.5%) of the protein, enables the use of visible fluorescence. Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins. By the use of these techniques, crystals that had previously been obscured in the crystallization drop can readily be identified and distinguished from amorphous precipitate or salt crystals. Additionally, lead conditions that may not have been obvious as such under white-light illumination can be identified. In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity

  14. Glucoamylase : green fluorescent protein fusions to monitor protein secretion in Aspergillus niger

    NARCIS (Netherlands)

    Gordon, C.L.; Khalaj, V.; Ram, A.F.J.; Archer, D.B.; Brookman, J.L.; Trinci, A.P.J.; Jeenes, D.J.; Doonan, J.H.; Wells, B.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Robson, G.D.

    2000-01-01

    A glucoamylase: :green fluorescent protein fusion (GLA: :sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA: :sGFP which employed slightly different len

  15. Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

    NARCIS (Netherlands)

    Bader, A.N.; Visser, N.V.; Amerongen, van H.; Visser, A.J.W.G.

    2014-01-01

    The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and ex

  16. Circular dichroism and polarized fluorescence characteristics of blue-green algal allophycocyanins

    Energy Technology Data Exchange (ETDEWEB)

    Canaani, O.D.; Gantt, E.

    1980-06-24

    Allophycocyanin, the terminal pigment in the phycobiliprotein transfer sequence, isolated from dissociated phycobilisomes of Nostoc sp., was fractionated on calcium phosphate columns into four spectral forms: APC I, II, III, and B. These forms had distinctive isoelectric points of 5.15, 4.68, 4.82, and 4.98, respectively. The APC forms differed in their secondary structure as suggested by the varying percentages of their ..cap alpha.. helix and ..beta..-pleated sheets. APC II and III are short-emitting forms with a fluorescence maximum at 660 nm, while APC I and B are long-emitting forms with a maximum at 681 nm. The maximum of APC I and B at -196/sup 0/C in 0.1 M phosphate and 20% glycerol shifted to 688 nm. Fluorescence polarization spectra suggest that there are at least two groups of chromophores responsible for the absorption of APC I and similarly of APC B. In APC II and III, the fluorescence was mostly depolarized. Circular dichroism revealed extensive positive and negative ellipticity band multiplicities in the chromophore absorption region of APC I and B, but not in APC II and III. Two main CD extrema in APC B, a negative band and a positive band, are probably the result of exciton coupling of phycocyanobilin chromophores absorbing at longer wavelength. In APC I three different peaks are revealed in the absorption spectrum and four ellipticity bands in the CD spectrum at -196/sup 0/C. These can best be explained as being due to the combined interactions of the chromophore with the protein and exciton coupling between chromophores.

  17. Measuring the bioactivity and molecular conformation of typically globular proteins with phenothiazine-derived methylene blue in solid and in solution: A comparative study using photochemistry and computational chemistry.

    Science.gov (United States)

    Ding, Fei; Xie, Yong; Peng, Wei; Peng, Yu-Kui

    2016-05-01

    Methylene blue is a phenothiazine agent, that possesses a diversity of biomedical and biological therapeutic purpose, and it has also become the lead compound for the exploitation of other pharmaceuticals such as chlorpromazine and the tricyclic antidepressants. However, the U.S. Food and Drug Administration has acquired cases of detrimental effects of methylene blue toxicities such as hemolytic anemia, methemoglobinemia and phototoxicity. In this work, the molecular recognition of methylene blue by two globular proteins, hemoglobin and lysozyme was characterized by employing fluorescence, circular dichroism (CD) along with molecular modeling at the molecular scale. The recognition of methylene blue with proteins appears fluorescence quenching via static type, this phenomenon does cohere with time-resolved fluorescence lifetime decay that nonfluorescent protein-drug conjugate formation has a strength of 10(4)M(-1), and the primary noncovalent bonds, that is hydrogen bonds, π-conjugated effects and hydrophobic interactions were operated and remained adduct stable. Meantime, the results of far-UV CD and synchronous fluorescence suggest that the α-helix of hemoglobin/lysozyme decreases from 78.2%/34.7% (free) to 58.7%/23.8% (complex), this elucidation agrees well with the elaborate description of three-dimensional fluorescence showing the polypeptide chain of proteins partially destabilized upon conjugation with methylene blue. Furthermore, both extrinsic fluorescent indicator and molecular modeling clearly exhibit methylene blue is situated within the cavity constituted by α1, β2 and α2 subunits of hemoglobin, while it was located at the deep fissure on the lysozyme surface and Trp-62 and Trp-63 residues are nearby. With the aid of computational analyses and combining the wet experiments, it can evidently be found that the recognition ability of proteins for methylene blue is patterned upon the following sequence: lysozyme

  18. Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Davidson Michael W

    2008-03-01

    Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

  19. A new series of two-photon blue/violet fluorescent trans-alkenes: Green synthesis and optical properties

    Science.gov (United States)

    Huang, Jiu-Qiang; Cai, Zhi-Bin; Jin, Fan; Li, Sheng-Li; Tian, Yu-Peng

    2016-02-01

    A new series of trans-alkenes (3a-3e) containing different electron-donating groups were synthesized by the solvent-free Horner-Wadsworth-Emmons reaction, and characterized by infrared, hydrogen nuclear magnetic resonance, mass spectrometry and elemental analysis. Their UV-visible absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence were systematically investigated in different solvents. Experimental results show different trends in linear and nonlinear optical properties with different donor units. 3a with triphenylamine donor exhibits the best optical properties. It emits strong blue up-converted fluorescence, and the two-photon absorption cross-section can be as large as 218 GM in DCM.

  20. Spectral characteristics and a possible topological assignment of blue green fluorescence excited by UV laser on leaves of untreated species

    International Nuclear Information System (INIS)

    For a number of years it has been known that a relatively strong fluorescence emission is produced by leaves when using UV radiation for excitation (Chappelle et al., 1984a); the apparent bandwidth is larger than 150 nm starting from 400 nm and includes several peaks; under excitation at 337 nm, their integrated energy is 6-11 times the energy released by chlorophyll (a) bands. The problem of bands assignment concerns relative contribution balance as several different emitters are located in chloroplasts, vacuoles, mitochondria, nuclei, cell wall, and epidermis. Within the sensitivity of a 4-lambda synchronous pulse amplified N2 laser photometer, and the experimental limits Of a continuous spectrofluorimeter, the conclusion we present here is that: a) The blue-green fluorescence emission comes mainly from other epidermal layers; b) it is transferable on a quartz lamina by quick dipping the leaves in organic solvents and subsequent solvent evaporation; c) it shows atypical diffusion controlled quenching of fluorescence intensity within a thermal window between 277 K (high fluorescence) and 310 K (low fluorescence); d) the fluorescence emissivity is not linked to short-term metabolic arrangements, but tends to follow long-term epidermis adaptations to drought, and excessive radiation

  1. Plasmon-controlled narrower and blue-shifted fluorescence emission in (Au-SiO2)SiC nanohybrids

    International Nuclear Information System (INIS)

    Fluorescent imaging is a key tool in biology. On one hand, organic dyes are subjected to bleaching; while, on the other hand II–VI semiconductor quantum dots are photostable, but may exhibit some toxicity. Silicon carbide nanoparticles (SiC NPs) are a good alternative as SiC is chemically inert and considered as biocompatible material. However, their fluorescence quantum yield is weak. Plasmon-controlled fluorescence appears as a mean to enhance SiC NPs fluorescence. To this aim, new fluorescent nanohybrids (NHs) involving a gold colloid surrounded by a silica shell and overcoated with SiC NPs have been engineered. Au-SiO2 particles were synthesized via a sol–gel method to obtain a controlled thickness of silica around gold colloid. Then, SiC NPs were immobilized onto silica by covalent bonding. TEM was used to study the structural properties of NHs. The influence of several parameters on NHs’ fluorescence properties was investigated. It reveals that excitation wavelength must be chosen as close as possible to the gold plasmon band to avoid quenching of emission due to energy transfer between gold absorption and SiC emission. Moreover, the silica thickness is a key parameter to obtain high enhancement; for gold colloids of 20 nm in diameter overcoated by a 25 nm shell, an enhancement factor as high as 12.5 was obtained with a narrower and blue-shifted emission band. This blue-shift can be attributed to the surface chemistry modification of SiC NPs when they are covalently bonded to silica.

  2. Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

    Directory of Open Access Journals (Sweden)

    Bryan Sands

    Full Text Available Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

  3. Rational design of enhanced photoresistance in a photoswitchable fluorescent protein

    International Nuclear Information System (INIS)

    Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions. (paper)

  4. Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors.

    Science.gov (United States)

    Yang, Meng; Reynoso, Jose; Jiang, Ping; Li, Lingna; Moossa, Abdool R; Hoffman, Robert M

    2004-12-01

    We report here the development of the transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives GFP expression in essentially all tissues. In crosses between nu/nu GFP male mice and nu/+ GFP female mice, the embryos fluoresced green. Approximately 50% of the offspring of these mice were GFP nude mice. Newborn mice and adult mice fluoresced very bright green and could be detected with a simple blue-light-emitting diode flashlight with a central peak of 470 nm and a bypass emission filter. In the adult mice, the organs all brightly expressed GFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum. The following systems were dissected out and shown to have brilliant GFP fluorescence: the entire digestive system from tongue to anus; the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart and major arteries and veins. The skinned skeleton highly expressed GFP. Pancreatic islets showed GFP fluorescence. The spleen cells were also GFP positive. Red fluorescent protein (RFP)-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma were transplanted to the transgenic GFP nude mice. All of these human tumors grew extensively in the transgenic GFP nude mouse. Dual-color fluorescence imaging enabled visualization of human tumor-host interaction by whole-body imaging and at the cellular level in fresh and frozen tissues. The GFP mouse model should greatly expand our knowledge of human tumor-host interaction. PMID:15574773

  5. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    International Nuclear Information System (INIS)

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

  6. Looking at the photodynamics of individual fluorescent molecules and proteins

    NARCIS (Netherlands)

    Garcia-Parajo, M.F.; Veerman, J.A.; Kuipers, L.; Hulst, van N.F.

    2001-01-01

    The photodynamics of individual molecules and fluorescent proteins has been investigated in real time. In the case of organic molecules, both the triplet state lifetime and intersystem crossing yield appear to vary in time and space. In the case of autofluorescent proteins, light-driven "on-off" flu

  7. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    Science.gov (United States)

    Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

    2014-09-01

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

  8. mKikGR, a monomeric photoswitchable fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Satoshi Habuchi

    Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

  9. Advanced Fluorescence Protein-Based Synapse-Detectors.

    Science.gov (United States)

    Lee, Hojin; Oh, Won Chan; Seong, Jihye; Kim, Jinhyun

    2016-01-01

    The complex information-processing capabilities of the central nervous system emerge from intricate patterns of synaptic input-output relationships among various neuronal circuit components. Understanding these capabilities thus requires a precise description of the individual synapses that comprise neural networks. Recent advances in fluorescent protein engineering, along with developments in light-favoring tissue clearing and optical imaging techniques, have rendered light microscopy (LM) a potent candidate for large-scale analyses of synapses, their properties, and their connectivity. Optically imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or microstructures enables the efficient, fine-resolution illumination of synaptic anatomy and function in large neural circuits. Here we review the latest progress in fluorescent protein-based molecular tools for imaging individual synapses and synaptic connectivity. We also identify associated technologies in gene delivery, tissue processing, and computational image analysis that will play a crucial role in bridging the gap between synapse- and system-level neuroscience. PMID:27445785

  10. Highly Selective Fluorescent Sensing of Proteins Based on a Fluorescent Molecularly Imprinted Nanosensor

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    2013-09-01

    Full Text Available A fluorescent molecularly imprinted nanosensor was obtained by grafting imprinted polymer onto the surface of multi-wall carbon nanotubes and post-imprinting treatment with fluorescein isothiocyanate (FITC. The fluorescence of lysozyme-imprinted polymer (Lys-MIP was quenched more strongly by Lys than that of nonimprinted polymer (NIP, which indicated that the Lys-MIP could recognize Lys. The resulted imprinted material has the ability to selectively sense a target protein, and an imprinting factor of 3.34 was achieved. The Lys-MIP also showed selective detection for Lys among other proteins such as cytochrome C (Cyt C, hemoglobin (HB and bovine serum albumin (BSA due to the imprinted sites in the Lys-MIP. This approach combines the high selectivity of surface molecular imprinting technology and fluorescence, and converts binding events into detectable signals by monitoring fluorescence spectra. Therefore, it will have further applications for Lys sensing.

  11. The photochemical and fluorescence properties of whole cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum.

    Science.gov (United States)

    Tel-or, E; Malkin, S

    1977-02-01

    The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured. Photosynthesis (O2 evolution) in whole cells; Hill reaction (O2 evolution) with Fe(CN)63- and NADP as electron acceptors (Photosystem II and photosystem II + Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern. On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90% (10%) of the chlorophyll a, 90% (10%) of the carotenoids and 15% (85%) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments; they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction. The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20--40%) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion. The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition

  12. Improved Coomassie Blue Dye-Based Fast Staining Protocol for Proteins Separated by SDS-PAGE

    OpenAIRE

    Májek, Pavel; Riedelová-Reicheltová, Zuzana; Pecánková, Klára; Dyr, Jan E.

    2013-01-01

    The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based...

  13. Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): Snipping the proton wire

    International Nuclear Information System (INIS)

    Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild-type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A* excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state

  14. Using Superfolder Green Fluorescent Protein for Periplasmic Protein Localization Studies ▿

    OpenAIRE

    Dinh, Thuy; Bernhardt, Thomas G

    2011-01-01

    Studies investigating the subcellular localization of periplasmic proteins have been hampered by problems with the export of green fluorescent protein (GFP). Here we show that a superfolding variant of GFP (sfGFP) is fluorescent following Sec-mediated transport and works best when the cotranslational branch of the pathway is employed.

  15. Comparative Study of Lettuce and Radish Grown Under Red and Blue Light-Emitting Diodes (LEDs) and White Fluorescent Lamps

    Science.gov (United States)

    Mickens, Matthew A.

    2012-01-01

    Growing vegetable crops in space will be an essential part of sustaining astronauts during long-term missions. To drive photosynthesis, red and blue light-emitting diodes (LEDs) have attracted attention because of their efficiency, longevity, small size, and safety. In efforts to optimize crop production, there have also been recent interests in analyzing the subtle effects of green light on plant growth, and to determine if it serves as a source of growth enhancement or suppression. A comparative study was performed on two short cycle crops of lettuce (Outredgeous) and radish (Cherry Bomb) grown under two light treatments. The first treatment being red and blue LEDs, and the second treatment consisting of white fluorescent lamps which contain a portion of green light. In addition to comparing biomass production, physiological characterizations were conducted on how the light treatments influence morphology, water use, chlorophyll content, and the production of A TP within plant tissues.

  16. Simultaneous improvement of emission color, singlet-triplet energy gap, and quantum efficiency of blue thermally activated delayed fluorescent emitters using a 1-carbazolylcarbazole based donor.

    Science.gov (United States)

    Kim, Mounggon; Choi, Jeong Min; Lee, Jun Yeob

    2016-08-21

    Blue thermally activated delayed fluorescent (TADF) emitters having 1-carbazolylcarbazole based donor moieties were developed to resolve the low quantum efficiency and large singlet-triplet energy splitting issues of the linker free TADF emitters. Investigation of the 1-carbazolylcarbazole derived donors as the donor units of two blue TADF emitters in comparison with 3-carbazolylcarbazole demonstrated that the 1-carbazolylcarbazole based donors increased the triplet energy, decreased the singlet-triplet energy gap, blue-shifted the emission color, and enhanced the quantum efficiency of the blue TADF devices. PMID:27443818

  17. Development of fiber optic spectroscopy for in-vitro and in-planta detection of fluorescent proteins

    Science.gov (United States)

    Liew, Oi Wah; Chen, Jun-Wei; Asundi, Anand K.

    2001-10-01

    The objective of this project is to apply photonics technology to bio-safety management of genetically modified (GM) plants. The conventional method for screening GM plants is through selection using antibiotic resistance markers. There is public concern with such approaches and these are associated with food safety issues, escape of antibiotic resistance genes to pathogenic microorganisms and interference with antibiotic therapy. Thus, the strategy taken in this project is to replace antibiotic resistance markers with fluorescent protein markers that allow for rapid and non-invasive optical screening of genetically modified plants. In this paper, fibre optic spectroscopy was developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in planta. In vitro detection was first carried out to optimize the sensitivity of the optical system. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fibre optic spectroscopy using different light sources, namely, blue LED (475 nm), tungsten halogen (350 - 1000 nm) and double frequency Nd:YAG green laser (532 nm). Fluorescence near the expected emission wavelengths could be detected up to 320X dilution for EGFP and DsRED with blue LED and 532 nm green laser, respectively, as the excitation source. Tungsten halogen was found to be unsuitable for excitation of both EGFP and DsRED. EGFP was successfully purified by size separation under non-denaturing electrophoretic conditions and quantified. The minimum concentration of EGFP detectable with blue LED excitation was 5 mg/ml. To determine the capability of spectroscopy detection in planta, transgenic potato hairy roots and whole modified plant lines expressing the

  18. Hybrid white organic light-emitting devices based on phosphorescent iridium–benzotriazole orange–red and fluorescent blue emitters

    International Nuclear Information System (INIS)

    We demonstrate that high color purity or efficiency hybrid white organic light-emitting devices (OLEDs) can be generated by integrating a phosphorescent orange–red emitter, bis[4-(2H-benzotriazol-2-yl)-N,N-diphenyl-aniline-N1,C3] iridium acetylacetonate, Ir(TBT)2(acac) with fluorescent blue emitters in two different emissive layers. The device based on deep blue fluorescent material diphenyl-[4-(2-[1,1′;4′,1″]terphenyl-4-yl-vinyl)-phenyl]-amine BpSAB and Ir(TBT)2(acac) shows pure white color with the Commission Internationale de L'Eclairage (CIE) coordinates of (0.33,0.30). When using sky-blue fluorescent dopant N,N′-(4,4′-(1E,1′E)-2,2′-(1,4-phenylene)bis(ethene-2,1-diyl) bis(4,1-phenylene))bis(2-ethyl-6-methyl-N-phenylaniline) (BUBD-1) and orange–red phosphor with a color-tuning phosphorescent material fac-tris(2-phenylpyridine) iridium (Ir(ppy)3 ), it exhibits peak luminance yield and power efficiency of 17.4 cd/A and 10.7 lm/W, respectively with yellow-white color and CIE color rendering index (CRI) value of 73. - Highlights: ► An iridium-based orange–red phosphor Ir(TBT)2(acac) was applied in hybrid white OLEDs. ► Duel- and tri-emitter WOLEDs were achieved with either high color purity or efficiency performance. ► Peak luminance yield of tri-emitter WOLEDs was 17.4 cd/A with yellow-white color and color rendering index (CRI) value of 73.

  19. Hybrid white organic light-emitting devices based on phosphorescent iridium-benzotriazole orange-red and fluorescent blue emitters

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Zhen-Yuan, E-mail: xiazhenyuan@hotmail.com [Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237 (China); Su, Jian-Hua [Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237 (China); Chang, Chi-Sheng; Chen, Chin H. [Display Institute, Microelectronics and Information Systems Research Center, National Chiao Tung University, Hsinchu, Taiwan 300 (China)

    2013-03-15

    We demonstrate that high color purity or efficiency hybrid white organic light-emitting devices (OLEDs) can be generated by integrating a phosphorescent orange-red emitter, bis[4-(2H-benzotriazol-2-yl)-N,N-diphenyl-aniline-N{sup 1},C{sup 3}] iridium acetylacetonate, Ir(TBT){sub 2}(acac) with fluorescent blue emitters in two different emissive layers. The device based on deep blue fluorescent material diphenyl-[4-(2-[1,1 Prime ;4 Prime ,1 Double-Prime ]terphenyl-4-yl-vinyl)-phenyl]-amine BpSAB and Ir(TBT){sub 2}(acac) shows pure white color with the Commission Internationale de L'Eclairage (CIE) coordinates of (0.33,0.30). When using sky-blue fluorescent dopant N,N Prime -(4,4 Prime -(1E,1 Prime E)-2,2 Prime -(1,4-phenylene)bis(ethene-2,1-diyl) bis(4,1-phenylene))bis(2-ethyl-6-methyl-N-phenylaniline) (BUBD-1) and orange-red phosphor with a color-tuning phosphorescent material fac-tris(2-phenylpyridine) iridium (Ir(ppy){sub 3} ), it exhibits peak luminance yield and power efficiency of 17.4 cd/A and 10.7 lm/W, respectively with yellow-white color and CIE color rendering index (CRI) value of 73. - Highlights: Black-Right-Pointing-Pointer An iridium-based orange-red phosphor Ir(TBT){sub 2}(acac) was applied in hybrid white OLEDs. Black-Right-Pointing-Pointer Duel- and tri-emitter WOLEDs were achieved with either high color purity or efficiency performance. Black-Right-Pointing-Pointer Peak luminance yield of tri-emitter WOLEDs was 17.4 cd/A with yellow-white color and color rendering index (CRI) value of 73.

  20. In vivo examination of membrane protein localization and degradation with green fluorescent protein.

    OpenAIRE

    Hampton, R Y; Koning, A.; Wright, R; Rine, J

    1996-01-01

    To test the utility of green fluorescent protein (GFP) as an in vivo reporter protein when fused to a membrane domain, we made a fusion protein between yeast hydroxymethylglutaryl-CoA reductase and GFP. Fusion proteins displayed spatial localization and regulated degradation consistent with the native hydroxymethylglutaryl-CoA reductase proteins. Thus, GFP should be useful in the study of both membrane protein localization and protein degradation in vivo.

  1. On the Design of Low-Cost Fluorescent Protein Biosensors

    Science.gov (United States)

    Tolosa, Leah

    , magnetic beads, nanoparticles or quantum dots designed to form covalent bonds with amino groups, sulfhydryl groups, carboxylic groups and other reactive functionalities in amino acids. It is not uncommon to conduct combinations of techniques, for example, the introduction of fluorescent labels or probes to proteins require in many cases, site-directed mutagenesis followed by covalent bonding of the fluorescent dye. Accordingly, two or more proteins can be combined to create hybrid or fusion proteins with multiple or altered functions. Indeed, research involving the green fluorescent protein and fluorescent proteins of a variety of colors has expanded by leaps and bounds in the last decade. Because these fluorescent proteins can be genetically encoded in cells, it is possible to observe various cellular processes in vivo. However, this topic has been reviewed extensively in the literature and, thus, will not be expounded on in this chapter.

  2. The coherent dynamics of photoexcited green fluorescent proteins

    CERN Document Server

    Cinelli, R A G; Pellegrini, V; Beltram, F; Cerullo, G; Zavelani-Rossi, M; De Silvestri, S; Tyagi, M; Giacca, M; Cinelli, Riccardo A. G.; Tozzini, Valentina; Pellegrini, Vittorio; Beltram, Fabio; Cerullo, Giulio; Zavelani-Rossi, Margherita; Silvestri, Sandro De; Tyagi, Mudit; Giacca, Mauro

    2001-01-01

    The coherent dynamics of vibronic wave packets in the green fluorescent protein is reported. At room temperature the non-stationary dynamics following impulsive photoexcitation displays an oscillating optical transmissivity pattern with components at 67 fs (497 cm-1) and 59 fs (593 cm-1). Our results are complemented by ab initio calculations of the vibrational spectrum of the chromophore. This analysis shows the interplay between the dynamics of the aminoacidic structure and the electronic excitation in the primary optical events of green fluorescent proteins.

  3. Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

    Science.gov (United States)

    Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada; Rebane, Aleksander; Lindquist, Jake R.; Santos, Sofia; Kats, Ilia; Davidson, Michael W.; Mazitschek, Ralph; Hughes, Thomas E.; Drobizhev, Mikhail; Knop, Michael; Shah, Jagesh V.

    2015-01-01

    We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. PMID:25877871

  4. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    Science.gov (United States)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  5. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    Science.gov (United States)

    Kim, Young Eun; Kim, Yu-Na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-05-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

  6. "Color Timer" mice: visualization of neuronal differentiation with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Kanki Hiroaki

    2010-02-01

    Full Text Available Abstract The molecular mechanisms governing the differentiation of neural stem cells (NSCs into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg mice in which production of the fluorescent protein Kusabira-Orange (KOr is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to

  7. Use of blue-green and chlorophyll fluorescence measurements for differentiation between nitrogen deficiency and pathogen infection in winter wheat.

    Science.gov (United States)

    Bürling, Kathrin; Hunsche, Mauricio; Noga, Georg

    2011-09-15

    In recent years, several sensor-based approaches have been established to early detect single plant stresses, but the challenge of discriminating between simultaneously occurring stressors still remains. Earlier studies on wheat plants strongly affected by pathogens and nitrogen deficiency indicated that chlorophyll fluorescence might be suited to distinguish between the two stressors. Nevertheless, there is lack of information on the pre-symptomatic detection of synchronized occurrence of slight N-deficiency and the early stages of pathogen infection. The usefulness of the blue, green, and yellow fluorescence signals in this context has not yet been explored. We hypothesized that differentiation between wheat plants' physiological reaction due to N-deficiency and leaf rust (Puccinia triticina) as well as N-deficiency and powdery mildew (Blumeria graminis f. sp. tritici) might be accomplished by means of UV laser-induced fluorescence spectral measurements between 370 and 620nm in addition to chlorophyll fluorescence (640-800nm). Plants were provided with either a normal or a modified Hoagland nutrient solution in order to induce a slight N deficit. Pathogen inoculation was carried out on the second fully developed leaf. Four experimental groups were evaluated: (a) N-full-supply [N+]; (b) N-deficiency [N-]; (c) N-full-supply+pathogen [N+/LR] or [N+/PM]; (d) N-deficiency+pathogen [N-/LR] or [N-/PM]. The results revealed that, in addition to the amplitude ratio of R/FR fluorescence, B/G fluorescence also facilitated reliable and robust discrimination among the four experimental groups. The discrimination among the experimental groups was accomplished as early as one and two days after inoculation for powdery mildew and leaf rust infection, respectively. During the 3days evaluation period, the differences among the treatment groups became more evident. Moreover, several other amplitude ratios and half-bandwidth ratios proved to be suited to early detect fungal

  8. Separation of Protein Oligomers by Blue Native Gel Electrophoresis

    OpenAIRE

    Braz, Valerie A.; Howard, Kathryn J.

    2009-01-01

    Native gel electrophoresis is used as a tool to assess structural differences in proteins. This note presents an application to separate oligomeric forms of proteins, such as HIV-1 reverse transcriptase monomers and homodimers. Technical difficulties encountered with various native gel techniques and ways to circumvent them are described.

