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Sample records for coral fluorescent proteins

  1. 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

  2. Patterns of fluorescent protein expression in Scleractinian corals.

    Science.gov (United States)

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  3. 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

    2016-05-26

    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.

  4. Effects of cold stress and heat stress on coral fluorescence in reef-building corals.

    Science.gov (United States)

    Roth, Melissa S; Deheyn, Dimitri D

    2013-01-01

    Widespread temperature stress has caused catastrophic coral bleaching events that have been devastating for coral reefs. Here, we evaluate whether coral fluorescence could be utilized as a noninvasive assessment for coral health. We conducted cold and heat stress treatments on the branching coral Acropora yongei, and found that green fluorescent protein (GFP) concentration and fluorescence decreased with declining coral health, prior to initiation of bleaching. Ultimately, cold-treated corals acclimated and GFP concentration and fluorescence recovered. In contrast, heat-treated corals eventually bleached but showed strong fluorescence despite reduced GFP concentration, likely resulting from the large reduction in shading from decreased dinoflagellate density. Consequently, GFP concentration and fluorescence showed distinct correlations in non-bleached and bleached corals. Green fluorescence was positively correlated with dinoflagellate photobiology, but its closest correlation was with coral growth suggesting that green fluorescence could be used as a physiological proxy for health in some corals.

  5. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.

    Science.gov (United States)

    Tsutsui, Hidekazu; Karasawa, Satoshi; Shimizu, Hideaki; Nukina, Nobuyuki; Miyawaki, Atsushi

    2005-03-01

    Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.

  6. Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral.

    Science.gov (United States)

    Smith-Keune, C; Dove, S

    2008-01-01

    Recent incidences of mass coral bleaching indicate that major reef building corals are increasingly suffering thermal stress associated with climate-related temperature increases. The development of pulse amplitude modulated (PAM) fluorometry has enabled rapid detection of the onset of thermal stress within coral algal symbionts, but sensitive biomarkers of thermal stress specific to the host coral have been slower to emerge. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to produce fingerprints of gene expression for the reef-building coral Acropora millepora exposed to 33 degrees C. Changes in the expression of 23 out of 399 putative genes occurred within 144 h. Down-regulation of one host-specific gene (AmA1a) occurred within just 6 h. Full-length sequencing revealed the product of this gene to be an all-protein chromatophore (green fluorescent protein [GFP]-homolog). RT-PCR revealed consistent down-regulation of this GFP-homolog for three replicate colonies within 6 h at both 32 degrees C and 33 degrees C but not at lower temperatures. Down-regulation of this host gene preceded significant decreases in the photosynthetic activity of photosystem II (dark-adapted F (v)/F (m)) of algal symbionts as measured by PAM fluorometry. Gene expression of host-specific genes such as GFP-homologs may therefore prove to be highly sensitive indicators for the onset of thermal stress within host coral cells.

  7. Development of Fluorescence Imaging Lidar for Boat-Based Coral Observation

    Directory of Open Access Journals (Sweden)

    Sasano Masahiko

    2016-01-01

    Full Text Available A fluorescence imaging lidar system installed in a boat-towable buoy has been developed for the observation of reef-building corals. Long-range fluorescent images of the sea bed can be recorded in the daytime with this system. The viability of corals is clear in these fluorescent images because of the innate fluorescent proteins. In this study, the specifications and performance of the system are shown.

  8. Development of a Regional Coral Observation Method by a Fluorescence Imaging LIDAR Installed in a Towable Buoy

    Directory of Open Access Journals (Sweden)

    Masahiko Sasano

    2016-01-01

    Full Text Available Coral bleaching and mortality is predicted to increase under global climate change. A new observation technique is required to monitor regional coral conditions. To this end, we developed a light detection and ranging (LIDAR system installed in a towable buoy for boat observations, which acquires continuous fluorescent images of the seabed during day-time. Most corals have innate fluorescent proteins in their tissue, and they emit fluorescence by ultraviolet excitation. This fluorescence distinguishes living coral from dead coral skeleton, crustose coralline algae, and sea algae. This paper provides a proof of concept for using the LIDAR system and fluorescence to map coral distribution within 1 km scale and coral cover within 100 m scale for a single reef in Japan.