  9. Solvent dependence of two-photon absorption spectra of the enhanced green fluorescent protein (eGFP) chromophore

    Science.gov (United States)

    Hosoi, Haruko; Tayama, Ryo; Takeuchi, Satoshi; Tahara, Tahei

    2015-06-01

    Two-photon absorption spectra of 4‧-hydroxybenzylidene-2,3-dimethylimidazolinone, a model chromophore of enhanced green fluorescent protein (eGFP), were measured in various solvents. The two-photon absorption band of its anionic form is markedly blue-shifted from the corresponding one-photon absorption band in all solvents. Moreover, the magnitude of the blue shift varies largely depending on the solvent, which does not accord with the assignment of the two-photon absorption band to the transitions to the vibrationally excited S1 state. Our finding is readily rationalized by considering overlapping contributions of the S1 ← S0 and S2 ← S0 transitions, suggesting the involvement of the S2 state also in two-photon fluorescence of eGFP.

  10. Folding and unfolding of a non-fluorescent mutant of green fluorescent protein

    International Nuclear Information System (INIS)

    Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and it remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to efficiently form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of guanidinium hydrochloride (GdnHCl). The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of 1,1'-bis(4-anilino-5-naphthalenesulphonic acid) (bis-ANS). Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is the formation of a molten globule, prone to aggregation

  11. Fluorescence studies of beer protein uptake by silica

    Science.gov (United States)

    Apperson, Kathleen; Birch, David J. S.; Leiper, Kenneth; McKeown, Ian P.

    2001-05-01

    Fluorescence has been investigated with respect to new methods for monitoring protein uptake by silica, with particular attention being given to haze forming proteins and foam proteins present in beer. These are of particular interest to the brewing industry as an important aspect of the brewing process is the prevention of chill haze formation. This is necessary in order to maintain the clarity of the beer and to extend the shelf life. Chill haze, which is a result of the interaction of certain proteins with some polyphenols, can be prevented by the removal of one or both of these constituents.

  12. Fluorescence microscopy of single autofluorescent proteins for cellular biology

    CERN Document Server

    Cognet, Laurent; Choquet, Daniel; Lounis, Brahim

    2002-01-01

    In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells.

  13. Two-photon directed evolution of green fluorescent proteins

    Science.gov (United States)

    Stoltzfus, Caleb R.; Barnett, Lauren M.; Drobizhev, Mikhail; Wicks, Geoffrey; Mikhaylov, Alexander; Hughes, Thomas E.; Rebane, Aleksander

    2015-07-01

    Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.

  14. Electrotransformation of Bacillus mojavensis with fluorescent protein markers

    Science.gov (United States)

    Gram-positive endophytic bacteria are difficult to transform. To study endophytic interactions between Bacillus mojavensis and maize, a method was developed to transform this species by electroporation with three fluorescent protein expressing integrative plasmids: pSG1154, pSG1192, and pSG1193. The...

  15. Development of a Green Fluorescent Protein-Based Laboratory Curriculum

    Science.gov (United States)

    Larkin, Patrick D.; Hartberg, Yasha

    2005-01-01

    A laboratory curriculum has been designed for an undergraduate biochemistry course that focuses on the investigation of the green fluorescent protein (GFP). The sequence of procedures extends from analysis of the DNA sequence through PCR amplification, recombinant plasmid DNA synthesis, bacterial transformation, expression, isolation, and…

  16. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    International Nuclear Information System (INIS)

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine

  17. Cathodoluminescence and Electron-Induced Fluorescence Enhancement of Enhanced Green Fluorescent Protein.

    Science.gov (United States)

    Nagayama, Kuniaki; Onuma, Tsubasa; Ueno, Ryosuke; Tamehiro, Katsuyuki; Minoda, Hiroki

    2016-02-18

    Becaues the spatial resolution of fluorescence microscopy is not high enough to study the molecular level of relationship between the structure and function of biological specimens, correlative light and electron microscopy has been used for this purpose. Another possibility for a high-resolution light microscopy is cathodoluminescence microscopy. Here, we report a new phenomenon, the electron-induced activation of luminescence (cathodoluminescence) and electron-enhanced fluorescence for the enhanced green fluorescent protein (EGFP). This was found using our recently developed hybrid fluorescence and electron microscopy. Contrary to the past reports, which showed a degradation of organic compounds by electron irradiation, stable cathodoluminescence emitted from an organic molecule, EGFP, has been observed using the hybrid microscopy. Addition of the glycerol promoted the fluorescence enhancement of EGFP probably due to the change in the electronic state density of excitation channels from the ground to the excited state or of relaxation channels from the excited to the emission state. Stable cathodoluminescence and enhanced fluorescence of the EGFP may introduce a cathodoluminescence microscopy, which will increase the variety of the imaging to investigate the biological compounds. PMID:26849242

  18. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms.

    Science.gov (United States)

    Pletneva, Nadya V; Pletnev, Sergei; Pakhomov, Alexey A; Chertkova, Rita V; Martynov, Vladimir I; Muslinkina, Liya; Dauter, Zbigniew; Pletnev, Vladimir Z

    2016-08-01

    The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement. PMID:27487823

  19. Protein immobilization and fluorescence quenching on polydopamine thin films.

    Science.gov (United States)

    Chen, Daqun; Zhao, Lei; Hu, Weihua

    2016-09-01

    Mussel inspired polydopamine (PDA) film has attracted great interest as a versatile functional coating for biomolecule immobilization in various bio-related devices. However, the details regarding the interaction between a protein and PDA film remain unclear. Particularly, there is very limited knowledge regarding the protein immobilization on PDA film, even though it is of essential importance in various fields. The situation is even more complicated if considering the fact that quite a number of approaches (e.g., different oxidizing reagent, buffer pH, grown time, grown media, etc.) have been developed to grow PDA films. In this work, protein attachment on PDA film was systematically investigated by using the real-time and label-free surface plasmon resonance (SPR) technique. The kinetics of protein-PDA interaction was explored and the influence of buffer pH and deposition media on the protein attachment was studied. Fluorescent protein microarray was further printed on PDA-coated glass slides for quantitative investigations and together with SPR data, the interesting fluorescence quenching phenomenon of PDA film was revealed. This work may deepen our understanding on the PDA-protein interaction and offer a valuable guide for efficient protein attachment on PDA film in various bio-related applications. PMID:27254254

  20. Murine leukemia virus (MLV replication monitored with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Bittner Alexandra

    2004-12-01

    Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

  1. A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

    Science.gov (United States)

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase chain…

  2. Greenish-blue fluorescence of 6-(4-aminophenyl)-4-(4-methoxyphenyl)-2-methoxynicotinonitrile: Synthesis, spectroscopy, crystal structure, and fluorescence property

    Science.gov (United States)

    Suwunwong, T.; Chantrapromma, S.; Fun, H.-K.

    2015-12-01

    The title compound, C20H17N3O2, was synthesized by the cyclization of a chalcone derivative with malononitrile, and was characterized by 1H NMR and FT-IR spectroscopy techniques. The crystal structure was determined by single crystal X-ray structure determination. In the crystal packing, the molecules are linked by N-H···N hydrogen bonds forming a two dimensional network parallel to the (1 0-2) plane. The crystal structure is further stabilized by weak C-H···π interactions. The compound exhibits greenish-blue fluorescence in acetonitrile with an emission wavelength at 476 nm when was excited at 365 nm.

  3. Resolving mitochondrial protein complexes using non-gradient blue native polyacrylamide gel electrophoresis

    OpenAIRE

    Yan, Liang-Jun; Forster, Michael J.

    2009-01-01

    Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomic analysis of high molecular weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this paper, we present an alternative BN-PAGE method that uses highly porous, non-gradient polyacrylamide gels for separation of rat brain mitochondrial protein...

  4. Identification of the mineralization front: comparison of a modified toluidine blue stain with tetracycline fluorescence.

    Science.gov (United States)

    Villanueva, A R; Kujawa, M; Mathews, C H; Parfitt, A M

    A modified toluidine blue method for identification of the mineralization front at the zone of demarcation between bone and osteoid in undecalcified, plastic-embedded sections of bone is described. The intensity of staining is increased both by increasing the pH of the solution and by increasing the duration of staining. The method is reproducible, since measurements of the extent of mineralization front on two nonadjacent sections from the same biopsy in 20 cases had a correlation coefficient of 0.98. The identification of the mineralization front by toluidine blue agrees closely with identification by means of in vivo tetracycline uptake, with a correlation coefficient of 0.97 between measurements of its extent using the two methods on adjacent sections from the same biopsy. It is likely that both toluidine blue staining at pH 6.5 and tetracycline uptake depend on some property of the most recently deposited bone mineral. With either method we find low values for the extent of mineralization front as a fraction of osteoid surface in many patients with osteoporosis. The uniformly normal values for this quantity in osteoporosis reported by other investigators may reflect different criteria for distinguishing osteoid-covered from quiescent bone surfaces. PMID:6200748

  5. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    Science.gov (United States)

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  6. Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

    Directory of Open Access Journals (Sweden)

    Qi Wang

    Full Text Available Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm and emits green fluorescence (525 nm. At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.

  7. Probabilistic mapping and image clustering for quantitative assessment of fluorescent protein localizations in Arabidopsis guard cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Takumi Higaki, Natsumaro Kutsuna & Seiichiro Hasezawa ### Abstract The protocol reported here describes a method to quantitatively evaluate fluorescently-tagged protein localizations from fluorescent microscopic images with a combination of probabilistic mapping and image clustering. We demonstrate the use of this protocol using kidney-shaped guard cells of plants. ### Introduction Microscopic assessment of protein localizations with fluorescent protein taggin...

  8. Monomeric red fluorescent protein variants used for imaging studies in different species

    NARCIS (Netherlands)

    Mueller-Taubenberger, Annette; Vos, Michel J.; Boettger, Angelika; Lasi, Margherita; Lai, Frank P. L.; Fischer, Markus; Rottner, Klemens

    2006-01-01

    Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation

  9. Rotational order–disorder structure of fluorescent protein FP480

    International Nuclear Information System (INIS)

    An analysis of the rotational order–disorder structure of fluorescent protein FP480 is presented. In the last decade, advances in instrumentation and software development have made crystallography a powerful tool in structural biology. Using this method, structural information can now be acquired from pathological crystals that would have been abandoned in earlier times. In this paper, the order–disorder (OD) structure of fluorescent protein FP480 is discussed. The structure is composed of tetramers with 222 symmetry incorporated into the lattice in two different ways, namely rotated 90° with respect to each other around the crystal c axis, with tetramer axes coincident with crystallographic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates a rotational OD structure with statistically averaged I422 symmetry, although the presence of very weak and diffuse additional reflections suggests that the randomness is only approximate

  10. The Cyan Fluorescent Protein (CFP Transgenic Mouse as a Model for Imaging Pancreatic Exocrine Cells

    Directory of Open Access Journals (Sweden)

    Hop S Tran Cao

    2009-03-01

    Full Text Available The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan. Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA. Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. Main outcome measure Determination of fluorescence intensity of different organs. Results Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

  11. Identification of the pigment responsible for the blue fluorescence band in the laser induced fluorescence (LIF) spectra of green plants, and the potential use of this band in remotely estimating rates of photosynthesis

    International Nuclear Information System (INIS)

    The laser-induced fluorescence (LIF) of vegetation is being investigated in this laboratory for use as a technique for the remote detection of the effects of environmental stress upon vegetation, as well as for plant identification. The fluorescence band with a maximum at 440 nm, in conjunction with the chlorophyll bands with maxima at 685 and 740 nm, has been found to be a critical band in the development of algorithms for detecting stress, and identifying plant types. The identification of the plant constituent responsible for this band is vital to understanding the mechanism underlying its fluorescence changes in response to environmental and physiological changes. The identification was achieved as follows: The laser induced fluorescence (LIF) spectra of pure plant pigments were determined. Fluorescence bands with maxima at 420 nm, 440 nm, 490 nm, and 525 nm were observed for vitamin K1, reduced nicotinamide adenine dinucleotide (NADPH), beta-carotene, and riboflavin, respectively. The LIF spectra of water extracts and acetone extracts of clover leaves were also measured. It was found that the blue fluorescence band was associated with the water extract. NADPH which is a water-soluble compound, and the water extract of clover had no fluorescence after oxidation by potassium ferricyanide, while the fluorescence of water insoluble vitamin K1 was unchanged by the oxidizing agent. It was also found that the absorption maximum of NADPH was the same as the absorption maximum of the aqueous extract of clover. The above findings indicated that the compound responsible for the blue fluorescence at 440 nm is in the reduced state and is water-soluble. It was concluded that NADPH was responsible for the blue fluorescence at 440 nm. The strong linear relationship between the fluorescence at 440 nm and the rate of photosynthesis suggests the possible use of LIF measurements in the remote estimation of photosynthetic rates. (author)

  12. Aptamer carbon nanodot sandwich used for fluorescent detection of protein.

    Science.gov (United States)

    Xu, Bailu; Zhao, Chuanqi; Wei, Weili; Ren, Jinsong; Miyoshi, Daisuke; Sugimoto, Naoki; Qu, Xiaogang

    2012-12-01

    Carbon nanodots (C-Dots) have attracted growing interest in recent years due to their low cost, ready scalability, excellent chemical stability, biocompatibility, colloidal stability, and resilience of photoluminescence. They have been employed as novel, ideal fluorescent probes for bio-imaging and smart sensing. In addition, taking advantage of their low-cytotoxicity, C-Dots have potential applications in biochemical and cell biological fields. Herein, we present the first assay with aptamer-functionalized C-Dots as a sensory platform for protein detection. The presence of thrombin can induce the aptamer-modified fluorescent C-Dots to form a sandwich structure with aptamer-functionalized silica nanoparticles through specific protein/aptamer interaction. The assay shows high specificity toward thrombin. A detection limit of 1 nM is obtained, which is significantly improved as compared to that of many previously reported fluorescence-based thrombin detection assays. Using other modified aptamers and antibodies instead of thrombin binding aptamers, this strategy may offer a suitable approach for detection of other proteins in biological, pharmaceutical and nano-mechanical applications. PMID:23050264

  13. Synthesis of highly fluorescent gold nanoclusters using egg white proteins.

    Science.gov (United States)

    Joseph, Dickson; Geckeler, Kurt E

    2014-03-01

    Gold nanoclusters (AuNCs) have gained interest during the recent years because of their low toxicity and finer size for the bioimaging and biolabeling applications in comparison to the semiconductor quantum dot analogues. Diverse materials such as sulfur compounds, peptides, dendrimers, proteins, etc., are exploited for the preparation of AuNCs. Henceforth, highly fluorescent, water-soluble, and few atom-containing gold nanoclusters are created using a rapid, straightforward, and green method. In this regard for the first time chicken egg white (CEW), one of the most unique materials, is utilized in an aqueous solution under basic conditions at physiological temperature for the preparation of AuNCs. Tyrosine and tryptophan amino acid residues are responsible for the conversion of Au ions to Au(0) under alkaline condtions. CEW contains four major proteins of which the main constituent protein, ovalbumin also leads to the formation of the AuNCs with a higher fluorescence emission compared to the CEW. The ratios between the different reaction partners are very crucial, along with temperature and time for the preparation of AuNCs with high photoluminescence emission. The limited vibrational motion of the proteins under alkaline condition and the bulkiness of the proteins help in the formation of AuNCs. PMID:24321847

  14. Near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Shcherbakova, Daria M; Baloban, Mikhail; Verkhusha, Vladislav V

    2015-08-01

    Near-infrared fluorescent proteins (NIR FPs), photoactivatable NIR FPs and NIR reporters of protein-protein interactions developed from bacterial phytochrome photoreceptors (BphPs) have advanced non-invasive deep-tissue imaging. Here we provide a brief guide to the BphP-derived NIR probes with an emphasis on their in vivo applications. We describe phenotypes of NIR FPs and their photochemical and intracellular properties. We discuss NIR FP applications for imaging of various cell types, tissues and animal models in basic and translational research. In this discussion, we focus on NIR FPs that efficiently incorporate endogenous biliverdin chromophore and therefore can be used as straightforward as GFP-like proteins. We also overview a usage of NIR FPs in different imaging platforms, from planar epifluorescence to tomographic and photoacoustic technologies. PMID:26115447

  15. Blue Light Hazard and Risk Group Classification of 8 W LED Tubes, Replacing Fluorescent Tubes, through Optical Radiation Measurements

    Directory of Open Access Journals (Sweden)

    Francesco Leccese

    2015-09-01

    Full Text Available In this paper, the authors discuss the results of a measurement survey of artificial optical radiation emitted by 8 W LED tubes suitable for the substitution of 18 W fluorescent lamps used for general lighting. For both types of lamps, three different color temperatures were chosen, 3000 K, 4000 K, and 6000 K. These measurements were performed to evaluate the photobiological safety of the sources. The radiance and irradiance values have been measured in a wide range of wavelengths (180–3000 nm. The measurement results obtained for the LED tubes have been compared to those of similar measurements obtained for fluorescent lamps. The analysis has been focused on the range of wavelengths 300–700 nm, the blue light range, which turned out to be defining for the risk groups of the lamps. This classification is a function of the maximum permissible exposure time as indicated in the European Standard EN 62471 on the photobiological safety of lamps and lamp systems.

  16. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  17. An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    Jun Zhou; Jian Lin; Cuihong Zhou; Xiaoyan Deng; Bin Xia

    2011-01-01

    Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein,which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 amino acid residue, and sfGFPC was mutated by site-directed gene mutagenesis to decrease the signal of negative control. Our results showed that mutations in sfGFPC (sfGFPC(m12)) can effectively decrease the signal of negative control. Thus, we provide an improved BiFC tool for the analysis of PPI. Further,since the self-assembly problem is a common shortcoming for application of BiFC, our research provides a feasible strategy for other BiFC candidate proteins with the same problem.

  18. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells. PMID:26649773

  19. Distribution and Spectroscopy of Green Fluorescent Protein and Acyl-CoA: Cholesterol Acytransferase in Sf21 Insect Cells

    Science.gov (United States)

    Richmond, R. C.; Mahtani, H.; Lu, X.; Chang, T. Y.; Malak, H.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    blue shift from 518nm obtained in neutral aqueous solution to 505nm obtained in localized regions within the cells. This blue shift indicates change in the fluorescence coupling of the GFP moiety of GCAT. It is hypothesized that change in tertiary environment of GCAT, coincident with intracellular deposition of GCAT, follows from intracellular trafficking of GCAT leading to membrane interactions with the ACAT moiety, and/or self-assembly of GCAT, that alters the chromophore environment of the GFP moiety of GCAT. These findings introduce a new technique of biophotonic imaging to studies of intracellular protein trafficking and interactions. This technique of hyperspectral imaging could contribute to advancing the emergent field of proteomics. Because of the noninvasive nature of this technique, kinetic processes associated with intracellular protein trafficking, and interactions of proteins within cellular domains, can be considered for investigation within a single cell as well as a cell population.

  20. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    OpenAIRE

    Morrell-Falvey, J. L.; Qi, H.; Doktycz, M. J.; Venkatraman, S.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features ...

  1. Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein-Ligand Reactions

    OpenAIRE

    Sun, Y.S.; Landry, J. P.; Fei, Y.Y.; Zhu, X.D.; Luo, J. T.; Wang, X.B.; Lam, K. S.

    2008-01-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Un-labeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity differ...

  2. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  3. Photophysics of the LOV-Based Fluorescent Protein Variant iLOV-Q489K Determined by Simulation and Experiment.

    Science.gov (United States)

    Davari, Mehdi D; Kopka, Benita; Wingen, Marcus; Bocola, Marco; Drepper, Thomas; Jaeger, Karl-Erich; Schwaneberg, Ulrich; Krauss, Ulrich

    2016-04-01

    Light, oxygen, voltage (LOV) based fluorescent proteins (FPs) represent a promising alternative to fluorescent reporters of the green fluorescent protein family. For certain applications like multicolor imaging or the design of FRET-based biosensors, the generation of spectrally shifted LOV-based FPs would be required. In a recent theoretical study ( Khrenova J. Phys. Chem. B 2015 , 119 ( 16 ), pp 5176 - 5183 ), the photophysical properties of a variant of the LOV-based fluorescent protein iLOV were predicted using quantum mechanics/molecular mechanics (QM/MM) approaches. The variant contained a lysine residue at the position of a highly conserved glutamine residue (Q489K), which directly interacts with the O4 and N5 atom of the flavin mononucleotide (FMN) chromophore. On the basis of QM/MM calculations, iLOV-Q489K was suggested to possess substantially red-shifted absorption and fluorescence-emission maxima with respect to parental iLOV. Here, we describe the experimental characterization of this variant, which, surprisingly contrary to the theoretical prediction, shows blue-shifted absorption and fluorescence-emission maxima. Using molecular dynamics (MD) simulations and QM/MM calculations, the molecular basis for the contradictory theoretical and experimental results is presented. Essentially, our computational analysis suggests that, in the Q489K variant, two possible side-chain conformers exist: (i) a least populated conformer K489in forming a hydrogen bond with the O4 atom of FMN chromophore and (ii) a most populated conformer K489out with the side-chain amino group flipped away from the FMN chromophore forming a new hydrogen bond with the backbone oxygen of G487. QM/MM calculated spectra of the K489out conformer are blue-shifted compared to the calculated spectra of parental iLOV, which is in accordance with experimental data. This suggests that the change in the conformation of K489 from K498in to K489out accounts for the change in the direction of the

  4. Nucleic acid encoding a self-assembling split-fluorescent protein system

    Energy Technology Data Exchange (ETDEWEB)

    Waldo, Geoffrey S; Cabantous, Stephanie

    2014-04-01

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  5. Nucleic acid encoding a self-assembling split-fluorescent protein system

    Energy Technology Data Exchange (ETDEWEB)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2015-07-14

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  6. Nucleic acid encoding a self-assembling split-fluorescent protein system

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-07

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  7. Pulsed Blue and Ultraviolet Laser System for Fluorescence Diagnostics based on Nonlinear Frequency Conversion

    DEFF Research Database (Denmark)

    Cheng, Haynes Pak Hay

    eliminates the need for prior preparation on the patient, and further reduces the cost of fluorescence imaging, and potentially, cancer diagnostic. By taking advantage of the relatively short wavelength of the 946 nm transition in a quasi-three-level Nd:YAG laser, the target 340 nm wavelength could....... The spatial and spectral properties of the pump source were also investigated individually, and it was concluded that a broad spectrum tapered diode pump source may be most stable and cost-effective. To generate high peak power pulsed output, Q-switched lasers were considered. In particular, synchronized Q......, which translates into a 79% temporal overlap when compared to the zero-delay scenario. The minimum timing jitter between the two pulses was 9 ns, which is one-standard deviation of the delay measurements. This is comparable to previously published results for an actively synchronized system if either...

  8. Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain

    International Nuclear Information System (INIS)

    The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FADox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FADox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FADox in the signalling state to the fully reduced hydroquinone form FADredH- and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed

  9. Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi, A. [Institut II - Experimentelle und Angewandte Physik, Universitaet Regensburg, Universitaetstrasse 31, D-93053 Regensburg (Germany); Penzkofer, A. [Institut II - Experimentelle und Angewandte Physik, Universitaet Regensburg, Universitaetstrasse 31, D-93053 Regensburg (Germany)], E-mail: alfons.penzkofer@physik.uni-regensburg.de; Griese, J.; Schlichting, I. [Max-Planck-Institut fuer medizinische Forschung, Abteilung Biomolekulare Mechanismen, Jahnstrasse 29, D-69120 Heidelberg (Germany); Kirienko, Natalia V.; Gomelsky, Mark [Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071 (United States)

    2008-12-10

    The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1{sub B}LUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FAD{sub ox}) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1{sub B}LUF and about 0.08 for BlrP1. The fluorescence efficiency of the FAD{sub ox} cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FAD{sub ox} in the signalling state to the fully reduced hydroquinone form FAD{sub red}H{sup -} and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.

  10. Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain

    Science.gov (United States)

    Tyagi, A.; Penzkofer, A.; Griese, J.; Schlichting, I.; Kirienko, Natalia V.; Gomelsky, Mark

    2008-12-01

    The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FAD ox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FAD ox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FAD ox in the signalling state to the fully reduced hydroquinone form FAD redH - and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.

  11. Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

    Science.gov (United States)

    Spitzer, Dirk; Dittmar, Kurt E. J.; Rohde, Manfred; Hauser, Hansjörg; Wirth, Dagmar

    2003-01-01

    Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions. PMID:12719600

  12. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  13. Highly Improved Efficiency of Deep-Blue Fluorescent Polymer Light-Emitting Device Based on a Novel Hole Interface Modifier with 1,3,5-Triazine Core.

    Science.gov (United States)

    Xia, Lianpeng; Xue, Yuyuan; Xiong, Kang; Cai, Chaosheng; Peng, Zuosheng; Wu, Ying; Li, Yuan; Miao, Jingsheng; Chen, Dongcheng; Hu, Zhanhao; Wang, Jianbin; Peng, Xiaobin; Mo, Yueqi; Hou, Lintao

    2015-12-01

    We present an investigation of deep-blue fluorescent polymer light-emitting diodes (PLEDs) with a novel functional 1,3,5-triazine core material (HQTZ) sandwiched between poly(3,4-ethylene dioxythiophene):poly(styrene sulfonic acid) layer and poly(vinylcarbazole) layer as a hole injection layer (HIL) without interface intermixing. Ultraviolet photoemission spectroscopy and Kelvin probe measurements were carried out to determine the change of anode work function influenced by the HQTZ modifier. The thin HQTZ layer can efficiently maximize the charge injection from anode to blue emitter and simultaneously enhance the hole mobility of HILs. The deep-blue device performance is remarkably improved with the maximum luminous efficiency of 4.50 cd/A enhanced by 80% and the maximum quantum efficiency of 4.93%, which is 1.8-fold higher than that of the conventional device without HQTZ layer, including a lower turn-on voltage of 3.7 V and comparable Commission Internationale de L'Eclairage coordinates of (0.16, 0.09). It is the highest efficiency ever reported to date for solution-processed deep-blue PLEDs based on the device structure of ITO/HILs/poly(9,9-dialkoxyphenyl-2,7-silafluorene)/CsF/AL. The results indicate that HQTZ based on 1,3,5-triazine core can be a promising candidate of interfacial materials for deep-blue fluorescent PLEDs. PMID:26422296

  14. Anaerobic green fluorescent protein as a marker of Bifidobacterium strains.

    Science.gov (United States)

    Landete, José M; Peirotén, Ángela; Rodríguez, Eva; Margolles, Abelardo; Medina, Margarita; Arqués, Juan L

    2014-04-01

    Some strains of Bifidobacterium are considered as probiotics and are being added as adjunct culture in food products due to their potential in maintaining a healthy intestinal microbial balance. However, despite these benefits, bifidobacteria still remain poorly understood at the genetic level compared with other microorganisms of industrial interest. In this work, we have developed a non-invasive green fluorescent based reporter system for real-time tracking of Bifidobacterium species in vivo. The reporter vector pNZ:Tu-GFPana is based on the pNZ8048 plasmid harboring a bifidobacterial promoter (elongation factor Tu from Bifidobacterium longum CECT 4551) and a fluorescent protein containing a flavin-mono-nucleotide-based cofactor (evoglow-Pp1) which is fluorescent under both aerobic and anaerobic conditions. pNZ:Tu-GFPana was constructed and found to stably replicate in B. longum CECT 4551 and in the intestinal strain Bifidobacterium breve INIA P734. The subsequent analysis of these strains allowed us to assess the functionality of this plasmid. Our results demonstrate the potential of pNZ:Tu-GFPana as a real-time reporter system for Bifidobacterium in order to track the behavior of this probiotic species in complex environments like food or intestinal microbiota, and to estimate their competition and colonization potential. PMID:24495586

  15. High-power-efficiency hybrid white organic light-emitting diodes with a single emitting layer doped with blue delayed fluorescent and yellow phosphorescent emitters

    International Nuclear Information System (INIS)

    High-efficiency hybrid white organic light-emitting diodes (HWOLEDs) with a blue thermally activated delayed fluorescent (TADF) emitter and a yellow phosphorescent emitter doped in a single emitting layer were developed. Exciton harvesting by the blue TADF and yellow phosphorescent emitters rendered both singlet and triplet excitons to contribute to the white emission, which leads to a high quantum efficiency of 22.4% and a power efficiency of 60.3 lm W−1 in the HWOLEDs. In addition, the electroluminescence spectra of the HWOLEDs were kept stable from 100 cd m−2 to 5, 000 cd m−2. (paper)

  16. Effect of Solvation on Electron Detachment and Excitation Energies of a Green Fluorescent Protein Chromophore Variant.

    Science.gov (United States)

    Bose, Samik; Chakrabarty, Suman; Ghosh, Debashree

    2016-05-19

    Hybrid quantum mechanics/molecular mechanics (QM/MM) is applied to the fluorinated green fluorescent protein (GFP) chromophore (DFHBDI) in its deprotonated form to understand the solvatochromic shifts in its vertical detachment energy (VDE) and vertical excitation energy (VEE). This variant of the GFP chromophore becomes fluorescent in an RNA environment and has a wide range of applications in biomedical and biochemical fields. From microsolvation studies, we benchmark (with respect to full QM) the accuracy of our QM/MM calculations with effective fragment potential (EFP) as the MM method of choice. We show that while the solvatochromic shift in the VEE is minimal (0.1 eV blue shift) and its polarization component is only 0.03 eV, the effect of the solvent on the VDE is quite large (3.85 eV). We also show by accurate calculations on the solvatochromic shift of the VDE that polarization accounts for ∼0.23 eV and therefore cannot be neglected. The effect of the counterions on the VDE of the deprotonated chromophore in solvation is studied in detail, and a charge-smearing scheme is suggested for charged chromophores. PMID:27116477

  17. Molecular Basis of Spectral Diversity in Near-Infrared Phytochrome-Based Fluorescent Proteins.

    Science.gov (United States)

    Shcherbakova, Daria M; Baloban, Mikhail; Pletnev, Sergei; Malashkevich, Vladimir N; Xiao, Hui; Dauter, Zbigniew; Verkhusha, Vladislav V

    2015-11-19

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are the probes of choice for deep-tissue imaging. Detection of several processes requires spectrally distinct NIR FPs. We developed an NIR FP, BphP1-FP, which has the most blue-shifted spectra and the highest fluorescence quantum yield among BphP-derived FPs. We found that these properties result from the binding of the biliverdin chromophore to a cysteine residue in the GAF domain, unlike natural BphPs and other BphP-based FPs. To elucidate the molecular basis of the spectral shift, we applied biochemical, structural and mass spectrometry analyses and revealed the formation of unique chromophore species. Mutagenesis of NIR FPs of different origins indicated that the mechanism of the spectral shift is general and can be used to design multicolor NIR FPs from other BphPs. We applied pairs of spectrally distinct point cysteine mutants to multicolor cell labeling and demonstrated that they perform well in model deep-tissue imaging. PMID:26590639

  18. Effect of transient expression of fluorescent protein probes in synovial and myoblast cell lines.

    Science.gov (United States)

    Shibasaki, Seiji; Fujita, Aika; Usui, Chihiro; Watanabe, Sachiko; Kitano, Sachie; Sano, Hajime; Iwasaki, Tsuyoshi

    2012-12-01

    Here, we investigate the appropriate fluorescent proteins for use in the culture of synovial MH7A and myoblast C2C12 cells. Fluorescent signal intensities of 3 different fluorescent proteins were examined in these cell lines. The fluorescent intensity of transiently expressed AcGFP, DsRed, and mStrawberry were examined in these cell lines, and the influence of the amount of plasmid used on transfection efficiency and cell viability were investigated. PMID:23503703

  19. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities

    OpenAIRE

    Bayraktar, Halil; Kiraz, Alper; Aas, Mehdi; Karadağ, Yasin; Manioğlu, Selen; Jonas, Alexandr; Anand, Suman; McGloin, David

    2014-01-01

    Fluorescent proteins are indispensable for selective, quantitative visualization of localization, dynamics, and interactions of key molecular constituents of live cells. Incorporation of fluorescent proteins into an optical cavity can lead to a significant increase in fluorescence signal levels due to stimulated emission and light amplification in the cavity, forming a laser with biological gain medium. Utilization of lasing emission from fluorescent biological molecules can then greatly enha...

  20. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    Science.gov (United States)

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. PMID:27451201

  1. Proton transfer and water exchange in the green fluorescent protein

    Science.gov (United States)

    Agmon, Noam

    2014-03-01

    The green fluorescent protein (GFP) is the only naturally occurring protein in which excited-state proton-transfer has been identified. Upon excitation, a proton is ejected from its chromophore, travelling through the ``privileged water molecule'' (PWM) and Ser205 to Glu222, on a 10 ps timescale or faster. However, time-resolved fluorescence from the chromophore exhibits a t-α power-law decay extending into the ns regime. With increasing temperature, α switches from 1/2 (below 230 K) to 3/2 (above it). This has been interpreted as pseudo one-dimensional proton hopping along an internal ``proton wire,'' with an activated process that opens a ``doorway'' for proton escape to solution at the higher temperatures. To identify such putative pathways, we have developed a computer code mapping all ``proton wires'' within a protein structure. Applying it to a X-ray GFP structure of 0.9 Angstrom resolution, a proton wire indeed continues from Glu222 along the axis of the GFP ``barrel,'' connecting to a negatively charged surface patch (a ``proton collecting antenna''?). This might explain the t- 1 / 2 behavior. However, a direct escape pathway opening from the chromophore to solution is not readily identified in the X-ray structure. Here we report molecular dynamics results showing that the PWM escapes to solution on the 100 ps timescale. This occurs by fluctuations of the beta-sheet, creating an opening through which water molecules can leave and enter the protein. The exact pathway of the PWM on its way in and out has been identified, as well as the water-exchange kinetics that follows a stretched-exponential time behavior. This research was supported by the ISRAEL SCIENCE FOUNDATION grant No. 766/12.

  2. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

    Directory of Open Access Journals (Sweden)

    Mueller-Roeber Bernd

    2010-05-01

    Full Text Available Abstract Background Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL. Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols

  3. Excited states of fluorescent proteins, mKO and DsRed: chromophore-protein electrostatic interaction behind the color variations.

    Science.gov (United States)

    Hasegawa, Jun-ya; Ise, Takehiko; Fujimoto, Kazuhiro J; Kikuchi, Akihiro; Fukumura, Eiko; Miyawaki, Atsushi; Shiro, Yoshitsugu

    2010-03-01

    The emitting states of green fluorescent protein (GFP), monomeric Kusabira orange (mKO), and Discosoma red (DsRed) were studied using QM/MM and SAC-CI methods. By comparing the electronic structures among the green-, orange-, and red-emitting states as well as their electrostatic and quantum mechanical interactions within the protein cavity, the basic mechanisms for determining emission colors have been clarified. We found that the orange and red emissions of mKO and DsRed, respectively, result from cancellation between two effects, the pi skeleton extension (red shift) and protein electrostatic potential (blue shift). The extension of the pi skeleton enhances the intramolecular charge-transfer character of the transition, which makes the fluorescence energy more sensitive to the protein's electrostatic potential. On the basis of this mechanism, we predicted amino acid mutations that could red shift the emission energy of DsRed. A novel single amino acid mutation, which was examined computationally, reduced the DsRed emission energy from 2.14 (579 nm) to 1.95 eV (636 nm), which is approaching near-infrared fluorescence. PMID:20131896

  4. Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP)☆

    OpenAIRE

    Coughlan, Kathleen; Huang, Xiangping; He, Xiangyuan; Chung, Charlotte H.Y.; Li, Guangpu; Tang, Jordan

    2013-01-01

    Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer’s disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells ex...

  5. Fluorescence aggregation assay for the protein biomarker mucin 1 using carbon dot-labeled antibodies and aptamers

    International Nuclear Information System (INIS)

    We have developed a sensitive fluorescence assay for the protein biomarker mucin 1 (MUC1). It is based on the aggregation of functionalized carbon dots (CDs) in the presence of MUC1. The CDs with a size of ∼2 nm display strong blue-green intrinsic fluorescence. Antibodies against MUC1 and the MUC1 aptamer were covalently conjugated to the CDs, and the immunoreaction between CD-labeled antibody to MUC1, and of the CD-labeled aptamer to MUC1 resulted in the formation of a sandwich structure that is accompanied by aggregation of CDs and quenching of fluorescence. The change in fluorescence is linearly related to the MUC1 concentration in the 5 to 100 nM range, with a detection limit at 2 nM. The assay was applied to the determination of MUC1 in (spiked) real serum samples, and good recoveries (ranging from 94.2 to 98.1 %) were obtained. This assay is cost-effective and convenient. It has a wide scope in that it, conceivably, can be extended to assays for other proteins and biomarkers of clinical interest. (author)

  6. Analysis of green fluorescent protein bioluminescence in vivo and in vitro using a glow discharge

    Science.gov (United States)

    Hernández, L.; Mandujano, L. A.; Cuevas, J.; Reyes, P. G.; Osorio-González, D.

    2015-03-01

    The discovery of fluorescent proteins has been a revolution in cell biology and related sciences because of their many applications, mainly emphasizing their use as cellular markers. The green fluorescent protein (GFP) is one of the most used as it requires no cofactors to generate fluorescence and retains this property into any organism when it is expressed by recombinant DNA techniques, which is a great advantage. In this work, we analyze the emission spectra of recombinant green fluorescent protein in vivo and in vitro exposed to a glow discharge plasma of nitrogen in order to relate electron temperature to fluorescence intensity.

  7. Complex Assembly Behavior During the Encapsulation of Green Fluorescent Protein Analogs in Virus Derived Protein Capsules

    NARCIS (Netherlands)

    Minten, Inge J.; Nolte, Roeland J.M.; Cornelissen, Jeroen J.L.M.

    2010-01-01

    Enzymes encapsulated in nanocontainers are a better model of the conditions inside a living cell than free enzymes in solution. In a first step toward the encapsulation of multiple enzymes inside the cowpea chlorotic mottle virus (CCMV) capsid, enhanced green fluorescent protein (EGFP) was attached

  8. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  9. White organic light-emitting devices based on blue fluorescent dye combined with dual sub-monolayer

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Huishan, E-mail: yanghuishan1697@163.com

    2013-10-15

    White organic light-emitting devices have been realized by using highly blue fluorescent dye 4,4′-Bis(2,2-diphenyl-ethen-1-yl)-4,4′-di-(tert-butyl)phenyl(p-TDPVBi) and [2-methyl-6-[2-(2, 3,6,7-tetrahydro-1H, red fluorescent dye 5H-benzo[ij] quinolizin-9-yl) ethenyl]-4H-pyran-4-ylidene] propane-dinitrile(DCM2), together with well known green fluorescent dye quinacridone (QAD). The fabrication of multilayer WOLEDs did not involve the hard-to-control doping process. The structure of the device is ITO/m-MTDATA (45 nm)/NPB(8 nm)/p-TDPVBi(15 nm)/DCM2(x nm)/Alq{sub 3} (5 nm)/QAD(y nm)/Alq{sub 3}(55 nm)/LiF(1 nm)/Al, where 4,4′,4′′-tris{N,-(3-methylphenyl)-N-phenylamine}triphenylamine (m-MTDATA) acts as a hole injection layer, N,N′-bis-(1-naphthyl)-N, N′-diphenyl-1, 1′-biph-enyl-4, 4′-diamine (NPB) acts as a hole transport layer, p-TDPVBi acts as a blue emitting layer, DCM2 acts as a red emitting layer, QAD acts as a green emitting layer, tris-(8-hydroxyquinoline) aluminum (Alq{sub 3}) acts as an electron transport layer, and WOLEDs of devices A, B, C and D are different in layer thickness of DCM2 and QAD, respectively. To change the thickness of dual sub-monolayer DCM2 and QAD, the WOLEDs were obtained. When x, y=0.05, 0.1, the Commission Internationale de 1’Eclairage (CIE) coordinates of the device change from (0.4458, 0.4589) at 3 V to (0.3137, 0.3455) at 12 V that are well in the white region, and the color temperature and color rendering index were 5348 K and 85 at 8 V, respectively. Its maximum luminance was 35260 cd/m{sup 2} at 12 V, and maximum current efficiency and maximum power efficiency were 13.54 cd/A at 12 V and 6.68 lm/W at 5 V, respectively. Moreover, the current efficiency is largely insensitive to the applied voltage. The electroluminescence intensity of white EL devices varied only little at deferent dual sub-monolayer. Device D exhibited relatively high color rendering index (CRI) in the range of 88–90, which was essentially

  10. White organic light-emitting devices based on blue fluorescent dye combined with dual sub-monolayer

    International Nuclear Information System (INIS)

    White organic light-emitting devices have been realized by using highly blue fluorescent dye 4,4′-Bis(2,2-diphenyl-ethen-1-yl)-4,4′-di-(tert-butyl)phenyl(p-TDPVBi) and [2-methyl-6-[2-(2, 3,6,7-tetrahydro-1H, red fluorescent dye 5H-benzo[ij] quinolizin-9-yl) ethenyl]-4H-pyran-4-ylidene] propane-dinitrile(DCM2), together with well known green fluorescent dye quinacridone (QAD). The fabrication of multilayer WOLEDs did not involve the hard-to-control doping process. The structure of the device is ITO/m-MTDATA (45 nm)/NPB(8 nm)/p-TDPVBi(15 nm)/DCM2(x nm)/Alq3 (5 nm)/QAD(y nm)/Alq3(55 nm)/LiF(1 nm)/Al, where 4,4′,4′′-tris{N,-(3-methylphenyl)-N-phenylamine}triphenylamine (m-MTDATA) acts as a hole injection layer, N,N′-bis-(1-naphthyl)-N, N′-diphenyl-1, 1′-biph-enyl-4, 4′-diamine (NPB) acts as a hole transport layer, p-TDPVBi acts as a blue emitting layer, DCM2 acts as a red emitting layer, QAD acts as a green emitting layer, tris-(8-hydroxyquinoline) aluminum (Alq3) acts as an electron transport layer, and WOLEDs of devices A, B, C and D are different in layer thickness of DCM2 and QAD, respectively. To change the thickness of dual sub-monolayer DCM2 and QAD, the WOLEDs were obtained. When x, y=0.05, 0.1, the Commission Internationale de 1’Eclairage (CIE) coordinates of the device change from (0.4458, 0.4589) at 3 V to (0.3137, 0.3455) at 12 V that are well in the white region, and the color temperature and color rendering index were 5348 K and 85 at 8 V, respectively. Its maximum luminance was 35260 cd/m2 at 12 V, and maximum current efficiency and maximum power efficiency were 13.54 cd/A at 12 V and 6.68 lm/W at 5 V, respectively. Moreover, the current efficiency is largely insensitive to the applied voltage. The electroluminescence intensity of white EL devices varied only little at deferent dual sub-monolayer. Device D exhibited relatively high color rendering index (CRI) in the range of 88–90, which was essentially voltage

  11. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    Science.gov (United States)

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  12. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

    DEFF Research Database (Denmark)

    Toddo, Stephen; Soderstrom, Bill; Palombo, Isolde;

    2012-01-01

    /periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.......A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps...... can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic...

  13. Synthesis, characterization and in vitro antimicrobial activity of a new blue fluorescent Cu(II) metal complex of bis-1,8-naphthalimide

    Science.gov (United States)

    Yordanova, Stanislava; Temiz, Havva Tümay; Boyaci, Ismail Hakki; Stoyanov, Stanimir; Vasileva-Tonkova, Evgenia; Asiri, Abdullah; Grabchev, Ivo

    2015-12-01

    A new blue fluorescent copper complex of bis-(4-N,N-dimetylaminoethoxy-N-ethyl-1,8- naphthalimidyl) amine has been synthesized and characterized by FT-IR, Raman and Surface enhanced Raman spectroscopy (SERS). Absorption and fluorescence spectral characteristics have been also used for the characterization of the copper complex. The antibacterial and antifungal activities of the new complex have been investigated and the minimum inhibitory concentrations have been determined. The metal complex has a good antibacterial and antifungal potential suggesting its prospective application in biomedicine for designing new effective antimicrobial preparations.

  14. Attenuation-Based Dual-Fluorescent-Protein Reporter for Screening Translation Inhibitors

    OpenAIRE

    Osterman, Ilya A.; Prokhorova, Irina V.; Sysoev, Vasily O.; Boykova, Yulia V.; Efremenkova, Olga V.; Svetlov, Maxim S.; Kolb, Vyacheslav A.; Bogdanov, Alexey A.; Sergiev, Petr V.; Dontsova, Olga A

    2012-01-01

    A reporter construct was created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon. Dual-fluorescent-protein genes for red fluorescent protein and cerulean fluorescent protein were used as a sensor and internal control of gene expression. The sequence of the attenuator was modified to avoid tryptophan sensitivity while preserving sensitivity to ribosome stalling. Antimicrobial compounds which cause translation arrest at the stage of elongation induc...

  15. A Diabatic Three-State Representation of Photoisomerization in the Green Fluorescent Protein Chromophore

    OpenAIRE

    Olsen, Seth; McKenzie, Ross H.

    2009-01-01

    We give a quantum chemical description of bridge photoisomerization reaction of green fluorescent protein (GFP) chromophores using a representation over three diabatic states. Bridge photoisomerization leads to non-radiative decay, and competes with fluorescence in these systems. In the protein, this pathway is suppressed, leading to fluorescence. Understanding the electronic structure of the photoisomerization is a prerequisite to understanding how the protein suppresses this pathway and pre...

  16. Photophysical studies on the interaction of amides with Bovine Serum Albumin (BSA) in aqueous solution: Fluorescence quenching and protein unfolding

    Energy Technology Data Exchange (ETDEWEB)

    Kumaran, R., E-mail: kumaranwau@rediffmail.com [Department of Chemistry, Dwaraka Doss Goverdhan Doss Vaishnav College, Arumbakkam, Chennai 600106 (India); Ramamurthy, P. [National Centre for Ultrafast Processes, University of Madras, Sekhizar Campus, Taramani, Chennai 600113 (India)

    2014-04-15

    Addition. of amides containing a H-CO(NH{sub 2}) or CH{sub 3}-CO(NH{sub 2}) framework to BSA results in a fluorescence quenching. On the contrary, fluorescence enhancement with a shift in the emission maximum towards the blue region is observed on the addition of dimethylformamide (DMF) (H-CON(CH{sub 3}){sub 2}). Fluorescence quenching accompanied initially with a shift towards the blue region and a subsequent red shift in the emission maximum of BSA is observed on the addition of formamide (H-CO(NH{sub 2})), whereas a shift in the emission maximum only towards the red region results on the addition of acetamide (CH{sub 3}-CONH{sub 2}). Steady state emission spectral studies reveal that amides that possess a free NH{sub 2} and N(CH{sub 3}){sub 2} moiety result in fluorescence quenching and enhancement of BSA respectively. The 3D contour spectral studies of BSA with formamide exhibit a shift in the emission towards the red region accompanied with fluorescence quenching, which indicates that the tryptophan residues of the BSA are exposed to a more polar environment. Circular Dichroism (CD) studies of BSA with amides resulted in a gradual decrease in the α-helical content of BSA at 208 nm, which confirms that there is a conformational change in the native structure of BSA. Time-resolved fluorescence studies illustrate that the extent of buried trytophan moieties exposed to the aqueous phase on the addition of amides follows the order DMFfluorescence methods. -- Highlights:

  17. Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method

    OpenAIRE

    Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J.; Saldi, Stephanie; Qiu, Jean

    2014-01-01

    The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometr...

  18. Fluorescent blue lights, injecting drug use and related health risk in public conveniences: findings from a qualitative study of micro-injecting environments.

    Science.gov (United States)

    Parkin, Stephen; Coomber, Ross

    2010-07-01

    This paper presents findings relating to injecting drug users' experiences and opinions of public toilets illuminated with fluorescent blue lights and presents an empirical assessment of the intended deterrent effect of such installations. Data analysis identified that blue lights deterred less than half the sample interviewed. Furthermore over half (18/31) of the sample were prepared to inject in conditions specifically designed to deter injecting practice. Of these, 11 respondents were completely undeterred and 7 individuals were only partially deterred by blue light environments. These findings are discussed within the interpretative frameworks of Pierre Bourdieu's theory of habitus and symbolic violence. The authors conclude that fluorescent blue lights contribute towards the development of situated resistance by injecting drug users within a public injecting habitus; a resistance that produces and reproduces drug-related harm and is a behaviour that opposes the symbolic violence of harm reduction intervention. The paper concludes with suggestions for theory-driven practical intervention that may seek to disrupt the harmful elements of the public injecting habitus. PMID:20167527

  19. Intrinsic Fluorescence as a Spectral Probe for Protein Denaturation Studies in the Presence of Honey

    Science.gov (United States)

    Wong, Y. H.; Kadir, H. A.; Tayyab, S.

    2015-11-01

    Honey was found to quench the intrinsic fluorescence of bovine serum albumin (BSA) in a concentration dependent manner, showing complete quenching in the presence of 5% (w/v) honey. Increasing the protein concentration up to 5.0 μM did not lead to the recovery of the protein fluorescence. Urea denaturation of BSA, which otherwise shows a two-step, three-state transition, using intrinsic fluorescence of the protein as the probe failed to produce any result in the presence of 5% (w/v) honey. Thus, intrinsic fluorescence cannot be used as a spectral probe for protein denaturation studies in the presence of honey.

  20. From jellyfish to biosensors: the use of fluorescent proteins in plants.

    Science.gov (United States)

    Voss, Ute; Larrieu, Antoine; Wells, Darren M

    2013-01-01

    The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors. PMID:24166435

  1. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  2. Development of transgenic chickens expressing enhanced green fluorescent protein

    International Nuclear Information System (INIS)

    In this work we demonstrated the successful production of transgenic chickens expressing the enhanced green fluorescence protein (EGFP) gene. Replication-defective recombinant retroviruses produced from vesicular stomatitis virus G glycoprotein pseudotyped retrovirus vector system were injected beneath the blastoderm of non-incubated chicken embryos (stage X). From 129 injected eggs, 13 chicks hatched after 21 days of incubation. All hatched chicks were found to express vector-encoded EGFP gene, which was under the control of the Rous sarcoma virus promoter and boosted post-transcriptionally by woodchuck hepatitis virus post-transcriptional regulatory element sequence. Green fluorescent signals, indicative of the EGFP gene expression, were detected in various body parts, including head, limb, eye, toe, and several internal organs. Genomic incorporation of the transgene was also proven by Southern blot assay. Our results show the exceptional versatile effectiveness of the EGFP gene as a marker in the gene expression-related studies which therefore would be very helpful in establishing a useful transgenic chicken model system for studies on embryo development and for efficient production of transgenic chickens as bioreactors

  3. Fluorescence microscopy methods in the study of protein structure and function.

    Science.gov (United States)

    Jensen-Smith, Heather; Currall, Benjamin; Rossino, Danielle; Tiede, LeAnn; Nichols, Michael; Hallworth, Richard

    2009-01-01

    As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions, and the Scanning Cysteine Accessibility Method (SCAM), used for the determination of protein conformation. The methods are readily adaptable to other proteins. PMID:18839359

  4. Electrochemical Study on the Interaction of Protein with Bromothymol Blue and Its Analytical Application

    Institute of Scientific and Technical Information of China (English)

    WANG Xue-liang; YANG Jie; JIAO Kui

    2008-01-01

    The interaction ofbromothymol blue(BB) with human serum albumin(HSA) was studied by electrochemical techniques and a sensitive method for proteins assay was developed.When BB interacted with HSA,the voltammetric peak current value of BB decreased linearly with the concentration of HSA in a range of 1.0-40.0 mg/L,and the peak potential shifted negatively.Based on the results,a sensitive assay method for proteins,such as HSA,bovine serum albumin(BSA),and egg albumin etc.was established.This method was further applied to determining the HSA in healthy human blood samples,and the results are not significantly different from those obtained by the classic Coomassie Brilliant Blue G-250 spectrophotometic method.The detecting conditions of this method were optimized and the interaction mechanism was discussed.The results show that the electrochemical parameters(formal potential E0,standard rate constant of the electrode reaction ks,parameter of kinetic na) of BB have no obvious changes before and after the interaction,which indicate that BB can interact with HSA,forming an electrochemical non-active complex.The equilibrium constant(βs) and the binding ratio(m) for this complex were calculated.The m is 4 and β,is 1.41 × 1019.This method is fast,simple,highly sensitive,and has good selectivity,which can be used in clinical measurements.

  5. Novel blue-emitting gold nanoclusters confined in human hemoglobin, and their use as fluorescent probes for copper(II) and histidine

    International Nuclear Information System (INIS)

    We report on the synthesis and characterization of highly fluorescent gold nanoclusters (Au-NCs) from a gold precursor using a single-step chemical reduction in the presence of human adult hemoglobin (Hb). The conformational changes of Hb before and after cluster formation were studied by various spectroscopic techniques. The Au-NCs in Hb display blue fluorescence with a peak centered at 450 nm (photoexcited at 365 nm) and a quantum yield of 2.8 %. The Au-NCs exhibit excellent photostability and long-term stability, and can be applied in the pH range 5–12 even in the presence of high electrolyte concentrations. The Au-NCs in Hb can act as a highly sensitive and selective fluorescent turn-off probe for Cu(II) ion. The observed reversible fluorescence recovery of Hb-AuNCs/Cu(II) aggregates was exploited to develop a selective and sensitive turn-on fluorescence assay for His. Under optimized conditions, the probe gives a fluorescent response that is linear in the 0.1 to 20.0 μM concentration range of Cu(II), with a limit of detection of 28 nM. The probe for His, in turn, has a linear range in the 1–21 μM concentration range, and the limit of detection is 0.6 μM. (author)

  6. Plasmon-controlled narrower and blue-shifted fluorescence emission in (Au-SiO{sub 2})SiC nanohybrids

    Energy Technology Data Exchange (ETDEWEB)

    Sui Ning; Monnier, Virginie, E-mail: virginie.monnier@ec-lyon.fr [Institut des Nanotechnologies de Lyon-INL, UMR CNRS 5270, Site Ecole Centrale de Lyon, Universite de Lyon (France); Zakharko, Yuriy [Institut des Nanotechnologies de Lyon-INL-UMR 5270-CNRS, Site INSA Lyon, Universite de Lyon (France); Chevolot, Yann [Institut des Nanotechnologies de Lyon-INL, UMR CNRS 5270, Site Ecole Centrale de Lyon, Universite de Lyon (France); Alekseev, Sergei [Kiev National Taras Schevchenko University, Faculty of Chemistry (Ukraine); Bluet, Jean-Marie; Lysenko, Vladimir [Institut des Nanotechnologies de Lyon-INL-UMR 5270-CNRS, Site INSA Lyon, Universite de Lyon (France); Souteyrand, Eliane [Institut des Nanotechnologies de Lyon-INL, UMR CNRS 5270, Site Ecole Centrale de Lyon, Universite de Lyon (France)

    2012-08-15

    Fluorescent imaging is a key tool in biology. On one hand, organic dyes are subjected to bleaching; while, on the other hand II-VI semiconductor quantum dots are photostable, but may exhibit some toxicity. Silicon carbide nanoparticles (SiC NPs) are a good alternative as SiC is chemically inert and considered as biocompatible material. However, their fluorescence quantum yield is weak. Plasmon-controlled fluorescence appears as a mean to enhance SiC NPs fluorescence. To this aim, new fluorescent nanohybrids (NHs) involving a gold colloid surrounded by a silica shell and overcoated with SiC NPs have been engineered. Au-SiO{sub 2} particles were synthesized via a sol-gel method to obtain a controlled thickness of silica around gold colloid. Then, SiC NPs were immobilized onto silica by covalent bonding. TEM was used to study the structural properties of NHs. The influence of several parameters on NHs' fluorescence properties was investigated. It reveals that excitation wavelength must be chosen as close as possible to the gold plasmon band to avoid quenching of emission due to energy transfer between gold absorption and SiC emission. Moreover, the silica thickness is a key parameter to obtain high enhancement; for gold colloids of 20 nm in diameter overcoated by a 25 nm shell, an enhancement factor as high as 12.5 was obtained with a narrower and blue-shifted emission band. This blue-shift can be attributed to the surface chemistry modification of SiC NPs when they are covalently bonded to silica.