  9. Wide field-of-view fluorescence imaging of coral reefs.

    Science.gov (United States)

    Treibitz, Tali; Neal, Benjamin P; Kline, David I; Beijbom, Oscar; Roberts, Paul L D; Mitchell, B Greg; Kriegman, David

    2015-01-13

    Coral reefs globally are declining rapidly because of both local and global stressors. Improved monitoring tools are urgently needed to understand the changes that are occurring at appropriate temporal and spatial scales. Coral fluorescence imaging tools have the potential to improve both ecological and physiological assessments. Although fluorescence imaging is regularly used for laboratory studies of corals, it has not yet been used for large-scale in situ assessments. Current obstacles to effective underwater fluorescence surveying include limited field-of-view due to low camera sensitivity, the need for nighttime deployment because of ambient light contamination, and the need for custom multispectral narrow band imaging systems to separate the signal into meaningful fluorescence bands. Here we describe the Fluorescence Imaging System (FluorIS), based on a consumer camera modified for greatly increased sensitivity to chlorophyll-a fluorescence, and we show high spectral correlation between acquired images and in situ spectrometer measurements. This system greatly facilitates underwater wide field-of-view fluorophore surveying during both night and day, and potentially enables improvements in semi-automated segmentation of live corals in coral reef photographs and juvenile coral surveys.

  10. Fluorescent bands in massive corals record centuries of coastal rainfall

    Science.gov (United States)

    Isdale, Peter

    1984-08-01

    Massive coral colonies on the Great Barrier Reef grow outwards at 5-25 mm yr-1 (ref. 1). Skeletal density varies seasonally in all massive corals from the Great Barrier Reef and an exact temporal record of growth can be obtained by X-radiography of appropriately cut sections of the colony2,3. Environmental influences on coral growth have been described from such analyses4-6. This is the first report from the skeletons of massive corals of yellow green fluorescent bands which appear under long-wave UV light. Such fluorescence is confined to corals growing within 20 km of the shore and is not present in massive corals from mid- and outer shelf reefs of the central Great Barrier Reef region. The timing, width and intensity of the fluorescent bands correlate strongly with summer, monsoonal rainfall and coastal runoff. Large colonies, several centuries old, can provide long records of the strength and periodicity of terrestrial runoff to the tropical nearshore environment. Such records are potentially important to climatology, meteorology, agriculture, civil engineering and management of the Great Barrier Reef.

  11. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    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.

  12. 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.

  13. Photoinduced activation of GFP-like proteins in tissues of reef corals

    Science.gov (United States)

    Salih, Anya; Wiedenmann, Joerg; Matz, Mikhail; Larkum, Anthony W.; Cox, Guy

    2006-02-01

    A variety of fluorescent and chromophoric proteins homologous to the green fluorescent protein (GFP) has been recently discovered and cloned from non-bioluminescent marine animals, such as corals, and now provide a multitude of colors for use in fluorescence imaging applications. Recently, a novel fluorescence imaging methodology has emerged that utilizes the unique photoactivatory property of several GFP-like proteins, which respond to irradiation by altering their optical properties, thereby providing a new spatio-temporal capability to the GFP-based imaging applications. During our studies of GFP-like proteins from the Great Barrier Reef corals, several novel photoactivatable (PA) GFP-like proteins have been discovered. These include fluorescence photo-amplifiers and reversible photoswitchers, similar to PA jelly-fish derived PA-GFP and Dronpa, that greatly increase their emissions following ultraviolet-A (UVA) irradiation; the red-to-green (R-to-G) converters, similar to DsRed, that rapidly change to green color following single- or 2-photon irradiation; the green-to-red (G-to-R) converters, that acquire bright red fluorescence following UV-violet irradiation, similar to Kaede-like proteins; and the kindling GFP-like proteins, that are non fluorescent, but rapidly acquire bright fluorescence after green light irradiation. We report on the various optical characteristics of these coral PA proteins that may be used to expand the scope of the available fluorescence bio-imaging technologies.

  14. Fluorescence of coral larvae predicts their settlement response to crustose coralline algae and reflects stress.

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    Kenkel, C D; Traylor, M R; Wiedenmann, J; Salih, A; Matz, M V