  7. A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria

    OpenAIRE

    Goh, Yan Y.; Ho, Bow; Ding, Jeak L.

    2002-01-01

    Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFPi proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide...

  8. Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy

    OpenAIRE

    Paez Segala, Maria G.; Sun, Mei G.; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A; Macklin, John J.; Patel, Ronak; Allen, John R.; Elizabeth S. Howe; Piszczek, Grzegorz; Hess, Harald F.; Davidson, Michael W.; Wang, Yalin; Looger, Loren L.

    2015-01-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

  9. Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy

    Science.gov (United States)

    Paez Segala, Maria G.; Sun, Mei G.; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A.; Macklin, John J.; Patel, Ronak; Allen, John R.; Howe, Elizabeth S.; Piszczek, Grzegorz; Hess, Harald F.; Davidson, Michael W.; Wang, Yalin; Looger, Loren L.

    2014-01-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. PMID:25581799

  10. Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins

    NARCIS (Netherlands)

    Visser, NV; Borst, JW; Hink, MA; van Hoek, A; Visser, AJWG

    2005-01-01

    Visible fluorescent proteins from Aequorea victoria contain next to the fluorophoric group a single tryptophan residue. Both molecules form a single donor-acceptor pair for resonance energy transfer (RET) within the protein. Time-resolved fluorescence experiments using tryptophan excitation have sho

  11. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes. PMID:27515076

  12. Tissue protein fluorescence for cancer diagnosis-a pilot study

    International Nuclear Information System (INIS)

    The present study deals with the application of fluorescent techniques in the characterization of normal and cancerous cervical tissues by measuring fluorescence emission spectra in the spectral range 300-500 nm at 280 nm excitation and fluorescence excitation spectra in the spectral range 250-320 nm at 340 nm emission

  13. R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.

    Directory of Open Access Journals (Sweden)

    You-Tzung Chen

    Full Text Available Green fluorescent protein (GFP and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry. This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

  14. Quinoline-Substituted 10-(naphthalene-7-yl)anthracene Derivatives for Blue Fluorescent Organic Light-Emitting Diodes.

    Science.gov (United States)

    Kim, Chanwoo; Park, Soo Na; Lee, Seul Bee; Kim, Young Seok; Lee, Ho Won; Kim, Young Kwan; Yoon, Seung Soo

    2016-02-01

    In this study, we have designed and synthesized blue emitters based on quinoline-substituted 10-(naphthalene-7-yl)anthracene. Particularly, a material exhibited highly efficient blue electroluminescence with CIE coordinates of (0.15, 0.18). PMID:27433688

  15. The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 A resolution.

    Science.gov (United States)

    Guss, J M; Merritt, E A; Phizackerley, R P; Freeman, H C

    1996-10-11

    The crystal structure of the cucumber basic protein (CBP), a type 1 or blue copper protein, has been refined at 1.8 A resolution. The molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a "beta-sandwich" or "beta-taco". The Cu atom has the normal blue copper NNSS' co-ordination with bond lengths Cu-N(His39) = 1.93 A, Cu-S(Cys79) = 2.16 A, Cu-N(His84) = 1.95 A, Cu-S(Met89) = 2.61 A. The Cu-S(Met) bond is the shortest so far observed in a blue copper protein. A disulphide link, (Cys52)-S-S-(Cys85), appears to play an important role in stabilising the molecular structure. It is suggested that the polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identify. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine. The structure of CBP was originally solved by the multiple wavelength anomalous scattering method, using data recorded at four wavelengths. All these data were included in the restrained least squares refinement. The final model comprises 96 amino acid residues, 122 solvent molecules and a copper atom. Several residues are modelled as having more than one conformation. The residual R is 0.141 for 41,910 observations (including Bijvoet-related observations) of 8.142 unique reflections in the resolution range 7 to 1.8 A. PMID:8876647

  16. BMAA detected as neither free nor protein bound amino acid in blue mussels.

    Science.gov (United States)

    Rosén, Johan; Westerberg, Erik; Schmiedt, Sebastian; Hellenäs, Karl-Erik

    2016-01-01

    The results of this study imply that β-methylamino-alanine (BMAA) obtained from extracts of blue mussels from the Swedish west coast is neither free nor protein bound. The results were obtained by separation (precipitation and ultrafiltration) of low and high molecular weight compounds from neutral extracts of blue mussels, and treatment of these extracts with low and high concentrations of acids, varying time and temperature. The main portion of BMAA was obtained from the low molecular weight fraction, released or formed at 95 °C in dilute acids. The measured amount of BMAA did not increase by strong acid treatment. Lysine was used as reference and was only released at significant amounts when treating the high molecular weight fraction with concentrated acid. The results also indicated that breakage of peptide bonds was not involved in the formation/release of BMAA in these extracts unless any BMAA peptide bond would be significantly more susceptible to dilute acid than e.g. the monitored lysine peptide bond. BMAA was measured using isotope dilution and detection of the underivatized compound by HILIC-UHPLC-MS/MS (Hydrophilic Interaction Liquid Chromatography, Ultra-High Performance Liquid Chromatography, tandem Mass Spectrometry). The findings might add to the understanding of conflicting data in the literature regarding the occurrence of BMAA, and have implications for studies on possible biomagnification of BMAA in the food chain and bioavailability from food. PMID:26577502

  17. Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

    DEFF Research Database (Denmark)

    Wojdyla, Katarzyna Iwona; Rogowska-Wrzesinska, Adelina; Wrzesinski, Krzysztof;

    2011-01-01

    We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our...... methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence...... were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis...

  18. Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

    DEFF Research Database (Denmark)

    Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina; Wrzesinski, Krzysztof;

    2011-01-01

    We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our...... methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence...... were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis of the...

  19. Blue-green fluorescence and visible-infrared reflectance of corn (Zea mays L.) grain for in situ field detection of nitrogen supply

    International Nuclear Information System (INIS)

    The sensing of spectral attributes of corn (Zea mays L.) grain from site specific areas of the field during the harvest process may be useful in managing agronomic inputs and production practices on those areas of the field in subsequent growing seasons. Eight levels of nitrogen (N) fertilization were applied to field grown corn at Beltsville, Maryland. These N treatments produced a range of chlorophyll levels, biomass and physiological condition in the live plant canopies. After harvest, spectra were obtained in the laboratory on whole grain samples. Fluorescence emissions were acquired from 400 to 600 nm and percent reflectance were measured in the visible (VIS) near infrared (NIR) and mid-infrared (MIR) regions from 400 nm to 2400 nm. A ultraviolet (UV) excitation band centered at 385 nm was the most effective in producing fluorescence emission differences in the blue-green region of the fluorescence spectrum with maxima centered from 430-470nm in the blue and with an intense shoulder centered at around 530-560 nm in the green region. Reflectance showed the most spectral differences in the NIR and MIR (970-2330 nm) regions

  20. Blue-Light-Independent Activity of Arabidopsis Cryptochromes in the Regulation of Steady-State Levels of Protein and mRNA Expression

    Institute of Scientific and Technical Information of China (English)

    Yue-Jun Yang; Xuan-Ming Liu; Chen-Tao Lin; Ze-Cheng Zuo; Xiao-Ying Zhao; Xu Li; John Klejnot; Yan Li; Ping Chen; Song-Ping Liang; Xu-Hong Yu

    2008-01-01

    Cryptochromes are blue-light receptors that mediate blue-light inhibition of hypocotyl elongation and bluelight stimulation of floral initiation in Arabidopsis. In addition to their blue-light-dependent functions, cryptochromes are also involved in blue-light-independent regulation of the circadian clock, cotyledon unfolding, and hypocotyl inhibition.However, the molecular mechanism associated with the blue-light-independent function of cryptochromes remains unclear. We reported here a comparative proteomics study of the light regulation of protein expression. We showed that, as expected, the protein expression of many metabolic enzymes changed in response to both blue light and red light. Surprisingly, some light-regulated protein expression changes are impaired in the cry1cry2 mutant in both blue light and red light. This result suggests that, in addition to mediating blue-light-dependent regulation of protein expression, cryptochromes are also involved in the blue-light-independent regulation of gene expression. Consistent with this hypothesis,the cry1cry2 mutant exhibited reduced changes of mRNA expression in response to not only blue light, but also red light,although the cryptochrome effects on the red-light-dependent gene expression changes are generally less pronounced.These results support a hypothesis that, in addition to their blue-light-specific functions, cryptochromes also play roles in the control of gene expression mediated by the red/far-red-light receptor phytochromes.

  1. Glow in the Dark: Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants

    Institute of Scientific and Technical Information of China (English)

    Wenzislava Ckurshumova; Adriana E. Caragea; Rochelle S. Goldstein; Thomas Berleth

    2011-01-01

    Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants,fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types,to monitor dynamic cell fate selection processes,and to obtain cell type-specific transcriptomes.Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes.The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms.In developmental studies,the use of fluorescent proteins has become critical,where morphological markers of tissues,cell types,or differentiation stages are either not known or not easily recognizable.In this review,we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

  2. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    Science.gov (United States)

    Tsekouras, Konstantinos

    2015-03-01

    Fluctuations are an inherent part of single molecule or few particle biophysical data sets. Traditionally, ``noise'' fluctuations have been viewed as a nuisance, to be eliminated or minimized. Here we look on how statistical inference methods - that take explicit advantage of fluctuations - have allowed us to draw an unexpected picture of single molecule diffusional dynamics. Our focus is on the diffusion of proteins probed using fluorescence correlation spectroscopy (FCS). First, we discuss how - in collaboration with the Bustamante and Marqusee labs at UC Berkeley - we determined using FCS data that individual enzymes are perturbed by self-generated catalytic heat (Riedel et al, Nature, 2014). Using the tools of inference, we found how distributions of enzyme diffusion coefficients shift in the presence of substrate revealing that enzymes performing highly exothermic reactions dissipate heat by transiently accelerating their center of mass following a catalytic reaction. Next, when molecules diffuse in the cell nucleus they often appear to diffuse anomalously. We analyze FCS data - in collaboration with Rich Day at the IU Med School - to propose a simple model for transcription factor binding-unbinding in the nucleus to show that it may give rise to apparent anomalous diffusion. Here inference methods extract entire binding affinity distributions for the diffusing transcription factors, allowing us to precisely characterize their interactions with different components of the nuclear environment. From this analysis, we draw key mechanistic insight that goes beyond what is possible by simply fitting data to ``anomalous diffusion'' models.

  3. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

    Science.gov (United States)

    Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

    2016-01-19

    This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

  4. Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

    Directory of Open Access Journals (Sweden)

    Padalkar Vikas S

    2011-11-01

    Full Text Available Abstract Background Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy-[2, 2'-bipyrimidine]-4, 6-diylbis(1H-pyrazol-3, 1-diyl dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375 and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

  5. Spectroscopic investigation on interaction and sonodynamic damage of Riboflavin to DNA under ultrasonic irradiation by using Methylene Blue as fluorescent probe

    Science.gov (United States)

    Wang, Qi; Wu, Qiong; Wang, Jun; Chen, Dandan; Fan, Ping; Wang, Baoxin

    2014-01-01

    In this paper, the Riboflavin (RF) as a sonosensitizer and Methylene Blue (MB) as a fluorescent probe were used to study the interaction and sonodynamic damage to Deoxyribonucleic Acid (DNA) by fluorescence and UV-vis spectroscopy. The results showed that the RF could efficiently bind to DNA in aqueous solution and exchange with the MB through competing reaction. And then, under ultrasonic irradiation, the RF could obviously damage the DNA. In addition, the influencing factors such as ultrasonic irradiation time and RF concentration on the sonodynamic damage to DNA were also considered. The experimental results showed that the sonodynamic damage degree increase with the increase of ultrasonic irradiation time and RF concentration. Perhaps, this paper may offer some important subjects for broadening the application of RF in sonodynamic therapy (SDT) technologies for tumor treatment.

  6. Ultrafast excited-state dynamics and fluorescence deactivation of near-infrared fluorescent proteins engineered from bacteriophytochromes

    Science.gov (United States)

    Zhu, Jingyi; Shcherbakova, Daria M.; Hontani, Yusaku; Verkhusha, Vladislav V.; Kennis, John T. M.

    2015-08-01

    Near-infrared fluorescent proteins, iRFPs, are recently developed genetically encoded fluorescent probes for deep-tissue in vivo imaging. Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties. Here we report the electronic excited state deactivation dynamics of the most red-shifted iRFPs: iRFP702, iRFP713 and iRFP720. Complementary measurements by ultrafast broadband fluorescence and absorption spectroscopy show that single exponential decays of the excited state with 600 ~ 700 ps dominate in all three iRFPs, while photoinduced isomerization was completely inhibited. Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility. On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.

  7. Attenuation-based dual-fluorescent-protein reporter for screening translation inhibitors.

    Science.gov (United States)

    Osterman, Ilya A; Prokhorova, Irina V; Sysoev, Vasily O; Boykova, Yulia V; Efremenkova, Olga V; Svetlov, Maxim S; Kolb, Vyacheslav A; Bogdanov, Alexey A; Sergiev, Petr V; Dontsova, Olga A

    2012-04-01

    A reporter construct was created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon. Dual-fluorescent-protein genes for red fluorescent protein and cerulean fluorescent protein were used as a sensor and internal control of gene expression. The sequence of the attenuator was modified to avoid tryptophan sensitivity while preserving sensitivity to ribosome stalling. Antimicrobial compounds which cause translation arrest at the stage of elongation induce the reporter both in liquid culture and on an agar plate. This reporter could be used for high-throughput screening of translation inhibitors. PMID:22252829

  8. Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

    Science.gov (United States)

    Wong, Chin Ken; Laos, Alistair J; Soeriyadi, Alexander H; Wiedenmann, Jörg; Curmi, Paul M G; Gooding, J Justin; Marquis, Christopher P; Stenzel, Martina H; Thordarson, Pall

    2015-04-27

    Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane. PMID:25736460

  9. [Comparison of histone-like proteins from blue-green algae with ribosomal basic proteins of alga and wheat germ histones].

    Science.gov (United States)

    Gofshteĭn, L V; Iurina, N P; Romashkin, V I; Oparin, A I

    1975-01-01

    Histone-like proteins was found in blue-green alga Anacystis nidulans, which has no nucleus. F2b2, F2a2, F2a1 fractions were found in histone-like algae proteins and no fraction F1. Content of basic amino acids (arginine being prevailing in algae protein) is quite identical in histone-like algae proteins and in wheat germs histones, while the content of acid amino acids is considerably higher in algae. The presence in procaryotic cells of basic proteins similar in a number of properties to histones of higher organisms suggests that these proteins are evolutionary precursors of eucaryotic histones. PMID:813782

  10. Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions

    OpenAIRE

    Kaufmann, Rainer; Schellenberger, Pascale; Seiradake, Elena; Dobbie, Ian M.; Jones, E. Yvonne; Davis, Ilan; Hagen, Christoph; Grünewald, Kay

    2014-01-01

    We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400–500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and...

  11. Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

    Science.gov (United States)

    Nishiuchi, Yuji; Inui, Tatsuya; Nishio, Hideki; Bódi, József; Kimura, Terutoshi; Tsuji, Frederick I.; Sakakibara, Shumpei

    1998-01-01

    The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor. PMID:9811837

  12. Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

    Science.gov (United States)

    Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

    2015-05-01

    The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods. PMID:24643390

  13. Postpartum Blues

    Medline Plus

    Full Text Available ... baby blues). What are the baby blues? The word "blues" is not really correct since women with ... baby blues). What are the baby blues? The word "blues" is not really correct since women with ...

  14. Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo

    Science.gov (United States)

    Lai, Hsien-Tsung; Chiang, Cheng-Ming

    2016-01-01

    Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1–158 amino acid residues, called Venus-N, and its C-terminal 159–239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is

  15. Organic Fluorescent Dyes Supported on Activated Boron Nitride: A Promising Blue Light Excited Phosphors for High-Performance White Light-Emitting Diodes

    Science.gov (United States)

    Li, Jie; Lin, Jing; Huang, Yang; Xu, Xuewen; Liu, Zhenya; Xue, Yanming; Ding, Xiaoxia; Luo, Han; Jin, Peng; Zhang, Jun; Zou, Jin; Tang, Chengchun

    2015-02-01

    We report an effective and rare-earth free light conversion material synthesized via a facile fabrication route, in which organic fluorescent dyes, i.e. Rhodamine B (RhB) and fluorescein isothiocyanate (FITC) are embedded into activated boron nitride (αBN) to form a composite phosphor. The composite phosphor shows highly efficient Förster resonance energy transfer and greatly improved thermal stability, and can emit at broad visible wavelengths of 500-650 nm under the 466 nm blue-light excitation. By packaging of the composite phosphors and a blue light-emitting diode (LED) chip with transparent epoxy resin, white LED with excellent thermal conductivity, current stability and optical performance can be realized, i.e. a thermal conductivity of 0.36 W/mk, a Commission Internationale de 1'Eclairage color coordinates of (0.32, 0.34), and a luminous efficiency of 21.6 lm.W-1. Our research opens the door toward to the practical long-life organic fluorescent dyes-based white LEDs.

  16. A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

    OpenAIRE

    Hughes Thomas E; Sheridan Douglas L

    2004-01-01

    Abstract Background There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one h...

  17. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  18. A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia

    Directory of Open Access Journals (Sweden)

    Klinger-Strobel M

    2016-02-01

    Full Text Available Mareike Klinger-Strobel,1,2,* Julia Ernst,3,* Christian Lautenschläger,4 Mathias W Pletz,1,2 Dagmar Fischer,3,5 Oliwia Makarewicz1,2 1Center for Infectious Diseases and Infection’s Control, 2Center for Sepsis Control and Care, Jena University Hospital, 3Department of Pharmaceutical Technology, Friedrich Schiller University Jena, 4Department of Internal Medicine IV, Jena University Hospital, 5Jena Center for Soft Matter (JCSM, Friedrich Schiller University Jena, Jena, Germany*These authors contributed equally to this work Abstract: Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO® 9, propidium iodide, fluorescein. Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(D,L-lactide-co-glycolide (PLGA-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA, after which blue fluorescent poly(ethylene glycol-block-PLGA (PEG-PLGA particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. Keywords: 7-amino-4

  19. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

    OpenAIRE

    Shen, Yi; Rosendale, Morgane; Robert E Campbell; Perrais, David

    2014-01-01

    Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultan...

  20. Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

    OpenAIRE

    Nobuaki Soh

    2008-01-01

    Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been stu...

  1. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy

    OpenAIRE

    Day, Richard N

    2013-01-01

    The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster Resonance Energy Transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained fro...

  2. Mass spectrometry data from a quantitative analysis of protein expression in gills of immuno-challenged blue mussels (Mytilus edulis).

    Science.gov (United States)

    Hörnaeus, K; Guillemant, J; Mi, J; Hernroth, B; Bergquist, J; Lind, S Bergström

    2016-09-01

    Here, we provide the dataset associated with our research article on the potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, "Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis)" [1]. Blue mussels were stimulated with lipopolysaccharides and samples were collected at different time points post injection. Protein extracts were prepared from the gills, digested using trypsin and a full in-depth proteome investigation was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed using the MaxQuant 1.5.1.2 software, "MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification" [2]. PMID:27358907

  3. A bistriphenylamine-substituted spirobifluorene derivative exhibiting excellent nonlinearity/transparency/thermal stability trade-off and strong two-photon induced blue fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Hongyao [Department of Chemistry, Shanghai Normal University, Shanghai 200234 (China); Xiao, Haibo, E-mail: xiaohb@shnu.edu.cn [Department of Chemistry, Shanghai Normal University, Shanghai 200234 (China); Ding, Lei [Department of Chemistry, Shanghai Normal University, Shanghai 200234 (China); Zhang, Chun; Ren, Aiming [State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023 (China); Li, Bo [Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai 200241 (China)

    2015-02-01

    A spirobifluorene-bridged donor/donor chromophore, 2,7-bis-(4-(N,N-diphenylamino)phen-1-yl)-9,9′-spirobifluorene (SPF-TP), was found to combine excellent transparency in the near UV–visible region (λ{sub cut-off} ≤ 420 nm), large two-photon absorption cross-section (4.5 × 10{sup 3}GM) and high thermal stability (T{sub d} = 501 °C). In comparison to the reported two-photon absorption molecules, SPF-TP represents the best thermal stability so far described in the literature. The main electronic factors explaining the high two-photon absorption activities of SPF-TP were analyzed by theoretical calculations. Cyclic voltammograms were employed to explore the causes of the excellent transparency of SPF-TP. It was found that the spiroconjugation effect is responsible for the excellent nonlinearity/transparency/thermal stability trade-off in SPF-TP. In addition, SPF-TP is also a good two-photon induced blue fluorescent material with high fluorescence quantum yield (Φ = 0.90, in THF). - Highlights: • We report a molecule exhibiting excellent transparency. • The two-photon absorption cross-section is as large as 4.5 × 10{sup 3}GM. • The molecule exhibits excellent thermal stability. • The molecule is a good two-photon induced blue fluorescent material. • The spiroconjugation effect explains the excellent properties.

  4. A bistriphenylamine-substituted spirobifluorene derivative exhibiting excellent nonlinearity/transparency/thermal stability trade-off and strong two-photon induced blue fluorescence

    International Nuclear Information System (INIS)

    A spirobifluorene-bridged donor/donor chromophore, 2,7-bis-(4-(N,N-diphenylamino)phen-1-yl)-9,9′-spirobifluorene (SPF-TP), was found to combine excellent transparency in the near UV–visible region (λcut-off ≤ 420 nm), large two-photon absorption cross-section (4.5 × 103GM) and high thermal stability (Td = 501 °C). In comparison to the reported two-photon absorption molecules, SPF-TP represents the best thermal stability so far described in the literature. The main electronic factors explaining the high two-photon absorption activities of SPF-TP were analyzed by theoretical calculations. Cyclic voltammograms were employed to explore the causes of the excellent transparency of SPF-TP. It was found that the spiroconjugation effect is responsible for the excellent nonlinearity/transparency/thermal stability trade-off in SPF-TP. In addition, SPF-TP is also a good two-photon induced blue fluorescent material with high fluorescence quantum yield (Φ = 0.90, in THF). - Highlights: • We report a molecule exhibiting excellent transparency. • The two-photon absorption cross-section is as large as 4.5 × 103GM. • The molecule exhibits excellent thermal stability. • The molecule is a good two-photon induced blue fluorescent material. • The spiroconjugation effect explains the excellent properties

  5. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    Directory of Open Access Journals (Sweden)

    Eriksen Jens

    2009-01-01

    Full Text Available Abstract DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

  6. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    Directory of Open Access Journals (Sweden)

    Hojman Pernille

    2009-04-01

    Full Text Available Abstract DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

  7. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yi; Keegan, Gemma L., E-mail: gemmakeegan@gmail.com [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland); Stranik, Ondrej [Leibniz Institute of Photonic Technology, Department of NanoBiophotonics (Germany); Brennan-Fournet, Margaret E. [CMP-EMSE, MOC, Department of Bioelectronics, Ecole Nationale Superieure des Mines (France); McDonagh, Colette [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland)

    2015-07-15

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs.

  8. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    International Nuclear Information System (INIS)

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs

  9. Inhibition of blue-light-dependent binding of 14-3-3 proteins to phototropins by hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao; SHIMAZAKI Kenichiro

    2005-01-01

    @@ Phototropins, following the discovery of phytochromes[1,2] and cryptochromes[3,4], are the most recently characterized blue-light (BL) receptors in plants. The N- terminal regions of the proteins contain two light oxygen and voltage (LOV)――LOV1 and LOV2, which belong to PAS domain involved in protein-protein interaction and ligand binding, possessing non-covalent binding sites for the chromophore FMN[5]. The C-terminal regions contain Ser/Thr kinase domains[6].

  10. Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

    Science.gov (United States)

    Flores, Rosa V.; Sola, Hilda M.; Torres, Juan C.; Torres, Rafael E.; Guzman, Ernick E.

    2013-01-01

    A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence,…

  11. Structural characterization of the photoswitchable fluorescent protein Dronpa-C62S

    International Nuclear Information System (INIS)

    The photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is increasingly recognized as a new technique for optical marking. Recently, Ando and his colleagues developed a new green fluorescent protein Dronpa, which possesses the unique photochromic property of being photoswitchable in a non-destructive manner. To better understand this mechanism, we determined the crystal structures of a new GFP Dronpa and its mutant C62S, at 1.9 A and 1.8 A, respectively. Determination of the structures demonstrates that a unique hydrogen-bonding network and the sulfur atom of the chromophore are critical to the photoswitching property of Dronpa. Reversible photoswitching was lost in cells expressing the Dronpa-C62S upon repetitive irradiation compared to the native protein. Structural and mutational analyses reveal the chemical basis for the functional properties of photoswitchable fluorescent proteins and provide the basis for subsequent coherent engineering of this subfamily of Dronpa homolog's

  12. A polarizable embedding DFT study of one-photon absorption in fluorescent proteins

    DEFF Research Database (Denmark)

    Beerepoot, Maarten; Steindal, Arnfinn H.; Kongsted, Jacob;

    2013-01-01

    A theoretical study of the one-photon absorption of five fluorescent proteins (FPs) is presented. The absorption properties are calculated using a polarizable embedding approach combined with density functional theory (PE-DFT) on the wild-type green fluorescent protein (wtGFP) and several of its...... shift from vacuum to protein. This is the first computational study of a range of fluorescent proteins using a polarizable embedding potential....... optimization of the chromophores within a frozen protein environment is needed in order to reproduce the experimental trends. Explicit account of polarization in the force field is not needed to yield the correct trend between the different FPs, but is necessary for reproducing the experimentally observed red...

  13. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    International Nuclear Information System (INIS)

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10-8 to 2.0 x 10-6 M with a detection limit of 1.6 x 10-8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

  14. Polarization-dependent fluorescence of proteins bound to nanopore-confined lipid bilayers

    Science.gov (United States)

    Li, R.-Q.; Marek, A.; Smirnov, Alex I.; Grebel, H.

    2008-09-01

    Lipid bilayers are essential structural component of biological membranes of all the living species: from viruses and bacteria to plants and humans. Biophysical and biochemical properties of such membranes are important for understanding physical mechanisms responsible for drug targeting. Binding events between proteins and the membrane may be ascertained by introducing fluorescence markers (chromophores) to the proteins. Here we describe a novel biosensing platform designed to enhance signals of these fluorescence markers. Nanoporous aluminum oxide membranes with and without gold (Au) surface coating have been employed for optical detection of bound conjugated streptavidin to biotinylated lipid bilayers-a model system that mimics protein docking to the membrane surface. Unexpectedly, it was found that fluorescence signals from such structures vary when pumped with E-polarized and H-polarized incident optical beams. The origin of the observed polarization-dependent effects and the implications for enhanced fluorescence detection in a biochip format are being discussed.

  15. Hidden photoinduced reactivity of the blue fluorescent protein mKalama1

    DEFF Research Database (Denmark)

    Vegh, Russell B.; Bloch, Dmitry A.; Bommarius, Andreas S.;

    2015-01-01

    absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We...

  16. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    Science.gov (United States)

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals. PMID:25265085

  17. Structural changes of soy proteins at the oil-water interface studied by fluorescence spectroscopy.

    Science.gov (United States)

    Keerati-u-rai, Maneephan; Miriani, Matteo; Iametti, Stefania; Bonomi, Francesco; Corredig, Milena

    2012-05-01

    Fluorescence spectroscopy was used to acquire information on the structural changes of proteins at the oil/water interface in emulsions prepared by using soy protein isolate, glycinin, and β-conglycinin rich fractions. Spectral changes occurring from differences in the exposure of tryptophan residues to the solvent were evaluated with respect to spectra of native, urea-denatured, and heat treated proteins. The fluorescence emission maxima of the emulsions showed a red shift with respect to those of native proteins, indicating that the tryptophan residues moved toward a more hydrophilic environment after adsorption at the interface. The heat-induced irreversible transitions were investigated using microcalorimetry. Fluorescence spectroscopy studies indicated that while the protein in solution underwent irreversible structural changes with heating at 75 and 95°C for 15 min, the interface-adsorbed proteins showed very little temperature-induced rearrangements. The smallest structural changes were observed in soy protein isolate, probably because of the higher extent of protein-protein interactions in this material, as compared to the β-conglycinin and to the glycinin fractions. This work brings new evidence of structural changes of soy proteins upon adsorption at the oil water interface, and provides some insights on the possible protein exchange events that may occur between adsorbed and unadsorbed proteins in the presence of oil droplets. PMID:22227018

  18. Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C

    DEFF Research Database (Denmark)

    Kristensen, B; Birkelund, Svend; Jørgensen, PL

    2003-01-01

    Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent...... along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein...... chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly...

  19. Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

    International Nuclear Information System (INIS)

    The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods—(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy—we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process (ΔGUN0) are obtained. The outcomes are compared to the global analysis results published previously. (paper)

  20. Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

    Science.gov (United States)

    Bader, Arjen N.; Visser, Nina V.; van Amerongen, Herbert; Visser, Antonie J. W. G.

    2014-12-01

    The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods—(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy—we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process ≤ft(Δ G\\text{UN}0\\right) are obtained. The outcomes are compared to the global analysis results published previously.

  1. Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions.

    Science.gov (United States)

    Sun, Y S; Landry, J P; Fei, Y Y; Zhu, X D; Luo, J T; Wang, X B; Lam, K S

    2008-12-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k(on) and k(off) for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as K(D) = k(off)/k(on), for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K(D) for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  2. Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.

    Science.gov (United States)

    Lubbeck, Jennifer L; Dean, Kevin M; Ma, Hairong; Palmer, Amy E; Jimenez, Ralph

    2012-05-01

    Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching. PMID:22424298

  3. Solid-state deep blue and UV fluorescent dyes based on para-bis(2-thienyl)phenylene

    Energy Technology Data Exchange (ETDEWEB)

    Krajčovič, Jozef; Kovalenko, Alexander, E-mail: kovalenko.alx@gmail.com; Heinrichová, Patricie; Vala, Martin; Weiter, Martin

    2015-11-15

    Despite the general rule of strong acceptor substituents having a tendency to quench fluorescence due to molecular stacking, it is shown how tetra-fluorination of the central phenylene unit of para-bis(2-thienyl)phenylene can augment the quantum yields of solid state fluorescent dyes. Another significant part of the present research was the study of the influence of the position of the solubilization alkyl chains position on the fluorescent properties of the abovementioned non- and tetra-fluorinated materials. Tenfold augmentation of quantum yields, depending on the position of the alkyl chains is reported. - Highlights: • Solid state luminescence was observed in para-bis(2-thienyl)phenylene molecules. • Quantum yields was augmented by polyfluorination of the central phenylene unit. • Tenfold augmentation of luminescence was observed by changing alkyls position. • Possibilities of steric hindrance and charge transfer were studied.

  4. Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

    Directory of Open Access Journals (Sweden)

    Siepert Eva-Maria

    2012-09-01

    Full Text Available Abstract Background Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP. Results As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv Ki-4(scFv and the anti-MucI single-chain fragment variable M12(scFv. During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal

  5. Preparation of multi-coloured different sized fluorescent gold clusters from blue to NIR, structural analysis of the blue emitting Au7 cluster, and cell-imaging by the NIR gold cluster

    Science.gov (United States)

    Roy, Subhasish; Baral, Abhishek; Bhattacharjee, Rameswar; Jana, Batakrishna; Datta, Ayan; Ghosh, Surajit; Banerjee, Arindam

    2015-01-01

    Blue, green, orange-red, red and NIR emitting gold quantum clusters have been prepared in aqueous media by using a bioactive peptide glutathione (reduced) at physiological pH. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses show that the core structure sizes of the five different gold clusters are Au7 (blue), Au16 (green), Au19 (orange-red), Au21 (red) and Au22 (NIR). The photo-stability and pH-stability of these quantum clusters have been measured, and these are photo-stable against continuous UV irradiation for a few hours. They also exhibit moderate to good pH-stability within the pH range of 5-12.5. A computational study reveals the organisation of gold atoms in the thiolate-protected blue quantum cluster and its several structural parameters, including the mode of interaction of ligand molecules with Au atoms in the Au7 cluster. Interestingly, it has been found that NIR emitting gold quantum cluster can easily be internalized into the adenocarcinomic human alveolar basal epithelial cell line (A549 cell line). Moreover, a MTT assay indicates that our NIR emitting gold quantum cluster show very low cytotoxicy to A549 cancer cells.Blue, green, orange-red, red and NIR emitting gold quantum clusters have been prepared in aqueous media by using a bioactive peptide glutathione (reduced) at physiological pH. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses show that the core structure sizes of the five different gold clusters are Au7 (blue), Au16 (green), Au19 (orange-red), Au21 (red) and Au22 (NIR). The photo-stability and pH-stability of these quantum clusters have been measured, and these are photo-stable against continuous UV irradiation for a few hours. They also exhibit moderate to good pH-stability within the pH range of 5-12.5. A computational study reveals the organisation of gold atoms in the thiolate-protected blue quantum cluster and its several

  6. Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.

    Science.gov (United States)

    Konold, Patrick E; Yoon, Eunjin; Lee, Junghwa; Allen, Samantha L; Chapagain, Prem P; Gerstman, Bernard S; Regmi, Chola K; Piatkevich, Kiryl D; Verkhusha, Vladislav V; Joo, Taiha; Jimenez, Ralph

    2016-08-01

    Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift. PMID:27447848

  7. Serum Protein Profile Study of Clinical Samples Using High Performance Liquid Chromatography-Laser Induced Fluorescence

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya;

    2009-01-01

    The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for...

  8. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    Science.gov (United States)

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  9. Rise-time of FRET-acceptor fluorescence tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Van Mierlo, C.P.M.; Visser, A.J.W.G.; Borst, J.W.

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no correspon

  10. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.;

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...... constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli...

  11. Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio.

    Science.gov (United States)

    Yoshimura, Hideaki; Inaguma, Asumi; Yamada, Toshimichi; Ozawa, Takeaki

    2012-06-15

    Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells. PMID:22387832

  12. Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

    Science.gov (United States)

    Siegel, Amanda P.; Baird, Michelle A.; Davidson, Michael W.; Day, Richard N.

    2013-01-01

    The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy. PMID:24129172

  13. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    Science.gov (United States)

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. PMID:21277981

  14. A fluence response study of lethality and mutagenicity of white, black, and blue fluorescent light, sunlamp, and sunlight irradiation in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Under a set of defined experimental conditions, the fluence response of Chinese hamster ovary (CHO) cells to various light sources was studied by measuring single-cell survival and mutation to 6-thioguanine (TG) resistance. Fluorescent white, black, and blue lights were slightly lethal and mutagenic. Sunlamp light was highly lethal and mutagenic, exhibiting these biological effects within 15 sec of exposure under conditions recommended by the manufacturer for human use. Lethal and mutagenic effects were observed after 5 min of sunlight exposure; responses varied with hourly and daily variations in solar radiation. Sunlight induced TG-resistant variants possessed <5% of parental cellular hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme activity, suggesting that the mutation induction occurs at this locus. The cell survival and mutation-induction curves generated by exposure of cells to both sunlamp and sunlight were similar to those obtained by the use of a standard far-UV lamp

  15. Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

    Science.gov (United States)

    Griffiths, N; Jaipargas, E-A; Wozny, M R; Barton, K A; Mathur, N; Delfosse, K; Mathur, J

    2016-08-01

    Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed. PMID:26820914

  16. Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

    Science.gov (United States)

    Pratt, Evan P S; Owens, Jake L; Hockerman, Gregory H; Hu, Chang-Deng

    2016-01-01

    Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software. PMID:27515079

  17. Time-Resolved Fluorescence Immunoassay for C-Reactive Protein Using Colloidal Semiconducting Nanoparticles

    Directory of Open Access Journals (Sweden)

    Pekka Hänninen

    2011-11-01

    Full Text Available Besides the typical short-lived fluorescence with decay times in the nanosecond range, colloidal II/VI semiconductor nanoparticles dispersed in buffer also possess a long-lived fluorescence component with decay times in the microsecond range. Here, the signal intensity of the long-lived luminescence at microsecond range is shown to increase 1,000-fold for CdTe nanoparticles in PBS buffer. This long-lived fluorescence can be conveniently employed for time-gated fluorescence detection, which allows for improved signal-to-noise ratio and thus the use of low concentrations of nanoparticles. The detection principle is demonstrated with a time-resolved fluorescence immunoassay for the detection of C-reactive protein (CRP using CdSe-ZnS nanoparticles and green light excitation.

  18. Deep blue fluorescent host materials based on a novel spiro[benzo[c]fluorene-7,9'-fluorene] core structure with side aromatic wings

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Inho; Seo, Jeonga; Gong, Myoungseon [Dankook Univ., Chunan (Korea, Republic of)

    2012-07-15

    Deep blue fluorescent host materials based on a novel spiro[benzo[c]fluorene-7,9'-fluorene] core structure with side aromatic wings in the 5- and 9-positions, namely, 5,9-di(naphthalen-2-yl)spiro[benzo[c]fluorene-7,9'-fluorene] (DN-SBFF), 5,9-bis(4-t-butylphenyl)spiro[benzo[c]fluorene-7,9'-fluorene] (BP-SBFF), and 5,9- bis(4-fluorophenyl)spiro[benzo[c]fluorene-7,9'-fluorene] (FP-SBFF), were designed and successfully prepared using the Suzuki reaction. The physical properties of these materials and their EL characteristics as blue host materials doped with N,N,N',N'-tetraphenyl spiro[benzo[c]fluorene-7,9'-fluorene]-5,9-diamine (TPA-SBFF) were investigated. The device used comprised ITO/N,N'-diphenyl-N,N'-bis[4-(phenyl-m-tolyl-amino)phenyl]- biphenyl-4,4'-diamine (DNTPD)/N,N'-di(1-naphthyl)-N,N'-diphenylbenzidine (NPB)/(FP-SBFF):dopant x%/tris(8-hydroxyquinoline)aluminum (Alq{sub 3})/LiF. The device obtained using FP-SBFF doped with TPA-SBFF showed high color purity (0.13, 0.18) and an efficiency of 6.61 cd/A at 7 V.

  19. Isolation and characterisation of 2-Tert-butyl-8-hydroxyquinoline as a crystalline solid and its blue fluorescent Li complex

    OpenAIRE

    Kathirgamanathan, Poopathy; Surendrakumar, Sivagnanasundram; Ravichandran, Seenivasagam; Blake, Alexander J.

    2014-01-01

    2-Tert-butyl-8-hydroxyquinoline (2-TB-8-hq) has been isolated as a crystalline solid and its X-ray structure elucidated, resolving three decades of controversy, since it was previously wrongly reported as yellow oil by some other workers. An improved synthetic method has been developed which increases the yield from 20% to 60%. The lithium complex of 2-TB-8-hq is blue emitting and the HOMO and LUMO levels are lowered by 0.86 eV and 0.74 eV, respectively, compared with the parent lithium 8-hyd...

  20. Isolation and characterisation of 2-Tert-butyl-8-hydroxyquinoline as a crystalline solid and its blue fluorescent Li complex

    OpenAIRE

    Poopathy Kathirgamanathan; Sivagnanasundram Surendrakumar; Seenivasagam Ravichandran; Blake, Alexander J.

    2014-01-01

    2-Tert-butyl-8-hydroxyquinoline (2-TB-8-hq) has been isolated as a crystalline solid and its X-ray structure elucidated, resolving three decades of controversy, since it was previously wrongly reported as yellow oil by some other workers. An improved synthetic method has been developed which increases the yield from 20% to 60%. The lithium complex of 2-TB-8-hq is blue emitting and the HOMO and LUMO levels are lowered by 0.86 eV and 0.74 eV, respectively, compared with the parent lithium 8-hyd...

  1. Isolation and Characterisation of 2-Tert-butyl-8-hydroxyquinoline as a Crystalline Solid and Its Blue Fluorescent Li Complex

    OpenAIRE

    Kathirgamanathan, Poopathy; Surendrakumar, Sivagnanasundram; Ravichandran, Seenivasagam; Blake, Alexander J.

    2014-01-01

    2-Tert-butyl-8-hydroxyquinoline (2-TB-8-hq) has been isolated as a crystalline solid and its X-ray structure elucidated, resolving three decades of controversy, since it was previously wrongly reported as yellow oil by some other workers. An improved synthetic method has been developed which increases the yield from 20% to 60%. The lithium complex of 2-TB-8-hq is blue emitting and the HOMO and LUMO levels are lowered by 0.86 eV and 0.74 eV, respectively, compared with the parent lithium 8-hyd...

  2. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.;

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom crystal structure of the oxidized protein revealed a disulfide bond- induced distortion of the beta -barrel, as well...... as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within...

  3. Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

    Directory of Open Access Journals (Sweden)

    Mann David GJ

    2012-05-01

    Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

  4. Synthesis of dental matrix proteins and viability of odontoblast-like cells irradiated with blue LED.

    Science.gov (United States)

    Alonso, Juliana Rosa Luiz; Turrioni, Ana Paula Silveira; Basso, Fernanda Gonçalves; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-04-01

    To evaluate the effect of irradiation with light-emitting diode (LED; 455 nm) on the viability and synthesis of dentin matrix proteins by odontoblast-like cells, MDPC-23 cells were cultivated (10(4) cells/cm(2)) in 24-well culture plates. After 12 h incubation in Dulbecco's modified Eagle's medium (DMEM), the cells were submitted to nutritional restriction by means of reducing the concentration of fetal bovine serum (FBS) for an additional 12 h. Cells were irradiated one single time with one of the following energy densities (EDs): 0.5, 2, 4, 10, or 15 J/cm(2) and irradiance fixed at 20 mW/cm(2). Non-irradiated cells served as control. After 72 h, cells were evaluated with regard to viability (methylthiazol tetrazolium technique (MTT)), mineralization nodule (MN) formation, total protein (TP) production, alkaline phosphatase activity (ALP), and collagen synthesis (Sircol), n = 8. The data were submitted to Kruskal-Wallis and Mann-Whitney tests (p > 0.05). There was no statistical difference between the viability of cells irradiated or not (control), for all the EDs. However, an increase in TP was observed for all the EDs when compared with the control group. A reduced ALP activity was seen in all irradiated groups, except for the ED of 0.5 J/cm(2), which did not differ from the control. There was no difference between the irradiated groups and control regarding collagen synthesis, with the exception of the ED of 10 J/cm(2), which inhibited this cell function. Significant reduction in MN occurred only for the EDs of 0.5 and 2 J/cm(2). The single irradiation with blue LED (455 nm), irradiance of 20 mW/cm(2), and energy densities ranging from 0.5 to 15 J/cm(2) exerted no effective biostimulatory capacity on odontoblast-like cells. PMID:26873499

  5. Human cytomegaloviruses expressing yellow fluorescent fusion proteins--characterization and use in antiviral screening.

    Directory of Open Access Journals (Sweden)

    Sarah Straschewski

    Full Text Available Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity. We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP fused with the viral proteins IE-2, ppUL32 (pp150, and ppUL83 (pp65. In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI. The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

  6. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

    Science.gov (United States)

    Bird, Louise E; Nettleship, Joanne E; Järvinen, Valtteri; Rada, Heather; Verma, Anil; Owens, Raymond J

    2016-01-01

    The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years. PMID:27553231

  7. How fluorescent labelling alters the solution behaviour of proteins

    OpenAIRE

    Quinn, M. K.; Gnan, N.; James, S; Ninarello, A.; Sciortino, F.; Zaccarelli, E.; McManus, J. J.

    2015-01-01

    A complete understanding of the role of molecular anisotropy in directing the self assembly of colloids and proteins remains a challenge for soft matter science and biophysics. For proteins in particular, the complexity of the surface at a molecular level poses a challenge for any theoretical and numerical description. A soft matter approach, based on patchy models, has been useful in describing protein phase behaviour. In this work we examine how chemical modification of the prot...

  8. Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat.

    Science.gov (United States)

    Schuster, M; Kilaru, S; Guo, M; Sommerauer, M; Lin, C; Steinberg, G

    2015-06-01

    The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent "cousins" show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta. Our data reveal that mCherry is the RFP of choice for investigation in Z. tritici, showing highest signal intensity in epi-fluorescence, when used with a Cy3 filter set, and laser scanning confocal microscopy. However, mCherry bleached significantly faster than mRFP, which favors this red tag in long-term observation experiments. Finally, we used dual-color imaging of eGFP and mCherry expressing wild-type strains in planta and show that pycnidia are formed by single strains. This demonstrates the strength of this method in tracking the course of Z. tritici infection in wheat. PMID:26092800

  9. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    OpenAIRE

    Jia-Xing Yue; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-01-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluo...

  10. Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

    OpenAIRE

    Leveau, Johan H. J.; Lindow, Steven E.

    2001-01-01

    We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual me...

  11. A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

    Science.gov (United States)

    Tiwari, Dhermendra K; Arai, Yoshiyuki; Yamanaka, Masahito; Matsuda, Tomoki; Agetsuma, Masakazu; Nakano, Masahiro; Fujita, Katsumasa; Nagai, Takeharu

    2015-06-01

    Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy. PMID:25894946

  12. Fluorescent properties of a blue-to green-emitting Ce3+, Tb3+ codoped amorphous calcium silicate phosphors

    International Nuclear Information System (INIS)

    Ce3+, Tb3+ codoped amorphous calcium silicate phosphor was prepared by heating (830 °C for 30 min) Ce3+, Tb3+ codoped calcium silicate hydrate phosphor formed by liquid-phase reaction. The excitation peak wavelength of the resulting phosphor was 330 nm and the emission peak wavelengths were at 544 nm, attributed to the 5D4→7F5 transition of Tb3+, and at 430–470 mm, attributed to Ce3+. The intensity ratio of the two peaks could be freely controlled by varying the Tb/Ca atomic ratio of the Ce3+, Tb3+ codoped amorphous calcium silicate phosphor, allowing light to be emitted over a wide range from blue to green. It was clarified that energy transfer exists from Ce3+ to Tb3+. - Graphical abstract: Ce3+, Tb3+ codoped calcium silicate hydrate phosphor was synthesized by liquid-phase reaction. This was heated at 830 °C to obtain a Ce3+, Tb3+ codoped amorphous calcium silicate phosphor. Under 330 nm excitation, this phosphor showed emission peaks at 430–470 nm and 542 nm. The luminescent color could be continuously changed blue to green with increasing Tb/Ca atomic ratio. It was clarified that electron transfer from Ce3+ to Tb3+ is occurring.

  13. Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes

    OpenAIRE

    Moon, C. Preston; Fleming, Karen G.

    2011-01-01

    Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these so...

  14. Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

    OpenAIRE

    Liu, Jing

    2005-01-01

    The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall po...

  15. Dual-color fluorescence-burst analysis to study pore formation and protein-protein interactions

    NARCIS (Netherlands)

    van den Bogaart, Geert; Kusters, Ilja; Velasquez, Jeanette; Mika, Jacek T.; Krasnikov, Victor; Driessen, Arnold J. M.; Poolman, Bert

    2008-01-01

    Dual-color fluorescence-burst analysis (DCBFA) enables to study leakage of fluorescently labeled (macro)molecules from liposomes that are labeled with a second, spectrally non-overlapping fluorophore. The fluorescent bursts that reside from the liposomes diffusing through the focal volume of a confo

  16. Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals.

    Science.gov (United States)

    Gilmore, Adam M; Larkum, Anthony W D; Salih, Anya; Itoh, Shigeru; Shibata, Yutaka; Bena, Chiaki; Yamasaki, Hideo; Papina, Marina; Van Woesik, Robert

    2003-05-01

    Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef-building coral tissue. We used a streak-camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (corals showed rapidly decaying species and reciprocal rises in greener emission components indicating Förster resonance energy transfer (FRET) between FP populations. The energy transfer modes were around 250 ps, and the main decay modes of the acceptor FP were typically 1900-2800 ps. All zooxanthellae emitted similar spectra and kinetics with peak emission (approximately 683 nm) mainly from photosystem II (PSII) chlorophyll (chl) a. Compared with the FP, the PSII emission exhibited similar rise times but much faster decay times, typically around 640-760 ps. The fluorescence kinetics and excitation versus emission mapping indicated that the FP emission played only a minor role, if any, in chl excitation. We thus suggest the FP could only indirectly act to absorb, screen and scatter light to protect PSII and underlying and surrounding animal tissue from excess visible and UV light. We conclude that our time-resolved spectral analysis and simulation revealed new FP emission components that would not be easily resolved at steady state because of their relatively rapid decays due to efficient FRET. We believe the methods show promise for future studies of coral bleaching and for potentially identifying FP species for use as genetic markers and FRET partners, like the related green FP from Aequorea spp. PMID:12812294

  17. Fluorescent Nanodiamonds with Bioorthogonally Reactive Protein-Resistant Polymeric Coatings

    Czech Academy of Sciences Publication Activity Database

    Řehoř, Ivan; Macková, Hana; Filippov, Sergey K.; Kučka, Jan; Proks, Vladimír; Šlegerová, Jitka; Turner, S.; van Tendeloo, G.; Ledvina, Miroslav; Hrubý, Martin; Cígler, Petr

    2014-01-01

    Roč. 79, č. 1 (2014), s. 21-24. ISSN 2192-6506 R&D Projects: GA ČR GAP108/12/0640; GA MŠk(CZ) LH11027 Grant ostatní: OPPK(CZ) CZ.2.16/3.1.00/24016; Seventh Framework Programme of the European Union(XE) FP7-262348; European Research Council(XE) FP7-246791 Institutional support: RVO:61388963 ; RVO:61389013 ; RVO:61389005 Keywords : click chemistry * fluorescence * nanoparticles * polymerization * solubilization Subject RIV: CC - Organic Chemistry ; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 2.997, year: 2014

  18. Photoacoustic imaging of the near-infrared fluorescent protein iRFP in vivo

    Science.gov (United States)

    Krumholz, Arie; Filonov, Grigory S.; Xia, Jun; Yao, Junjie; Verkhusha, Vladislav V.; Wang, Lihong V.

    2012-02-01

    Genetically encoded probes powerfully and non-invasively target specific tissues, cells, and subcellular locations. iRFP, a novel near-infrared fluorescent protein with low quantum yield whose absorption and fluorescence maxima are located at wavelengths longer than the Q-band of hemoglobin absorption, is ideal for PAT. Here, we report on an in vitro comparison of iRFP with other far-red fluorescent proteins, and its use in imaging a mouse tumor xenograft model. In an in vivo experiment, we stably transfected iRFP into MTLn3 adenocarcinoma cells and injected them into the mammary fat pad of female SCID/NCr mice, then imaged the resulting tumors two and three weeks post injection. The contrast increase from the protein expression was high enough to clearly separate the tumor region from the rest of the animal.

  19. A Diabatic Three-State Representation of Photoisomerization in the Green Fluorescent Protein Chromophore

    CERN Document Server

    Olsen, Seth

    2009-01-01

    We give a quantum chemical description of bridge photoisomerization reaction of green fluorescent protein (GFP) chromophores using a representation over three diabatic states. Bridge photoisomerization leads to non-radiative decay, and competes with fluorescence in these systems. In the protein, this pathway is suppressed, leading to fluorescence. Understanding the electronic structure of the photoisomerization is a prerequisite to understanding how the protein suppresses this pathway and preserves the emitting state of the chromophore. We present a solution to the state-averaged complete active space problem, which is spanned at convergence by three fragment-localized orbitals. We generate the diabatic-state representation by applying a block diagonalization transformation to the Hamiltonian calculated for the anionic chromophore model HBDI with multi-reference, multi-state perturbation theory. The diabatic states that emerge are charge-localized structures with a natural valence-bond interpretation. At plan...

  20. Improving the sensitivity of protein microarray by evanescent-field-induced fluorescence

    Institute of Scientific and Technical Information of China (English)

    WANG Li-qiang; LU Zu-kang

    2005-01-01

    To improve the sensitivity of protein microarray, a prism surface replaces the surface of the common microscope slide.The protein targets arrayed on the surface are hybridized and labelled by fluorescent probes. Evanescent excitation occurs when the convergent laser reaches the surface, and a photomultiplier tube detects the emitted fluorescent signal. A two-dimensional actuator scans the whole surface to achieve planar laser excitation and fluorescence collection. The penetration depth of the evanescent field into the protein targets is only some hundred nanometers and can be controlled by different incident angle of the laser beam, so the undesired background signals are reduced dramatically and the detection sensitivity is improved by a factor of 50 to 100 comparing to confocal excitation. This approach can detect low abundance analytes without signal amplification.

  1. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

    Science.gov (United States)

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

    2016-04-01

    A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants. PMID:27041337

  2. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with...... Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain...... Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals...

  3. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    Science.gov (United States)

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  4. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    Directory of Open Access Journals (Sweden)

    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  5. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

    Directory of Open Access Journals (Sweden)

    Shingo Sotoma

    2016-03-01

    Full Text Available The impeccable photostability of fluorescent nanodiamonds (FNDs is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

  6. Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers.

    OpenAIRE

    Nag, K.; Perez-Gil, J.; Cruz, A; Keough, K M

    1996-01-01

    Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescen...

  7. The structure of the chromophore within DsRed, a red fluorescent protein from coral

    OpenAIRE

    Gross, Larry A.; Baird, Geoffrey S.; Hoffman, Ross C.; Baldridge, Kim K; Tsien, Roger Y.

    2000-01-01

    DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolut...

  8. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

    Science.gov (United States)

    Chu, Jun; Oh, Younghee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J; Laviv, Tal; Welf, Erik S; Dean, Kevin M; Zhang, Feijie; Kim, Benjamin B; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A; Davidson, Michael W; Kay, Mark A; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S; Ng, Ho-Leung; Lin, Michael Z

    2016-07-01

    Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

  9. DNA binding fluorescent proteins for the direct visualization of large DNA molecules.

    Science.gov (United States)

    Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon; Choe, Sojeong; Lim, Sangyong; Lee, Hyun Soo; Jo, Kyubong; Schwartz, David C

    2016-01-01

    Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely 'paints' entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells. PMID:26264666

  10. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Directory of Open Access Journals (Sweden)

    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  11. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples.

    Directory of Open Access Journals (Sweden)

    Joachim Goedhart

    Full Text Available BACKGROUND: Fluorescence Resonance Energy Transfer (FRET between the green fluorescent protein (GFP variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells.

  12. Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

    Science.gov (United States)

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-01-01

    Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB. PMID:24145242

  13. Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

    Science.gov (United States)

    Rolinski, Olaf J.; McLaughlin, Damien; Birch, David J. S.; Vyshemirsky, Vladislav

    2016-06-01

    The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496–502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

  14. Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

    Science.gov (United States)

    Rolinski, Olaf J.; McLaughlin, Damien; Birch, David J. S.; Vyshemirsky, Vladislav

    2016-09-01

    The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496–502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

  15. β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

    Science.gov (United States)

    Bubnell, Jaclyn; Pfister, Patrick; Sapar, Maria L.; Rogers, Matthew E.; Feinstein, Paul

    2013-01-01

    Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality. PMID:24086401

  16. Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    By using silkworms,Bombyx mori, fluorescent cocoon sex identification (FCSI) as an experimental material, direct fluorescence spectrometry of the cocoon surface indicates that the fluorescent color of silkworm cocoons is made up of two peaks of yellow and blue-purple fluorescence emission. The fluorescent difference between male and female cocoons is attributed to the differential absorption of yellow fluorescent substances by the midgut tissue of 5th instar female silkworms. Thin layer chromatography (TLC) and fluorescent spectra indicate that blue-purple fluorescent substances are composed of at least five blue-purple fluorescent pigments, and yellow fluorescent substances are made up of at least three. UV spectra and AlCl3 color reaction show that the three fluorescent yellow pigments are flavonoids or their glycosides. Silkworm FCSI is due to selective absorption or accumulation of the yellow fluorescent pigments by the posterior midgut cells of female 5th instar larvae. The cells of the FCSI silkworm midgut, especially the cylinder intestinal cells of the posterior midgut have a component which is a yellow fluorescent pigment-specific binding protein that may be vigorously expressed in the 5th instar larvae.

  17. Investigation on Molecular Non-covalent Interaction in the Sodium Dodecyl Benzene Sulfonatepolychrome Blue B-protein Replacement Reaction

    Institute of Scientific and Technical Information of China (English)

    GAO,Hong-Wen(郜洪文); WU,Ji-Rong(邬继荣); SHEN,Rong(沈荣)

    2004-01-01

    The molecular non-covalent interaction often originates from the electrostatic attraction and accords with the Langmuir isothermal adsorption. The sodium dodecyl benzene sulfonate (SDBS)-polychrome blue B (PCB)-protein [bovine serum albumin (BSA), ovalbumin (OVA) and myoglobin (MB)] ternary reaction has been investigated at Ph 3.88. Protein to replace PCB from the PCB-SDBS binding product was used to characterize the assembly of an invisible-spectral compound, SDBS, on proteins by measuring the variation of PCB light-absorption by the microsurface adsorption-spectral correction (MSASC) technique. The effect of ionic strength and temperature on the aggregation was studied. Results showed that the aggregates SDBS92·BSA, SDBS58·OVA and SDBS15·MB at 30 ℃ and SDBS83·BSA, SDBS39·OVA and SDBS10·MB at 50 ℃ are formed.

  18. Accessing gelling ability of vegetable proteins using rheological and fluorescence techniques.

    Science.gov (United States)

    Batista, Ana Paula; Portugal, Carla A M; Sousa, Isabel; Crespo, João G; Raymundo, Anabela

    2005-08-01

    This work aims to present a comprehensive study about the macroscopic characteristics of globular vegetable proteins, in terms of their gelling ability, by understanding their molecular behaviour, when submitted to a thermal gelling process. The gels of soy, pea and lupin proteins were characterized by rheological techniques. Gelation kinetics, mechanical spectra, as well as the texture of these gels were analyzed and compared. Additionally, capillary viscometry, steady-state fluorescence and fluorescence anisotropy were used to monitor the structural changes induced by the thermal denaturation, which constitutes the main condition for the formation of a gel structure. Based on these techniques it was possible to establish a relationship between the gelling ability of each protein isolate and their structural resistance to thermal unfolding, enabling us to explain the weakest and the strongest gelling ability observed for lupin and soy proteins isolates, respectively. PMID:15996729

  19. Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis

    Directory of Open Access Journals (Sweden)

    Lin Qiu

    2014-01-01

    Full Text Available In this report, fluorescence detection coupled capillary electrophoresis (CE-FL was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

  20. Secretion of human interleukin-2 fused with green fluorescent protein in recombinant Pichia pastoris.

    Science.gov (United States)

    Cha, Hyung Joon; Dalal, Nimish N; Bentley, William E

    2005-07-01

    Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. PMID:16014994

  1. Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging

    Science.gov (United States)

    Yamanaka, Masahito; Saito, Kenta; Smith, Nicholas I.; Arai, Yoshiyuki; Uegaki, Kumiko; Yonemaru, Yasuo; Mochizuki, Kentaro; Kawata, Satoshi; Nagai, Takeharu; Fujita, Katsumasa

    2015-10-01

    The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.

  2. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    International Nuclear Information System (INIS)

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm2. FEL irradiation at a wavelength of 5.75 and 6.1 μm, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 μm. The maximum transfer efficiency was about 0.5%

  3. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    CERN Document Server

    Awazu, K; Tamiya, E

    2002-01-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 mu m. The maximum transfer efficiency was about 0.5%.

  4. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    Science.gov (United States)

    Awazu, Kunio; Kinpara, Takeshi; Tamiya, Eiichi

    2002-05-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm 2. FEL irradiation at a wavelength of 5.75 and 6.1 μm, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 μm. The maximum transfer efficiency was about 0.5%.

  5. Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

    Directory of Open Access Journals (Sweden)

    Amar B. T. Ghisaidoobe

    2014-12-01

    Full Text Available F resonance energy transfer (FRET occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (\\(\\uplambda_{\\textsc{ex}}\\sim\\ nm, \\(\\uplambda_{\\textsc{em}}\\sim\\ 350 nm, in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the proteinlocal environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic F resonance energy transfer (iFRET, a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins.

  6. Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

    Directory of Open Access Journals (Sweden)

    Kiyotaka Sakai

    2009-10-01

    Full Text Available Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A caused by the binding with immunoglobulin G (IgG in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 μg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

  7. Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2010-08-17

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  8. Femtosecond lasing from a fluorescent protein in a one dimensional random cavity

    CERN Document Server

    Drane, T M; Shapiro, M; Milner, V

    2015-01-01

    We present evidence of ultrafast random lasing from the fluorescent protein DsRed2 embedded in a random one-dimensional cavity. Lasing is achieved when a purified protein solution, placed inside a layered random medium, is optically excited with a femtosecond pump pulse in the direction perpendicular to the plane of random layers. We demonstrate that pumping with ultrashort pulses resulted in a lasing threshold two orders of magnitude lower than that found for nanosecond excitation.

  9. Site-Specific Analysis of Protein Hydration Based on Unnatural Amino Acid Fluorescence

    Czech Academy of Sciences Publication Activity Database

    Amaro, Mariana; Brezovský, J.; Kováčová, S.; Sýkora, Jan; Bednář, D.; Němec, V.; Lišková, V.; Kurumbang, N. P.; Beerens, K.; Chaloupková, R.; Paruch, K.; Hof, Martin; Damborský, J.

    2015-01-01

    Roč. 137, č. 15 (2015), s. 4988-4992. ISSN 0002-7863 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : analysis * fluorescence * hydration of proteins Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 12.113, year: 2014

  10. Study of the glucoamylase promoter in Aspergillus niger using green fluorescent protein

    NARCIS (Netherlands)

    Santerre Henriksen, A.L.; Even, S.; Müller, C.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Nielsen, J.

    1999-01-01

    An Aspergillus niger strain expressing a red-shifted green fluorescent protein (GFP) in the cytoplasm under the control of the glucoamylase promoter (PglaA) was characterized with respect to its physiology and morphology. Although xylose acted as a repressor carbon source during batch cultivations,

  11. Ab initio study of the optical properties of green fluorescent protein

    NARCIS (Netherlands)

    Zaccheddu, Maurizio

    2008-01-01

    In the present we focus on the optical properties of the Green Fluorescent Protein (GFP), which are modelled using the state-of-the-art computational tools availeable up to date: the Density Functional Theory (DFT) in the Hybrid QM/MM approach is employed to access the ground state configuration of

  12. A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici☆

    Science.gov (United States)

    Kilaru, S.; Schuster, M.; Studholme, D.; Soanes, D.; Lin, C.; Talbot, N.J.; Steinberg, G.

    2015-01-01

    Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue. PMID:26092799

  13. A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.

    Science.gov (United States)

    Kilaru, S; Schuster, M; Studholme, D; Soanes, D; Lin, C; Talbot, N J; Steinberg, G

    2015-06-01

    Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue. PMID:26092799

  14. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    Science.gov (United States)

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  15. Membrane Binding of Peripheral Proteins and its Characterisation Applying Fluorescence Methods

    Czech Academy of Sciences Publication Activity Database

    Hutterer, R.; Hof, Martin

    Trivandrum : Transworld Research Network, 2002 - (Pandalai, S.), s. 277-288 ISBN 81-7895-063-4 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : membrane binding * proteins * fluorescence Subject RIV: CF - Physical ; Theoretical Chemistry

  16. Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.

    Science.gov (United States)

    Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S; Smorodinsky, Nechama I; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

    2011-10-01

    We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection. PMID:21795382

  17. Structural Determinants of Improved Fluorescence in a Family of Bacteriophytochrome-Based Infrared Fluorescent Proteins: Insights from Continuum Electrostatic Calculations and Molecular Dynamics Simulations.

    Science.gov (United States)

    Feliks, Mikolaj; Lafaye, Céline; Shu, Xiaokun; Royant, Antoine; Field, Martin

    2016-08-01

    Using X-ray crystallography, continuum electrostatic calculations, and molecular dynamics simulations, we have studied the structure, protonation behavior, and dynamics of the biliverdin chromophore and its molecular environment in a series of genetically engineered infrared fluorescent proteins (IFPs) based on the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. Our study suggests that the experimentally observed enhancement of fluorescent properties results from the improved rigidity and planarity of the biliverdin chromophore, in particular of the first two pyrrole rings neighboring the covalent linkage to the protein. We propose that the increases in the levels of both motion and bending of the chromophore out of planarity favor the decrease in fluorescence. The chromophore-binding pocket in some of the studied proteins, in particular the weakly fluorescent parent protein, is shown to be readily accessible to water molecules from the solvent. These waters entering the chromophore region form hydrogen bond networks that affect the otherwise planar conformation of the first three rings of the chromophore. On the basis of our simulations, the enhancement of fluorescence in IFPs can be achieved either by reducing the mobility of water molecules in the vicinity of the chromophore or by limiting the interactions of the nearby protein residues with the chromophore. Finally, simulations performed at both low and neutral pH values highlight differences in the dynamics of the chromophore and shed light on the mechanism of fluorescence loss at low pH. PMID:27471775

  18. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    International Nuclear Information System (INIS)

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  19. Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato

    International Nuclear Information System (INIS)

    Polyclonal antibodies against pea phytochrome detect 2 protein bands (about 116 and 120 kDa) on blots of crude protein extracts and protein of microsomal preparations of dark-grown tomato seedlings. Both protein bands are undetectable in Western blots of the aurea mutant extracts. Neither protein band is detectable after isogenic wild-type seedlings are illuminated with 3 h of red light, either in the crude extract or in the membrane fraction of the irradiated seedlings; this result is consistent with the hypothesis that both bands are phytochrome. When dark-grown wild-type seedlings are illuminated with 3 h of red light or blue light against a red light background, the transcript levels for chlorophyll a/b-binding proteins of photosystem I and II, plastocyanin, and the subunit II of photosystem I increase. In all cases, the same fluence rate of blue light is much more effective than red light alone, a result that indicates the involvement of a blue/UV-A light photoreceptor in addition to the involvement of the far-red-absorbing form of phytochrome, Pfr. The aurea mutant responds neither to red light nor to blue light. Thus, no Pfr-independent induction of the four transcripts by a blue/UV-A light photoreceptor can be measured in the aurea mutant

  20. Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

    Science.gov (United States)

    Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

    2014-04-01

    At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)

  1. Upconversion fluorescence metal-organic frameworks thermo-sensitive imprinted polymer for enrichment and sensing protein.

    Science.gov (United States)

    Guo, Ting; Deng, Qiliang; Fang, Guozhen; Gu, Dahai; Yang, Yukun; Wang, Shuo

    2016-05-15

    A novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein was successfully prepared by combining molecular imprinting technology with upconversion nanoparticles (UCNPs) and metal-organic frameworks (MOFs). Herein, the UCNPs acted as signal reporter for composite materials because of its excellent fluorescence property and chemical stability. MOFs were introduced to molecularly imprinted polymer (MIP) due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive imprinted material which allows for swelling and shrinking with response to temperature changes was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the temperature-sensitive functional monomer and N,N-methylenebisacrylamide (MBA) as the cross-linker. The recognition characterizations of imprinted material-coated UCNPs/MOFs (UCNPs/MOFs/MIP) were evaluated, and the results showed that the fluorescence intensity of UCNPs/MOFs/MIP reduced gradually with the increase of BHB concentration. The fluorescence material was response to the temperature. The adsorption capacity was as much as 167.6 mg/g at 28°C and 101.2mg/g at 44°C, which was higher than that of traditional MIP. Therefore, this new fluorescence material for enrichment and sensing protein is very promising for future applications. PMID:26722764

  2. Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection.

    Science.gov (United States)

    Kaneta, Takashi; Yamamoto, Daisuke; Imasaka, Totaro

    2009-11-01

    The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection. PMID:19862753

  3. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    Science.gov (United States)

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

  4. Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals

    International Nuclear Information System (INIS)

    The effectiveness of using ultraviolet microscopes to illuminate protein crystals in high throughput screens is evaluated. The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with ∼84% of their surface area buried) and to be surrounded by partially polar microenvironments (with ∼43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third (∼18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed

  5. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing

    Science.gov (United States)

    Shi, Siyuan; Thomas, Abu; Corzo, Neil V.; Kumar, Prem; Huang, Yuping; Lee, Kim Fook

    2016-01-01

    Recent studies in quantum biology suggest that quantum mechanics help us to explore quantum processes in biological system. Here, we demonstrate generation of photon pairs through spontaneous four-wave mixing process in naturally occurring fluorescent proteins. We develop a general empirical method for analyzing the relative strength of nonlinear optical interaction processes in five different organic fluorophores. Our results indicate that the generation of photon pairs in green fluorescent proteins is subject to less background noises than in other fluorophores, leading to a coincidence-to-accidental ratio ~145. As such proteins can be genetically engineered and fused to many biological cells, our experiment enables a new platform for quantum information processing in a biological environment such as biomimetic quantum networks and quantum sensors. PMID:27076032

  6. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing

    Science.gov (United States)

    Shi, Siyuan; Thomas, Abu; Corzo, Neil V.; Kumar, Prem; Huang, Yuping; Lee, Kim Fook

    2016-04-01

    Recent studies in quantum biology suggest that quantum mechanics help us to explore quantum processes in biological system. Here, we demonstrate generation of photon pairs through spontaneous four-wave mixing process in naturally occurring fluorescent proteins. We develop a general empirical method for analyzing the relative strength of nonlinear optical interaction processes in five different organic fluorophores. Our results indicate that the generation of photon pairs in green fluorescent proteins is subject to less background noises than in other fluorophores, leading to a coincidence-to-accidental ratio ~145. As such proteins can be genetically engineered and fused to many biological cells, our experiment enables a new platform for quantum information processing in a biological environment such as biomimetic quantum networks and quantum sensors.

  7. Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

    DEFF Research Database (Denmark)

    Spetzler, J C; Westphal, V; Winther, Jakob R.; Meldal, M

    1998-01-01

    Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz...... group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide...

  8. Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

    Directory of Open Access Journals (Sweden)

    Juan A. González-Vera

    2015-11-01

    Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

  9. Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential

    Directory of Open Access Journals (Sweden)

    Thomas Knopfel

    2009-06-01

    Full Text Available Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs, referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.

  10. Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.

    Science.gov (United States)

    Colletier, Jacques-Philippe; Sliwa, Michel; Gallat, François-Xavier; Sugahara, Michihiro; Guillon, Virginia; Schirò, Giorgio; Coquelle, Nicolas; Woodhouse, Joyce; Roux, Laure; Gotthard, Guillaume; Royant, Antoine; Uriarte, Lucas Martinez; Ruckebusch, Cyril; Joti, Yasumasa; Byrdin, Martin; Mizohata, Eiichi; Nango, Eriko; Tanaka, Tomoyuki; Tono, Kensuke; Yabashi, Makina; Adam, Virgile; Cammarata, Marco; Schlichting, Ilme; Bourgeois, Dominique; Weik, Martin

    2016-03-01

    Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins. PMID:26866390

  11. New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Alexandra A Kuznetsova

    Full Text Available Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu, pyrrolocytosine (Cpy and 1,3-diaza-2-oxophenoxazine (tCO. For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU, which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

  12. Excitation energy migration in yellow fluorescent protein (citrine) layers adsorbed on modified gold surfaces

    Science.gov (United States)

    Yusoff, Hanis Mohd; Rzeźnicka, Izabela I.; Hoshi, Hirotaka; Kajimoto, Shinji; Horimoto, Noriko Nishizawa; Sogawa, Kazuhiro; Fukumura, Hiroshi

    2013-09-01

    The nature of functional proteins adsorbed on solid surfaces is interesting from the perspective of developing of bioelectronics and biomaterials. Here we present evidence that citrine (one of yellow fluorescent protein variants) adsorbed on modified gold surfaces would not undergo denaturation and energy transfer among the adsorbed citrine molecules would occur. Gold substrates were chemically modified with 3-mercaptopropionic acid and tert-butyl mercaptan for the preparation of hydrophilic and hydrophobic surfaces, respectively. A pure solution of citrine was dropped and dried on the modified gold substrates and their surface morphology was studied with scanning tunnelling microscopy (STM). The obtained STM images showed multilayers of citrine adsorbed on the modified surfaces. On hydrophobic surfaces, citrine was adsorbed more randomly, formed various non-uniform aggregates, while on hydrophilic surfaces, citrine appeared more aligned and isolated uniform protein clusters were observed. Fluorescence lifetime and anisotropy decay of these dried citrine layers were also measured using the time correlated single photon counting method. Fluorescence anisotropy of citrine on the hydrophobic surface decayed faster than citrine on the hydrophilic surface. From these results we concluded that fluorescence energy migration occurred faster among citrine molecules which were randomly adsorbed on the hydrophobic surface to compare with the hydrophilic surface.

  13. New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

    Science.gov (United States)

    Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Vorobjev, Yuri N; Barthes, Nicolas P F; Michel, Benoît Y; Burger, Alain; Fedorova, Olga S

    2014-01-01

    Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified. PMID:24925085

  14. Fluorescence properties of organic dyes: quantum chemical studies on the green/blue neutral and protonated DMA-DPH emitters in polymer matrices.

    Science.gov (United States)

    Kerkines, Ioannis S K; Petsalakis, Ioannis D; Argitis, Panagiotis; Theodorakopoulos, Giannoula

    2011-12-28

    The absorption and fluorescence spectra of the green emitter DMA-DPH {1-[4-(dimethylamino)phenyl]-6-phenylhexa-1,3,5-triene} and its protonated blue-emitter form have been studied theoretically through time-dependent density functional theory (TD-DFT) and resolution-of-identity 2nd order perturbative coupled cluster (RI-CC2) calculations with basis sets up to augmented triple-ζ quality, in the gas phase and in solvents of different polarity. These systems dispersed in a polymer matrix are of interest for applications in organic light emitting diode devices (OLEDs). Calculations show that the observed absorption and emission spectra correspond to transitions between the S(0) and S(1) states, in both systems. The nature and characteristics of these transitions are discussed. Excellent agreement with experimental data is obtained, both for absorption and emission, provided that the state-specific polarized continuum model (SS-PCM) method is employed for the inclusion of the solvent. PMID:22025129

  15. Application of green fluorescent protein for monitoring phenol-degrading strains

    Directory of Open Access Journals (Sweden)

    Ana Milena Valderrama F.

    2011-12-01

    Full Text Available Several methods have been developed for detecting microorganisms in environmental samples. Some systems for incorporating reporter genes, such as lux or the green fluorescent protein (GFP gene, have been developed recently This study describes gfp gene marking of a phenol degrading strain, its evaluation and monitoring in a bioreactor containing refinery sour water. Tagged strains were obtained having the same physiological and metabolic characteristics as the parent strain. Fluorescent expression was kept stable with no selection for more than 50 consecutive generations and tagged strains were recovered from the bioreactor after forty-five days of phenol-degradation treatment. 

  16. Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

    OpenAIRE

    Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S.; Smorodinsky, Nechama I.; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

    2011-01-01

    We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibitio...

  17. Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses[OPEN

    Science.gov (United States)

    Kudla, Jörg

    2016-01-01

    Techniques to detect and verify interactions between proteins in vivo have become invaluable tools in functional genomic research. While many of the initially developed interaction assays (e.g., yeast two-hybrid system and split-ubiquitin assay) usually are conducted in heterologous systems, assays relying on bimolecular fluorescence complementation (BiFC; also referred to as split-YFP assays) are applicable to the analysis of protein-protein interactions in most native systems, including plant cells. Like all protein-protein interaction assays, BiFC can produce false positive and false negative results. The purpose of this commentary is to (1) highlight shortcomings of and potential pitfalls in BiFC assays, (2) provide guidelines for avoiding artifactual interactions, and (3) suggest suitable approaches to scrutinize potential interactions and validate them by independent methods. PMID:27099259

  18. Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

    International Nuclear Information System (INIS)

    Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections

  19. Effects of L1-ORF2 fragments on green fluorescent protein gene expression

    OpenAIRE

    Xiu-Fang Wang; Xia Jin; Xiaoyan Wang; Jing Liu; Jingjing Feng; QinQing Yang; Wenli Mu; Xiaojuan Shi; Zhanjun Lu

    2009-01-01

    The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vect...

  20. Silicon biochips for dual label-free and fluorescence detection: Application to protein microarray development

    OpenAIRE

    Cretich M.; Reddington A.; Monroe M.; Bagnati M.; Damin F.; Sola L.; Unlu M.S.; Chiari M.

    2011-01-01

    A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500 nm SiO2 layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100 nm SiO2 providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fl...

  1. Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome.

    Directory of Open Access Journals (Sweden)

    Ling Huang

    Full Text Available Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2, a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1, while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.

  2. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Day, Richard N

    2014-03-15

    The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster resonance energy transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. PMID:23806643

  3. Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.

    Science.gov (United States)

    Kaufmann, Rainer; Schellenberger, Pascale; Seiradake, Elena; Dobbie, Ian M; Jones, E Yvonne; Davis, Ilan; Hagen, Christoph; Grünewald, Kay

    2014-07-01

    We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement. PMID:24884378

  4. Production of transgenic mice carrying green fluorescence protein gene by a lentiviral vector-mediated approach

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jingzhi; GUO Xinbing; XIE Shuyang; ZHU Yiwen; HUANG Ying; WANG Shu; REN Zhaorui

    2006-01-01

    A pseudo-lentivirus, which carries green fluorescence protein (GFP) expressing cassette, was injected into the perivitelline space of murine fertilized oocytes before transplanting into the oviducts of the foster mothers. The GFP transgenic pups were then obtained. By PCR amplification, fluorescent microscopy and flow assisted cytometry sorting analysis, we found that the integration rate of the transgene was estimated at above 40%. Real-time PCR analysis indicated that the copy number of the integrated GFP cassette was around 40. Fluorescent in situ hybridization analysis demonstrated that the integration pattern was random but inheritable. The transgenic mice with multi-integration sites and various expression levels possessed a great value in practice as well as research. The approach reported herein provides an efficient way to generate and screen the transgenic mouse strains.

  5. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    Science.gov (United States)

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-01

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates. PMID:25517425

  6. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    International Nuclear Information System (INIS)

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λemmax ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP

  7. Use of liposomal amplifiers in total internal reflection fluorescence fiber-optic biosensors for protein detection.

    Science.gov (United States)

    Chang, Ying-Feng; Fu, Chen; Chen, Yi-Ting; Fang-Ju Jou, Amily; Chen, Chii-Chang; Chou, Chien; Annie Ho, Ja-An

    2016-03-15

    Evanescent-wave excited fluorescence technology has been demonstrated to enhance sensitivity and reduce matrix effects, making it suitable for biosensor development. In this study, we developed a liposome-based, total internal reflection fluorescence, fiber-optic biosensor (TIRF-FOB) for protein detection, which integrates a liposomal amplifier and sandwich immunoassay format with TIRF-FOB. In addition, the antibody-tagged and fluorophore-entrapped liposomes for heterogeneous detection of target molecules were designed and synthesized. This biosensor successfully detected the target protein (model analyzed here is IgG) with a limit of detection (LOD) of 2.0 attomoles for the target protein (equivalent to 2.0 pg/mL of protein presented in 150 μL of sample solution). The features of this ultra-sensitive liposomal TIRF-FOB are (i) fluorescence is excited via evanescent waves and amplified via liposomes; (ii) the use of two polyclonal antibodies in the sandwich assay format increases the specificity and lowers the cost of our assay. Based on the exceptional detection sensitivity and cost-effectiveness, we believe that the proposed biosensor has great potential as a practical, clinical diagnostic tool in the near future. PMID:26595485

  8. Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.

    Science.gov (United States)

    Krahn, Katy Nash; Bouten, Carlijn V C; van Tuijl, Sjoerd; van Zandvoort, Marc A M J; Merkx, Maarten

    2006-03-15

    Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods. PMID:16476406

  9. Fluorescence studies with malate dehydrogenase from rhizobium japonicum 3I1B-143 bacteroids: a two-tryptophan containing protein

    Science.gov (United States)

    Ghiron, Camillo A.; Eftink, Maurice R.; Waters, James K.; Emerich, David W.

    1990-05-01

    A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine MDH. The large number of trp residues (6) complicates the interpretation of some studies. To circumvent this we have performed studies with a two tryptophan (per subunit) MDH from Rhizobium japonicum 311B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.3 ns (blue) and 6.6 ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both tip residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malonate.

  10. Circularly permuted monomeric red fluorescent proteins with new termini in the beta-sheet.

    Science.gov (United States)

    Carlson, Haley J; Cotton, Darrel W; Campbell, Robert E

    2010-08-01

    Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca(2+) and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole-dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th beta-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca(2+) biosensors. PMID:20521333

  11. The radioprotective effect against gamma-irradiation of methylene blue in the rat with reference to serum enzymes and pancreatic protein fractions examined by isoelectric focussing

    International Nuclear Information System (INIS)

    The Sprague-Dawley male rats were given 360 rads of single whole-body gamma-irradiation following an intraperitoneal injection of methylene blue (40 mg/kg). Serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum amylase, serum lipase, and serum lecithinase A activities, and isoelectric focussing pattern of pancreatic juice proteins were determined at various time intervals after exposure. Methylene blue reduced generally the rise of SGOT, sGPT, amylase, and lipase activities. Methylene blue delayed also the serum lecithinase A fall after exposure. Mean number of protein bands as revealed by isoelectric focussing of pancreatic juice were significantly altered in both the control and the methylene blue-treated group after exposure. Especially methylene blue-treated group showed a marked delay in the decrease in number of protein bands after exposure. The possibility of using the SGOT, the SGPT, the serum amylase, the serum lipase, and the serum lecithinase A levels, and the number of protein bands in isoelectric focussing pattern of pancreatic juice as an early index of radiation injury is suggested. (auth.)

  12. Postpartum Blues

    Medline Plus

    Full Text Available ... blues The postpartum blues E-mail to a friend Please fill in all fields. Please enter a ... blues: Talk to your partner or a good friend about how you feel Get plenty of rest ...

  13. Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti

    Science.gov (United States)

    Leal, Gabriel M.; Leal, Walter S.

    2015-01-01

    Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules. PMID:25671088

  14. The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites.

    Science.gov (United States)

    Adisa, Akinola; Rug, Melanie; Klonis, Nectarios; Foley, Michael; Cowman, Alan F; Tilley, Leann

    2003-02-21

    The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane. PMID:12456681

  15. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    Science.gov (United States)

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  16. Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Jianke Ren; Long Wang; Guoxiang Liu; Wen Zhang; Zhejin Sheng; Zhugang Wang; Jian Fei

    2008-01-01

    Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibodypreparation.

  17. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

  18. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen; Sommer, Morten O A

    2015-12-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different GFP variants, yeast-enhanced GFP, GFP+ and superfolder GFP to yield a sequence-diverged triple GFP molecule 3vGFP with 74-84% internal repeat identity. Unlike a single GFP, the brightness of 3vGFP allowed characterization of a weak promoter in S. cerevisiae. Utilizing 3vGFP, we further engineered a less leaky Cu(2+)-inducible promoter based on CUP1. The basal expression level of the new promoter was approximately 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu(2+)-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms. PMID:26392044

  19. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    Science.gov (United States)

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis. PMID:26807518

  20. Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence.

    Science.gov (United States)

    Wang, Siyang; Circu, Magdalena L; Zhou, Hu; Figeys, Daniel; Aw, Tak Y; Feng, June

    2011-09-23

    S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions. PMID:21820121

  1. Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina

    Directory of Open Access Journals (Sweden)

    Nam Kyu Kang

    2015-12-01

    Full Text Available Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals.

  2. Hydrogen Bond Fluctuations Control Photochromism in a Reversibly Photo-Switchable Fluorescent Protein.

    Science.gov (United States)

    Morozov, Dmitry; Groenhof, Gerrit

    2016-01-11

    Reversibly switchable fluorescent proteins (RSFPs) are essential for high-resolution microscopy of biological samples, but the reason why these proteins are photochromic is still poorly understood. To address this problem, we performed molecular dynamics simulations of the fast switching Met159Thr mutant of the RSFP Dronpa. Our simulations revealed a ground state structural heterogeneity in the chromophore pocket that consists of three populations with one, two, or three hydrogen bonds to the phenolate moiety of the chromophore. By means of non-adiabatic quantum mechanics/molecular dynamics simulations, we demonstrated that the subpopulation with a single hydrogen bond is responsible for off-switching through photo-isomerization of the chromophore, whereas two or more hydrogen bonds inhibit the isomerization and promote fluorescence instead. While rational design of new RSFPs has so far focused on structure alone, our results suggest that structural heterogeneity must be considered as well. PMID:26612709

  3. Determination of Protein by Fluorescence Enhancement of Curcumin in Lanthanum-Curcumin-Sodium Dodecyl Benzene Sulfonate-Protein System

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Zaozhuang University, People' s Republic of China; Huang, Wei [Zaozhuang University, People' s Republic of China; Zhang, Yunfeng [Zaozhuang University, People' s Republic of China; Wang, Mingyin [Zaozhuang University, People' s Republic of China; Sun, Lina [Zaozhuang University, People' s Republic of China; Tang, Bo [Shandong University, Jinan, China; Wang, Wei [ORNL

    2011-01-01

    We found that the fluorescence intensity of the lanthanum (La(3+))-curcumin (CU) complex can be highly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this finding, a new fluorimetric method for the determination of protein was developed. Under optimized conditions, the enhanced intensities of fluorescence are quantitatively in proportion to the concentrations of proteins in the range 0.0080-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1) for human serum albumin (HSA) with excitation of 425 nm, and 0.00020-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1)for human serum albumin (HSA) with excitation of 280 nm, while corresponding qualitative detection limits (S/N 3) are as low as 5.368, 0.573, 0.049, 0.562 g mL(-1), respectively. Study on reaction mechanism reveals that proteins can bind with La(3+), CU and SDBS through self-assembling function with electrostatic attraction, hydrogen bonding, hydrophobic interaction and van der Waals forces, etc. The proteins form a supermolecular association with multilayer structure, in which La(3+)-CU is clamped between BSA and SDBS. The unique high fluorescence enhancement of CU is resulted through synergic effects of favorable hydrophobic microenvironment provided by BSA and SDBS, and efficient intermolecular energy transfer among BSA, SDBS and CU. In energy transfer process, La(3+) plays a crucial role because it not only shortens the distance between SDBS and CU, but also acts as a "bridge" for transferring the energy from BSA to CU.

  4. Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research.

    Directory of Open Access Journals (Sweden)

    Laura H Comley

    Full Text Available BACKGROUND: Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that thy1-driven expression of yellow fluorescent protein (YFP in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury. CONCLUSIONS/SIGNIFICANCE: We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.

  5. Post-mortem re-cloning of a transgenic red fluorescent protein dog

    OpenAIRE

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

    2011-01-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Althoug...

  6. Targeting retinal dopaminergic neurons in tyrosine hydroxylase-driven green fluorescent protein transgenic zebrafish

    OpenAIRE

    Meng, Shi; Ryu, Soojin; Zhao, Bin; Zhang, Dao-Qi; Driever, Wolfgang; McMahon, Douglas G.

    2008-01-01

    Purpose Dopamine plays key roles in a variety of basic functions in the central nervous system. To study developmental and functional roles of dopaminergic cells in zebrafish, we have generated a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (th1) promoter. Methods A 12 kb gene fragment that contains the th1 promoter was isolated and ligated to the MmGFP coding sequence, linearized, microinjected into 1–2 cell stage embry...

  7. Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

    OpenAIRE

    Liyue Wang; Kao Zhang; Hongyu Lin; Wenyan Li; Jiexia Wen; Jianlou Zhang; Yonghong Zhang; Xiujin Li; Fei Zhong

    2014-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene wa...

  8. An improved Coomassie Brilliant Blue (CBB R-250) staining to proteins in gels

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    An improved CBB staining with higher sensitivity than that of the typical CBB staining was reported. The main improvement was using a fixing step of 25% trichloroacetic acid (TCA) before CBB staining. For most proteins studied, the sensitivity of the improved CBB staining was about twice as high as that of the typical method. For basic and low molecular weight proteins such as ribosomal proteins, the sensitivity of this improved staining method was about 3.5-28 times that of the typical method. It was speculated that the improved procedure would be suitable for exact quantitative analysis of proteins fractionated by SDS-PAGE, especially for basic and low molecular weight proteins. On the other hand, this new modified method might be also applied to multidisciplinary studies, such as biological researches and nuclear sciences.

  9. A sulfhydryl-reactive ruthenium (II complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays.

    Directory of Open Access Journals (Sweden)

    Jing-Tang Lin

    Full Text Available To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate. The synthesized Ru(II complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The emission peak wavelength of the Ru(II-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II complex, indicating that Ru(II-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG binding assay was conducted. The result showed that Ru(II-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

  10. Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport*

    OpenAIRE

    Johnson, Danielle E.; Ai, Hui-wang; Wong, Peter; Young, James D.; Campbell, Robert E.; Casey, Joseph R

    2009-01-01

    Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluor...

  11. Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

    DEFF Research Database (Denmark)

    Brøndsted Nielsen, Mogens; Andersen, Lars Henrik; Rinza, Tomás Rocha

    2011-01-01

    The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase...... and its response in particular to hydrogen bond interactions. The overall goal is to understand how the protein binding pocket influences the absorption behavior, and the current status of our ongoing efforts is presented....

  12. Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

    Directory of Open Access Journals (Sweden)

    Merabet Samir

    2011-01-01

    Full Text Available Abstract Background Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Results Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Conclusion Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

  13. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  14. Purification, crystallization and preliminary X-ray diffraction of fluorescence recovery protein from Synechocystis PCC 6803

    International Nuclear Information System (INIS)

    Fluorescence recovery protein from Synechocystis PCC 6803 plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. The full-length form and a truncated form were overexpressed, purified and crystallized, and diffraction was observed to 2.75 Å resolution. Fluorescence recovery protein (FRP), which is encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. As the crystal structure of FRP may provide information about the biological functions and mechanism of action of the protein, recombinant full-length FRP and a truncated form were overexpressed, purified and crystallized at 291 K using ethylene imine polymer as the precipitant. An FRP data set was collected to a resolution of 2.75 Å at low temperature (100 K). The crystal belonged to space group P41212, with unit-cell parameters a = b = 61.9, c = 160.7 Å, α = β = γ = 90°. Assuming that the asymmetric unit contains three molecules, the Matthews coefficient was calculated to be 2.1 Å3 Da−1

  15. The past, present and future of fluorescent protein tags in anaerobic protozoan parasites.

    Science.gov (United States)

    Morin-Adeline, Victoria; Šlapeta, Jan

    2016-03-01

    The world health organization currently recognizes diarrhoeal diseases as a significant cause of death in children globally. Protozoan parasites such as Giardia and Entamoeba that thrive in the oxygen-deprived environment of the human gut are common etiological agents of diarrhoea. In the urogenital tract of humans, the anaerobic protozoan parasite Trichomonas vaginalis is notorious as the most common non-viral, sexually transmitted pathogen. Even with high medical impact, our understanding of anaerobic parasite physiology is scarce and as a result, treatment choices are limited. Fluorescent proteins (FPs) are invaluable tools as genetically encoded protein tags for advancing knowledge of cellular function. These FP tags emit fluorescent colours and once attached to a protein of interest, allow tracking of parasite proteins in the dynamic cellular space. Application of green FPs-like FPs in anaerobic protozoans is hindered by their oxygen dependency. In this review, we examine aspects of anaerobic parasite biology that clash with physio-chemical properties of FPs and limit their use as live-parasite protein tags. We expose novel FPs, such as miniSOG that do not require oxygen for signal production. The potential use of novel FPs has the opportunity to leverage the anaerobe parasitologist toolkit to that of aerobe parasitologist. PMID:26653973

  16. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions.

    Science.gov (United States)

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. PMID:27436096

  17. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    International Nuclear Information System (INIS)

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified 194LRMEKLNI201 as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES

  18. Marked changes in electron transport through the blue copper protein azurin in the solid state upon deuteration

    CERN Document Server

    Amdursky, Nadav; Sheves, Mordechai; Cahen, David

    2012-01-01

    Measuring electron transport (ETp) across proteins in the solid-state offers a way to study electron transfer (ET) mechanism(s) that minimizes solvation effects on the process. Solid state ETp is sensitive to any static (conformational) or dynamic (vibrational) changes in the protein. Our macroscopic measurement technique extends the use of ETp meas-urements down to low temperatures and the concomitant lower current densities, because the larger area still yields measurable currents. Thus, we reported previously a surprising lack of temperature-dependence for ETp via the blue copper protein azurin (Az), from 80K till denaturation, while ETp via apo-(Cu-free) Az was found to be temperature de-pendent \\geq 200K. H/D substitution (deuteration) can provide a potentially powerful means to unravel factors that affect the ETp mechanism at a molecular level. Therefore, we measured and report here the kinetic deuterium isotope effect (KIE) on ETp through holo-Az as a function of temperature (30-340K). We find that deu...

  19. Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ

    Science.gov (United States)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2009-02-01

    Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

  20. Elucidation of the new generation fluorescent protein tdTomato for space related radiobiological research

    Science.gov (United States)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Reitz, Guenther

    Astronauts in space are exposed to a potentially harmful radiation field, which does not exist in its quality and quantity on earth. Radiation exposure in space can lead to delayed or acute health effects. A successful long-term space mission requires better risk estimation and development of appropriate countermeasures, therefore study of the cellular radiation response is necessary. Ionizing radiation can provoke active cellular responses (cell cycle arrest, DNA repair, apoptosis or other forms of cell type). Exposure to ionizing radiation also activates various signaling pathways in human cells. In the cellular radiation-response, two pivotal signal transduction pathways have to be comprehensively studied i.e. the p53-pathway and NF-κB-pathway. Discovery of fluorescent proteins has revolutionized biological research by making it possible to carry out functional studies in living cells and understanding complex signaling pathways. Previously the green fluorescent proteins EGFP and d2EGFP were used for signaling pathway studies. In this work the new red fluorescent protein tdTomato will be used for comprehensive investigation of NF-κB and other transcription factor activation after exposure of human cells to ionizing radiation (X-rays, heavy ions; space conditions). tdTomato has many advantages over EGFP because of its high fluorescence signals and a better signal/noise ratio in human cells. The previously constructed reporter system with d2EGFP was used to evaluate NF-kB activation after exposure to heavy ion particles of different biological effectiveness. The sensitivity threshold of this system was determined to be 2 particle traversals per cell nucleus. In the current study a more sensitive reporter assay will be constructed using a GAL4-VP16 turbo system that comprises a receptor plasmid and a reporter plasmid. This reporter assay will be designed and constructed with tdTomato and evaluation will be done with different molecular techniques.

  1. Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

    Science.gov (United States)

    Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

    2001-05-01

    In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

  2. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    Science.gov (United States)

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

  3. Significance of the expression of green fluorescent protein on detection of glioma invasion in vivo

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To investigate the invasion and metastasis of gliomain vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma.

  4. Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Lars Boenicke; Bradley D. Howard; Ilka Vogel; Hoiger Kalthoff

    2003-01-01

    AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

  5. Signal Discrimination Between Fluorescent Proteins in Live Cells by Long-wavelength Optical Modulation.

    Science.gov (United States)

    Jablonski, Amy E; Hsiang, Jung-Cheng; Bagchi, Pritha; Hull, Nathan; Richards, Chris I; Fahrni, Christoph J; Dickson, Robert M

    2012-12-01

    Fluorescent proteins (FPs) have revolutionized molecular and cellular biology; yet, discrimination over cellular autofluorescence, spectral deconvolution, or detection at low concentrations remain challenging problems in many biological applications. By optically depopulating a photoinduced dark state with orange secondary laser co-excitation, the higher-energy green AcGFP fluorescence is dynamically increased. Modulating this secondary laser then modulates the higher-energy, collected fluorescence; enabling its selective detection by removing heterogeneous background from other FPs. Order-of-magnitude reduction in obscuring fluorophore background emission has been achieved in both fixed and live cells. This longwavelength modulation expands the dimensionality to discriminate FP emitters based on dark state lifetimes and enables signal of interest to be recovered by removing heterogeneous background emitter signals. Thus, AcGFP is not only useful for extracting weak signals from systems plagued by high background, but it is a springboard for further FP optimization and utilization for improving sensitivity and selectivity in biological fluorescence imaging. PMID:23419973

  6. Identification of Transmembrane Protein 134 as a Novel LMP1-Binding Protein by Using Bimolecular Fluorescence Complementation and an Enhanced Retroviral Mutagen

    OpenAIRE

    Talaty, Pooja; Emery, Amanda; Holthusen, Kirsten; Everly, David N.

    2012-01-01

    Latent membrane protein 1 (LMP1) of Epstein-Barr virus induces constitutive signaling in infected cells. LMP1 signaling requires oligomerization of LMP1 via its transmembrane domain, localization to lipid rafts in the membrane, and association of the LMP1 cytoplasmic domain to adaptor proteins, such as the tumor necrosis factor receptor-associated factors (TRAFs). Protein complementation is a novel technique to examine protein-protein interaction through the assembly of functional fluorescent...

  7. Amaltheys: A fluorescence-based analyzer to assess cheese milk denatured whey proteins.

    Science.gov (United States)

    Lacotte, Pierre; Gomez, Franck; Bardeau, Floriane; Muller, Sabine; Acharid, Abdelhaq; Quervel, Xavier; Trossat, Philippe; Birlouez-Aragon, Inès

    2015-10-01

    The cheese industry faces many challenges to optimize cheese yield and quality. A very precise standardization of the cheese milk is needed, which is achieved by a fine control of the process and milk composition. Thorough analysis of protein composition is important to determine the amount of protein that will be retained in the curd or lost in the whey. The fluorescence-based Amaltheys analyzer (Spectralys Innovation, Romainville, France) was developed to assess pH 4.6-soluble heat-sensitive whey proteins (sWP*) in 5 min. These proteins are those that can be denatured upon heat-treatment and further retained in the curd after coagulation. Monitoring of sWP* in milk and subsequent adaptation of the process is a reliable solution to achieve stable cheese yield and quality. Performance of the method was evaluated by an accredited laboratory on a 0 to 7 g/L range. Accuracy compared with the reference Kjeldahl method is also provided with a standard error of 0.25 g/L. Finally, a 4-mo industrial trial in a cheese plant is described, where Amaltheys was used as a process analytical technology to monitor sWP* content in ingredients and final cheese milk. Calibration models over quality parameters of final cheese were also built from near-infrared and fluorescence spectroscopic data. The Amaltheys analyzer was found to be a rapid, compact, and accurate device to help implementation of standardization procedures in the dairy industry. PMID:26210276

  8. Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

    International Nuclear Information System (INIS)

    Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrPC) into a disease associated form (PrPSc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrPC or a β-sheet rich, protease resistant form similar to PrPSc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrPSc from PrPC. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.

  9. Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

    Science.gov (United States)

    Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

    2016-03-01

    K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution. PMID:26894407

  10. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

    Science.gov (United States)

    Rodriguez, Erik A; Tran, Geraldine N; Gross, Larry A; Crisp, Jessica L; Shu, Xiaokun; Lin, John Y; Tsien, Roger Y

    2016-09-01

    Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP. PMID:27479328

  11. Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

    Science.gov (United States)

    Drobizhev, Mikhail; Stoltzfus, Caleb; Topol, Igor; Collins, Jack; Wicks, Geoffrey; Mikhaylov, Alexander; Barnett, Lauren; Hughes, Thomas E; Rebane, Aleksander

    2014-08-01

    Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue. PMID:25004113

  12. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues.

    OpenAIRE

    Reshetnyak, Y K; Koshevnik, Y; Burstein, E A

    2001-01-01

    In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710-1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of ...

  13. Effects of Dietary Protein Concentration and L-carnitine on Growth, Processing Yield, and Body Composition of Channel X Blue Catfish Hybrids

    Science.gov (United States)

    A study was conducted in earthen ponds to evaluate effects of dietary protein concentration and L-carnitine supplementation on production and processing traits of channel catfish × blue catfish hybrids. Hybrid fingerlings, mean initial weight = 66 g, were stocked into 20, 0.04-ha earthen ponds at a...

  14. Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice

    Directory of Open Access Journals (Sweden)

    Papaioannou Virginia E

    2004-12-01

    Full Text Available Abstract Background Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications. Results We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis. Conclusions Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate.

  15. Non-adiabatic dynamics of isolated green fluorescent protein chromophore anion

    International Nuclear Information System (INIS)

    On-the-fly ab initio molecular dynamics calculations have been performed to investigate the relaxation mechanism of green fluorescent protein chromophore anion under vacuum. The CASSCF surface hopping simulation method based on Zhu-Nakamura theory is applied to present the real-time conformational changes of the target molecule. The static calculations and dynamics simulation results suggest that not only the twisting motion around bridging bonds between imidazolinone and phenoxy groups but the strength mode of C=O and pyramidalization character of bridging atom are major factors on the ultrafast fluorescence quenching process of the isolated chromophore anion. The abovementioned factors bring the molecule to the vicinity of conical intersections on its potential energy surface and to finish the internal conversion process. A Hula-like twisting pattern is displayed during the relaxation process and the entire decay process disfavors a photoswitching pattern which corresponds to cis-trans photoisomerization

  16. Non-adiabatic dynamics of isolated green fluorescent protein chromophore anion

    Science.gov (United States)

    Zhao, Li; Zhou, Pan-Wang; Li, Bin; Gao, Ai-Hua; Han, Ke-Li

    2014-12-01

    On-the-fly ab initio molecular dynamics calculations have been performed to investigate the relaxation mechanism of green fluorescent protein chromophore anion under vacuum. The CASSCF surface hopping simulation method based on Zhu-Nakamura theory is applied to present the real-time conformational changes of the target molecule. The static calculations and dynamics simulation results suggest that not only the twisting motion around bridging bonds between imidazolinone and phenoxy groups but the strength mode of C=O and pyramidalization character of bridging atom are major factors on the ultrafast fluorescence quenching process of the isolated chromophore anion. The abovementioned factors bring the molecule to the vicinity of conical intersections on its potential energy surface and to finish the internal conversion process. A Hula-like twisting pattern is displayed during the relaxation process and the entire decay process disfavors a photoswitching pattern which corresponds to cis-trans photoisomerization.

  17. Non-adiabatic dynamics of isolated green fluorescent protein chromophore anion

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Li, E-mail: zhaoli282@dicp.ac.cn, E-mail: pwzhou@dicp.ac.cn, E-mail: libinsnet@dicp.ac.cn, E-mail: aihuagao@dicp.ac.cn; Gao, Ai-Hua, E-mail: zhaoli282@dicp.ac.cn, E-mail: pwzhou@dicp.ac.cn, E-mail: libinsnet@dicp.ac.cn, E-mail: aihuagao@dicp.ac.cn [State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); University of the Chinese Academy of Sciences, Beijing 100049 (China); Zhou, Pan-Wang, E-mail: zhaoli282@dicp.ac.cn, E-mail: pwzhou@dicp.ac.cn, E-mail: libinsnet@dicp.ac.cn, E-mail: aihuagao@dicp.ac.cn; Li, Bin, E-mail: zhaoli282@dicp.ac.cn, E-mail: pwzhou@dicp.ac.cn, E-mail: libinsnet@dicp.ac.cn, E-mail: aihuagao@dicp.ac.cn; Han, Ke-Li, E-mail: klhan@dicp.ac.cn [State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China)

    2014-12-21

    On-the-fly ab initio molecular dynamics calculations have been performed to investigate the relaxation mechanism of green fluorescent protein chromophore anion under vacuum. The CASSCF surface hopping simulation method based on Zhu-Nakamura theory is applied to present the real-time conformational changes of the target molecule. The static calculations and dynamics simulation results suggest that not only the twisting motion around bridging bonds between imidazolinone and phenoxy groups but the strength mode of C=O and pyramidalization character of bridging atom are major factors on the ultrafast fluorescence quenching process of the isolated chromophore anion. The abovementioned factors bring the molecule to the vicinity of conical intersections on its potential energy surface and to finish the internal conversion process. A Hula-like twisting pattern is displayed during the relaxation process and the entire decay process disfavors a photoswitching pattern which corresponds to cis-trans photoisomerization.

  18. Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein

    DEFF Research Database (Denmark)

    Björnberg, Olof; Østergaard, Henrik; Winther, Jakob R

    2006-01-01

    Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we...... have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction with...... separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity....

  19. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    International Nuclear Information System (INIS)

    Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light (λmax=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-, which buried into β-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol-1), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol-1. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in β-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes

  20. Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

    Directory of Open Access Journals (Sweden)

    Wüstner Daniel

    2012-11-01

    Full Text Available Abstract Background Fluorescence loss in photobleaching (FLIP is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp function is fitted to fluorescence loss (FL inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP, we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ disease proteins like mutant huntingtin (mtHtt can form large aggregates called inclusion bodies (IB’s. The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and

  1. Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing

    DEFF Research Database (Denmark)

    Teilum, Kaare; Maki, Kosuke; Kragelund, Birthe B;

    2002-01-01

    Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue alpha-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 micros to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence...... energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant tau = 80 micros, indicating formation of an intermediate with only slightly enhanced fluorescence of...... partially collapsed states with approximately 1/3 of the solvent-accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms....

  2. First molecular identification of the transgene red fluorescent protein (RFP in transgenic ornamental zebrafish (Danio rerio introduced in Peru

    Directory of Open Access Journals (Sweden)

    Carlos Scotto

    2013-09-01

    Full Text Available In this paper the transgenic fluorescent red, orange and pink zebra fish (Danio rerio, found in local aquariums in Peru, were identified using the PCR technique to amplify the transgene RFP sea anemone belonging to Discosoma spp. The gene expression of the red fluorescent protein (RFP transgene was found to determine different gradients-of-bioluminescence (shades in color in each GMO fish analyzed. We performed sequence analysis of the two variants of the RFP along with six variants of the existing fluorescent protein GFP from the Genbank, this could help identify quickly if they are new genes or variants thereof as these novel fluorescent proteins may be introduced in aquatic GMO in the future. Thus, developing and improving biosecurity measures through its timely detection at the molecular genetic level.

  3. Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy

    Indian Academy of Sciences (India)

    Anoop M Saxena; G Krishnamoorthy; Jayant B Udgaonkar; N Periasamy

    2007-03-01

    In protein-folding studies it is often required to differentiate a system with only two-states, namely the native (N) and unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s) could also be present. This differentiation of a two-state from a multi-state structural transition is non-trivial when studied by the several steady-state spectroscopic methods that are popular in protein-folding studies. In contrast to the steady-state methods, time-resolved fluorescence has the capability to reveal the presence of heterogeneity of structural forms due to the `fingerprint’ nature of fluorescence lifetimes of various forms. In this work, we establish this method by quantitative analysis of amplitudes associated with fluorescence lifetimes in multiexponential decays. First, we show that we can estimate, accurately, the relative population of species from two-component mixtures of non-interacting molecules such as fluorescent dyes, peptides and proteins. Subsequently, we demonstrate, by analysing the amplitudes of fluorescence lifetimes which are controlled by fluorescence resonance energy transfer (FRET), that the equilibrium folding-unfolding transition of the small singledomain protein barstar is not a two-step process.

  4. Improvement of the Fluorescence Intensity during a Flow Cytometric Analysis for Rice Protoplasts by Localization of a Green Fluorescent Protein into Chloroplasts

    Directory of Open Access Journals (Sweden)

    Min Kyoung You

    2014-12-01

    Full Text Available Protoplasts have been a useful unicellular system for various molecular biological analyses based on transient expression and single cell analysis using fluorescence-activated cell sorting (FACS, widely used as a powerful method in functional genomics. Despite the versatility of these methods, some limits based on low fluorescence intensity of a flow cytometric analysis (FCA using protoplasts have been reported. In this study, the chloroplast targeting of fluorescent proteins (FPs led to an eight-fold increase in fluorescence intensity and a 4.5-fold increase of transfection ratio from 14.7% to 65.7% as compared with their targeting into the cytoplasm. Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals. To investigate the reason underlying this finding, mRNA/protein levels and transfection efficiency were analyzed, and results suggest that mRNA/protein levels and transfection ratio are not much different between K-sGFP and KR-sGFP. From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency. Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population. Our study could be usefully applicable for highly sensitive FACS and FCA-investigations of green tissue.

  5. Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing.

    Science.gov (United States)

    Hughes, Alex J; Tentori, Augusto M; Herr, Amy E

    2012-10-24

    We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the amino acid 222 E-to-G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF was employed to monitor dark (nonfluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3-4 formal charges distinguish the bright and dark populations of avGFP, as compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen-bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events. PMID:23017083

  6. Development of an X-ray fluorescence holographic measurement system for protein crystals

    Science.gov (United States)

    Sato-Tomita, Ayana; Shibayama, Naoya; Happo, Naohisa; Kimura, Koji; Okabe, Takahiro; Matsushita, Tomohiro; Park, Sam-Yong; Sasaki, Yuji C.; Hayashi, Kouichi

    2016-06-01

    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α2β2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

  7. A double-emulsion microfluidic platform for in vitro green fluorescent protein expression

    International Nuclear Information System (INIS)

    Microfluidic droplet technology has gained popularity due to the advantages over conventional emulsion techniques and capabilities for a wide range of applications. In this paper, the development of a simple microfluidic-based double-emulsion system is reported. Such a system could be potentially used for in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single-emulsion droplets. Then, the single-emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double-emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser-induced fluorescence. The effect of the flow rates of the internal aqueous phase and outer continuous aqueous phase on the formation of the double-emulsion droplets is investigated to provide information for system optimization. On-chip storage of double-emulsion droplets is also investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation kits or crude Escherichia coli S30 extracts. In vitro expression of the green fluorescent protein is successfully demonstrated in this system

  8. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    International Nuclear Information System (INIS)

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH3CN and the tyramine then labeled with 125I. Dilactitol-125I-tyramine (DLT) and inulin-125I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol)2-N-CH2-CH2-NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins

  9. Fluorescence based assessment of SDS induced hydrophobic collapse in globular proteins

    Science.gov (United States)

    Manjunath, S.; Makani, Venkata Krishna Kanth; Satyamoorthy, Kapaettu; Rao, Bola Sadashiva Satish; Bhat, Gopalkrishna; Kanth, Akriti Baby; Mahato, Krishna Kishore

    2016-03-01

    The molecular mechanism of interaction between SDS and proteins is not clearly understood so far. According to the current knowledge SDS is known to interact with the hydrophobic regions of the proteins. Tryptophan and tyrosine are hydrophobic and hydrophilic aromatic amino acids respectively, which are also known for their intrinsic fluorescence nature in proteins. By observing the autofluorescence of both these hydrophobic and hydrophilic amino acids upon SDS treatment, information about SDS-protein interactions could be obtained. In the present study we have recorded the autofluorescence spectra of five globular proteins [Bovine serum albumin (BSA), Human serum albumin (HSA), Ribonuclease A (RNase A), Lysozyme and Trypsin] by the sequential excitation from 260nm to 295nm at every 5nm intervals. The results obtained clearly indicated BSA and HSA undergone hydrophobic collapse around their tryptophan moieties due to the increased folding of their secondary and tertiary structures upon SDS treatment. Trypsin on the other hand showed complete unfolding upon treatment with SDS. Lysozyme and RNase A did not show any difference in their autofluorescence upon SDS treatment may be due to the stability and fluorophores composition in them. The above results obtained with specific UV excitations clearly shown the tertiary folding and ensembles of the secondary and tertiary structures upon SDS treatment is governed by their stability and bonds stabilizing the proteins.

  10. A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

    Directory of Open Access Journals (Sweden)

    Hughes Thomas E

    2004-08-01

    Full Text Available Abstract Background There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1 they only produce one kind, or color, of fluorescent fusion protein and (2 one half of all GFP insertions within the target coding sequence are in the wrong orientation. Results We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R. Conclusions These new designs increase the efficiency of random fusion protein generation in one of two ways: (1 by creating two different fusion proteins from each insertion or (2 by being independent of orientation.

  11. Multi-state lasing in self-assembled ring-shaped green fluorescent protein microcavities

    International Nuclear Information System (INIS)

    We demonstrate highly efficient lasing from multiple photonic states in microcavities filled with self-assembled rings of recombinant enhanced green fluorescent protein (eGFP) in its solid state form. The lasing regime is achieved at very low excitation energies of 13 nJ and occurs from cavity modes dispersed in both energy and momentum. We attribute the momentum distribution to very efficient scattering of incident light at the surface of the eGFP rings. The distribution of lasing states in energy is induced by the large spectral width of the gain spectrum of recombinant eGFP (FWHM ≅ 25 nm)

  12. Multi-state lasing in self-assembled ring-shaped green fluorescent protein microcavities

    Science.gov (United States)

    Dietrich, Christof P.; Höfling, Sven; Gather, Malte C.

    2014-12-01

    We demonstrate highly efficient lasing from multiple photonic states in microcavities filled with self-assembled rings of recombinant enhanced green fluorescent protein (eGFP) in its solid state form. The lasing regime is achieved at very low excitation energies of 13 nJ and occurs from cavity modes dispersed in both energy and momentum. We attribute the momentum distribution to very efficient scattering of incident light at the surface of the eGFP rings. The distribution of lasing states in energy is induced by the large spectral width of the gain spectrum of recombinant eGFP (FWHM ≅ 25 nm).

  13. A Dark Excited State of Fluorescent Protein Chromophores, Considered as Brooker Dyes

    CERN Document Server

    Olsen, Seth

    2010-01-01

    The green fluorescent protein (GFP) chromophore is an asymmetric monomethine dye system. In the resonance color theory of dyes, a strong optical excitation arises from interactions of two valence-bond structures with a third, higher structure. We use correlated quantum chemistry to show that the anionic chromophore is a resonant Brooker dye, and that the third structure corresponds to a higher stationary electronic state of this species. The excitation energy of this state should be just below the first excitation energy of the neutral form. This has implications for excited state mechanism in GFPs, which we discuss.

  14. Multi-state lasing in self-assembled ring-shaped green fluorescent protein microcavities

    Energy Technology Data Exchange (ETDEWEB)

    Dietrich, Christof P., E-mail: cpd3@st-andrews.ac.uk; Höfling, Sven; Gather, Malte C., E-mail: mcg6@st-andrews.ac.uk [SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews KY16 9SS (United Kingdom)

    2014-12-08

    We demonstrate highly efficient lasing from multiple photonic states in microcavities filled with self-assembled rings of recombinant enhanced green fluorescent protein (eGFP) in its solid state form. The lasing regime is achieved at very low excitation energies of 13 nJ and occurs from cavity modes dispersed in both energy and momentum. We attribute the momentum distribution to very efficient scattering of incident light at the surface of the eGFP rings. The distribution of lasing states in energy is induced by the large spectral width of the gain spectrum of recombinant eGFP (FWHM ≅ 25 nm)

  15. Fluorescence study of protein immobilization on poly(4-hydroxyphenyl thiophene-3-carboxylate)-coated electrodes

    International Nuclear Information System (INIS)

    Synthesis of poly(4-hydroxyphenyl thiophene-3-carboxylate) (PHPT) was carried out in acetonitrile(ACN)/TBAClO4(0.05 M) solvent-electrolyte medium using different working electrodes (glassy carbon and ITO) via potentiodynamic electrolysis. Characterization of the resulting polymer was performed using cyclic voltammetry (CV), scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescence microscopy (FS). The polymer exhibits well-defined and reversible redox behavior in an organic solvent. Flourescence microscopy indicates that protein immobilizes better onto the surface of PHPT-modified electrodes than onto those without such modification.

  16. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies.

    Science.gov (United States)

    Grefen, Christopher; Donald, Naomi; Hashimoto, Kenji; Kudla, Jörg; Schumacher, Karin; Blatt, Michael R

    2010-10-01

    Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant

  17. Fluorescent proteins function as a prey attractant: experimental evidence from the hydromedusa Olindias formosus and other marine organisms

    Directory of Open Access Journals (Sweden)

    Steven H. D. Haddock

    2015-09-01

    Full Text Available Although proteins in the green fluorescent protein family (GFPs have been discovered in a wide array of taxa, their ecological functions in these organisms remain unclear. Many hypothesized roles are related to modifying bioluminescence spectra or modulating the light regime for algal symbionts, but these do not explain the presence of GFPs in animals that are non-luminous and non-symbiotic. Other hypothesized functions are unrelated to the visual signals themselves, including stress responses and antioxidant roles, but these cannot explain the localization of fluorescence in particular structures on the animals. Here we tested the hypothesis that fluorescence might serve to attract prey. In laboratory experiments, the predator was the hydromedusa Olindias formosus (previously known as O. formosa, which has fluorescent and pigmented patches on the tips of its tentacles. The prey, juvenile rockfishes in the genus Sebastes, were significantly more attracted (P<1×10−5 to the medusa's tentacles under lighting conditions where fluorescence was excited and tentacle tips were visible above the background. The fish did not respond significantly when treatments did not include fluorescent structures or took place under yellow or white lights, which did not generate fluorescence visible above the ambient light. Furthermore, underwater observations of the behavior of fishes when presented with a brightly illuminated point showed a strong attraction to this visual stimulus. In situ observations also provided evidence for fluorescent lures as supernormal stimuli in several other marine animals, including the siphonophore Rhizophysa eysenhardti. Our results support the idea that fluorescent structures can serve as prey attractants, thus providing a potential function for GFPs and other fluorescent proteins in a diverse range of organisms.

  18. Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA, protein and lipid.

    Science.gov (United States)

    Sulaiman, Saba A J; Al-Rasbi, Ghalia S; Abou-Zied, Osama K

    2016-05-01

    Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein-ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein-binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26910188

  19. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2007-06-01

    Full Text Available A novel fluorescence sensing system for branched-chain amino acids (BCAAswas developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPsconjugated with environmentally sensitive fluorescence probes. LIVBP was cloned fromEscherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated bygenetic engineering. The mutant LIVBPs were then modified with environmentallysensitive fluorophores. Based on the fluorescence intensity change observed upon thebinding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M showedthe highest and most sensitive response. The BCAAs Leu, Ile, and Val can each bemonitored at the sub-micromolar level using Gln149Cys-M. Measurements were alsocarried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurementis not significantly affected by the change in the molar ratio of Leu, Ile and Val in thesample. Its high sensitivity and group-specific molecular recognition ability make the newsensing system ideally suited for the measurement of BCAAs and the determination of theFischer ratio, an indicator of hepatic disease involving metabolic dysfunction.

  20. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    Science.gov (United States)

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-06-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

  1. Postpartum Blues

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    Full Text Available ... Quality Collaboratives Launch Prematurity research centers What is team science? More than 75 years of solving problems ... delivery cause the postpartum blues. How can you manage the baby blues? The American College of Obstetricians ...

  2. Postpartum Blues

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    Full Text Available ... with this condition are happy most of the time. But compared to how she usually feels, the ... the "blues" usually lessens and goes away over time. What causes the baby blues? Medical experts believe ...

  3. Postpartum Blues

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    Full Text Available ... Postpartum care > The postpartum blues The postpartum blues E-mail to a friend Please fill in all fields. Please enter a valid e-mail address. Your information: Your recipient's information: Your ...

  4. Postpartum Blues

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    Full Text Available ... usually lessens and goes away over time. What causes the baby blues? Medical experts believe that changes ... usually lessens and goes away over time. What causes the baby blues? Medical experts believe that changes ...

  5. Postpartum Blues

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    Full Text Available ... Feels sad Feels confused The postpartum blues peak three to five days after delivery. They usually end ... Feels sad Feels confused The postpartum blues peak three to five days after delivery. They usually end ...

  6. Postpartum Blues

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    Full Text Available ... postpartum blues Now playing: E-mail to a friend Please fill in all fields. Please enter a ... blues: Talk to your partner or a good friend about how you feel Get plenty of rest ...

  7. Postpartum Blues

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    Full Text Available ... The postpartum blues The postpartum blues Now playing: E-mail to a friend Please fill in all fields. Please enter a valid e-mail address. Your information: Your recipient's information: Your ...

  8. Postpartum Blues

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    Full Text Available ... of Obstetricians and Gynecologists recommends that women do these things to help relieve the baby blues: Talk ... of Obstetricians and Gynecologists recommends that women do these things to help relieve the baby blues: Talk ...

  9. Postpartum Blues

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    Full Text Available ... Medical experts believe that changes in the woman's hormones after delivery cause the postpartum blues. How can ... Medical experts believe that changes in the woman's hormones after delivery cause the postpartum blues. How can ...

  10. Postpartum Blues

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    Full Text Available ... can you manage the baby blues? The American College of Obstetricians and Gynecologists recommends that women do ... can you manage the baby blues? The American College of Obstetricians and Gynecologists recommends that women do ...

  11. Postpartum Blues

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    Full Text Available ... postpartum blues The postpartum blues E-mail to a friend Please fill in all fields. Please enter ... hear about breakthroughs for babies and families. Ask a question Our health experts can answer questions about ...

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  13. Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

    Directory of Open Access Journals (Sweden)

    Tobias Haase

    Full Text Available BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC Förster resonance energy transfer (FRET was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP was used as FRET donor and yellow fluorescent protein (YFP as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat, Winger and Marletta (Biochemistry 2005, 44:4083-90 have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a

  14. Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria.

    Science.gov (United States)

    Leveau, J H; Lindow, S E

    2001-12-01

    We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP

  15. Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

    NARCIS (Netherlands)

    Overkamp, Wout; Beilharz, Katrin; Weme, Ruud Detert Oude; Solopova, Ana; Karsens, Harma; Kovacs, Akos T.; Kok, Jan; Kuipers, Oscar P.; Veening, Jan-Willem

    2013-01-01

    Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental c

  16. Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta.

    Science.gov (United States)

    Martin, Kathleen; Kopperud, Kristin; Chakrabarty, Romit; Banerjee, Rituparna; Brooks, Robert; Goodin, Michael M

    2009-07-01

    Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community. PMID:19309457

  17. Simulation of Far-Field Superresolution Fluorescence Imaging with Two-Color One-Photon Excitation of Reversible Photoactivatable Protein

    Institute of Scientific and Technical Information of China (English)

    WANG Chen; QIAO Ling-Ling; MAO Zheng-Le

    2011-01-01

    We propose to achieve far-field super-resolution imaging by using offset two-color one-photon (2C1P) excitation of reversible photoactivatable fluorescence proteins. Due to the distinctive photoswitching performance of the proteins, such as dronpa, the fluorescence emission will only come from the overlapped region of activation beam and excitation beam. The analysis solution of rate equation shows that the resolution of offset 2C1P microscope is "engineered" by laser power of excitation and activation beams and the power ratio between them. Superior lateral and transverse resolution is theoretically demonstrated compared with conventional fluorescence scanning microscopy.%@@ We propose to achieve far-field super-resolution imaging by using offset two-color one-photon(2C1P) excitation of reversible photoactivatable fluorescence proteins.Due to the distinctive photoswitching performance of the proteins,such as dronpa,the fluorescence emission will only come from the overlapped region of activation beam and excitation beam.The analysis solution of rate equation shows that the resolution of offset 2C1P microscope is "engineered" by laser power of excitation and activation beams and the power ratio between them.Superior lateral and transverse resolution is theoretically demonstrated compared with conventional fluorescence scanning microscopy.

  18. Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

    Institute of Scientific and Technical Information of China (English)

    滕路; 孟国良; 刑阳; 尚克刚; 王小珂; 顾军

    2003-01-01

    Objective To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy.Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418r6TGr cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated. They have normal properties, such as karyotype, viability and di fferentiation ability. The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells. Cultured in suspension, the "green" ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges. The "green" embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence. Conclusions This generation of "green" targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical a nd research purposes.

  19. An individually coated near-infrared fluorescent protein as a safe and robust nanoprobe for in vivo imaging

    Science.gov (United States)

    Yang, Yu; Xiang, Kun; Yang, Yi-Xin; Wang, Yan-Wen; Zhang, Xin; Cui, Yangdong; Wang, Haifang; Zhu, Qing-Qing; Fan, Liqiang; Liu, Yuanfang; Cao, Aoneng

    2013-10-01

    A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging.A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging. Electronic supplementary information (ESI

  20. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    Energy Technology Data Exchange (ETDEWEB)

    Kovalev, Valeri I [School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); Bartona, James S [School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); Richardson, Patricia R [School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ (United Kingdom); Jones, Anita C [School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ (United Kingdom)

    2006-07-15

    There is a risk of contamination of surgical instruments by infectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect {approx}10 attomole/cm{sup 2} with a scan speed of {approx}3-10 cm{sup 2}/s of the test instrument's surface. A theoretical analysis and experimental measurements will be discussed.

  1. Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties

    International Nuclear Information System (INIS)

    Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties

  2. Ultraviolet photodissociation enhances top-down mass spectrometry as demonstrated on green fluorescent protein variants.

    Science.gov (United States)

    Dang, Xibei; Young, Nicolas L

    2014-05-01

    Ultraviolet photodissociation (UVPD) is a compelling fragmentation technique with great potential to enhance proteomics generally and top-down MS specifically. In this issue, Cannon et al. (Proteomics 2014, 14, XXXX-XXXX) use UVPD to perform top-down MS on several sequence variants of green fluorescent protein and compare the results to CID, higher energy collision induced dissociation, and electron transfer dissociation. As compared to the other techniques UVPD produces a wider variety of fragment ion types that are relatively evenly distributed across the protein sequences. Overall, their results demonstrate enhanced sequence coverage and higher confidence in sequence assignment via UVPD MS. Based on these and other recent results UVPD is certain to become an increasingly widespread and valuable tool for top-down proteomics. PMID:24723542

  3. Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluorescens for secretion and fluorescence of green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Park Yeonwoo

    2012-05-01

    Full Text Available Abstract Background TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs. Each LARD was fused to epidermal growth factor (EGF or green fluorescent protein (GFP, and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter. Results Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens ▵tliA, a knock-out mutant of tliA. Conclusion LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ.

  4. Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

    Science.gov (United States)

    Hare, Joanna E; Goodchild, Sophia C; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

    2016-07-12

    Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35. PMID:27299171

  5. Assembly and intracellular delivery of quantum dot-fluorescent protein bioconjugates

    Science.gov (United States)

    Medintz, Igor L.; Pons, Thomas; Delehanty, James B.; Susumu, Kimihiro; Dawson, Philip E.; Mattoussi, Hedi

    2008-02-01

    We have previously assembled semiconductor quantum dot (QD)-based fluorescence resonance energy transfer (FRET) sensors that can specifically detect nutrients, explosives or enzymatic activity. These sensors utilized the inherent benefits of QDs as FRET donors to optimize signal transduction. In this report we functionalize QDs with the multi-subunit multi-chromophore b-phycoerythrin (b-PE) light harvesting complex using biotin-Streptavidin binding. FRET and gel electrophoretic analyses were used to characterize and confirm the QD-b-PE self-assembly. We found that immobilizing additional cell-penetrating peptides on the nanocrystal surface along with the b-PE was the key factor allowing the mixed surface QD-cargos to undergo endocytosis and intracellular delivery. Our findings on the intracellular uptake promoted by CPP were compared to those collected using microinjection technique, where QD-assemblies were delivered directly into the cytoplasm; this strategy allows bypassing of the endocytic uptake pathway. Intracellular delivery of multifunctional QD-fluorescent protein assemblies has potential applications for use in protein tracking, sensing and diagnostics.

  6. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

    Science.gov (United States)

    Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

    2014-02-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

  7. Functional incorporation of green fluorescent protein into hepatitis B virus envelope particles

    International Nuclear Information System (INIS)

    The envelope of hepatitis B virus (HBV), containing the L, M, and S proteins, is essential for virus entry and maturation. For direct visualization of HBV, we determined whether envelope assembly could accommodate the green fluorescent protein (GFP). While the C-terminal addition of GFP to S trans-dominant negatively inhibited empty envelope particle secretion, the N-terminal GFP fusion to S (GFP.S) was co-integrated into the envelope, giving rise to fluorescent particles. Microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of GFP.S, required for particle export were rescued by interprotein interactions with wild-type S. Thereby, a dual location of GFP, inside and outside the envelope, was observed. GFP.S was also efficiently packaged into the viral envelope, and these GFP-tagged virions retained the capacity for attachment to HBV receptor-positive cells in vitro. Together, GFP-tagged virions should be suitable to monitor HBV uptake and egress in live hepatocytes

  8. Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

    Science.gov (United States)

    Moseyko, N.; Feldman, L. J.

    2001-01-01

    This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non-invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH-sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH-sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole-root tissues of A. thaliana is reported. The utility of pH-sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.

  9. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    Science.gov (United States)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  10. Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide

    Directory of Open Access Journals (Sweden)

    Venukumar Vemula

    2015-08-01

    Full Text Available Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated.

  11. High accuracy of mesoscopic epi-fluorescence tomography for non-invasive quantitative volume determination of fluorescent protein-expressing tumours in mice

    International Nuclear Information System (INIS)

    To compare mesoscopic epi-fluorescence tomography (MEFT) and EPRI-illumination reflectance imaging (EPRI) for quantitative tumour size assessment in mice. Tumour xenografts of green/red fluorescent protein (GFP/RFP)-expressing colon cancer cells were measured using MEFT, EPRI, ultrasound (US) and micro computed tomography (μCT) at day 14 post-injection (n = 6). Results from MEFT and EPRI were correlated with each other and with US and μCT (reference methods). Tumour volumes were measured ex vivo by GFP and RFP fluorescence imaging on cryoslices and compared with the in vivo measurements. High correlation and congruency were observed between MEFT, US and μCT (MEFT/US: GFP: r 2 = 0.96; RFP: r 2 = 0.97, both P 2 = 0.93; RFP: r 2 = 0.90; both P 2 = 0.96; RFP: r 2 = 0.99; both P 2 = 0.95; RFP: r 2 = 0.94; both P 2 = 0.86; RFP: r 2 = 0.86; both P < 0.05). Fluorescence distribution reconstruction using MEFT affords highly accurate three-dimensional (3D) tumour volume data showing superior accuracy compared to EPRI. Thus, MEFT is a very suitable technique for quantitatively assessing fluorescence distribution in superficial tumours at high spatial resolution. (orig.)

  12. Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: A comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies

    International Nuclear Information System (INIS)

    Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor

  13. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs inSaccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen;

    2015-01-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP...... functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different...... cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms....

  14. Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress.

    Science.gov (United States)

    Hashemi, Amenehsadat; Gharechahi, Javad; Nematzadeh, Ghorbanali; Shekari, Faezeh; Hosseini, Seyed Abdollah; Salekdeh, Ghasem Hosseini

    2016-08-01

    To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress. PMID:27362847

  15. An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

    OpenAIRE

    van Remmerden Yvonne; Xu Fang; van Eldik Mandy; Heldens Jacco GM; Huisman Willem; Widjojoatmodjo Myra N

    2012-01-01

    Abstract Background Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. Methods Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. Results The fluorescence plaque reduction a...

  16. Multicolor Fluorescence Nanoscopy by Photobleaching : Concept Verification and its Application to Resolve Selective Storage of Proteins in Platelets

    OpenAIRE

    Rönnlund, Daniel; Xu, Lei; Perols, Anna; Eriksson Karlström, Amelie; Auer, Gert; Widengren, Jerker

    2014-01-01

    Fluorescence nanoscopy provides means to discernthe finer details of protein localization and interaction in cells by offeringan order of magnitude higher resolution than conventional optical imagingtechniques. However, these super resolution techniques put higher demands onthe optical system as well as on the fluorescent probes, making multicolorfluorescence nanoscopy a challenging task. Here we present a new and simpleprocedure which exploits the photostability and excitation spectra of dye...

  17. Plasmonic Non-linear Conversion of Continuous Wave Light by Gold Nanoparticle Clusters withFluorescent Protein Loaded Gaps

    CERN Document Server

    Salakhutdinov, Ildar; Abak, Musa Kurtulus; Turkpence, Deniz; Piantanida, Luca; Fruk, Ljiljana; Tasgin, Mehmet Emre; Lazzarino, Marco; Bek, Alpan

    2014-01-01

    We propose and demonstrate a method which is feasible for deterministic activation of few molecules. Our method relies on non-linear optical excitation of few enhanced yellow fluorescent protein molecules that are sandwiched between gaps of asymmetrically constructed plasmonic gold nanoparticle clusters. We observe that as infrared photons, which cannot get absorbed by fluorescent molecules, are converted through efficient second harmonic generation activity of gold nanoparticles to visible photons, the molecules absorb them and fluoresce. Our numerical simulations demonstrate that observation of SHG with cw laser becomes possible owing to the cooperative action of conversion enhancement through Fano resonance, hybridization in the plasmon absorption spectrum and the size asymmetry of nanoparticle dimers.

  18. Ultrafast quenching of tryptophan fluorescence in proteins: Interresidue and intrahelical electron transfer

    International Nuclear Information System (INIS)

    Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl- and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications

  19. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    International Nuclear Information System (INIS)

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLSSV40) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLSSV40 in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLSSV40 formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLSSV40 likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLSSV40 can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus

  20. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.