    2011-09-07

    Multi-coloured homologues of the green fluorescent protein generate some of the most striking visual phenomena in the ocean. Despite their natural prominence in reef-building corals and widespread use in biotechnology, their biological role remains obscure. Here, we experimented with larvae of Acropora millepora to determine what can be learned about a coral larva or recruit from its fluorescent colour. We performed 12 crosses between seven A. millepora colonies representing differing fluorescence phenotypes, the larvae of which were exposed to a natural settlement cue (crustose coralline algae) and heat-light stress. Parental effects explained 18 per cent of variation in colour and 47 per cent of variation in settlement. The colour of the larval family emerged as a predictor of the settlement success: redder families were significantly less responsive to the provided settlement cue (p = 0.006). This relationship was owing to a correlation between parental effects on settlement and colour (r(2) = 0.587, p = 0.045). We also observed pronounced (16%) decline in settlement rate, as well as subtle (2%), but a statistically significant decrease in red fluorescence, as a consequence of heat-light stress exposure. Variation in settlement propensity in A. millepora is largely owing to additive genetic effects, and is thought to reflect variation in dispersal potential. Our results suggest an optical signature to discriminate between long- and short-range dispersing genotypes, as well as to evaluate stress. Further research in this direction may lead to the development of field applications to trace changes in coral life history and physiology caused by global warming.

  15. 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

    of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples...

  16. Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea

    Science.gov (United States)

    Eyal, Gal; Wiedenmann, Jörg; Grinblat, Mila; D’Angelo, Cecilia; Kramarsky-Winter, Esti; Treibitz, Tali; Ben-Zvi, Or; Shaked, Yonathan; Smith, Tyler B.; Harii, Saki; Denis, Vianney; Noyes, Tim; Tamir, Raz; Loya, Yossi

    2015-01-01

    The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50–60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40–100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging. PMID:26107282

  17. 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

  18. Using X-Ray Fluorescence Technique to Quantify Metal Concentration in Coral Cores from Belize

    Science.gov (United States)

    Kingsley, C.; Bhattacharya, A.; Hangsterfer, A.; Carilli, J.; Field, D. B.

    2016-12-01

    Caribbean coral reefs are some of the most threatened marine ecosystems in the world. Research appears to suggest that environmental stressors of local origin, such as sediment run off, can reduce the resilience of these reefs to global threats such as ocean warming. Sedimentation can stunt coral growth, reduce its resilience, and it is possible that trapped material could render coral skeletons brittle (personal discussions). Material trapped in coral skeletons can provide information on the sources of particulate matter in the ocean ecosystem. Despite the importance of quantifying sources and types of materials trapped in corals, the research community is yet to fully develop techniques that allow accurate representation of trapped matter, which is potentially a major source of metal content in reef building coral skeletons. The dataset presented here explores the usefulness of X-Ray Fluorescence (XRF), a widely used tool in environmental studies (but generally not in corals), to estimate metal content in coral cores collected from four locations near Belize, with varying degrees of impact from coastal processes. The coral cores together cover a period of 1862-2006. Trace, major, and minor metal content from these cores have been well-studied using solution-based ICP-MS, providing us with the unique opportunity to test the efficacy of XRF technique in characterizing metal content in these coral cores. We have measured more than 50 metals using XRF every two millimeters along slabs removed from the middle of a coral core to characterize materials present in coral skeletons. We compared the results from XRF to solution-based ICP-MS - that involves dissolving subsamples of coral skeleton to measure metal content. Overall, it appears that the non-destructive XRF technique is a viable supplement in determining sediment and metal content in coral cores, and may be particularly helpful for assessing resistant phases such as grains of sediment that are not fully

  19. FLUORESCENCE LIFETIME DISTRIBUTIONS IN PROTEINS

    OpenAIRE

    ALCALA, JR; Gratton, E; PRENDERGAST, FG

    1987-01-01

    The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most ...

  20. Lasing from fluorescent protein crystals.

    Science.gov (United States)

    Oh, Heon Jeong; Gather, Malte C; Song, Ji-Joon; Yun, Seok Hyun

    2014-12-15

    We investigated fluorescent protein crystals for potential photonic applications, for the first time to our knowledge. Rod-shaped crystals of enhanced green fluorescent protein (EGFP) were synthesized, with diameters of 0.5-2 μm and lengths of 100-200 μm. The crystals exhibit minimal light scattering due to their ordered structure and generate substantially higher fluorescence intensity than EGFP or dye molecules in solutions. The magnitude of concentration quenching in EGFP crystals was measured to be about 7-10 dB. Upon optical pumping at 485 nm, individual EGFP crystals located between dichroic mirrors generated laser emission with a single-mode spectral line at 513 nm. Our results demonstrate the potential of protein crystals as novel optical elements for self-assembled, micro- or nano-lasers and amplifiers in aqueous environment.

  1. Going Viral with Fluorescent Proteins.

    Science.gov (United States)

    Costantini, Lindsey M; Snapp, Erik L

    2015-10-01

    Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  2. Identification of Scleractinian Coral Recruits Using Fluorescent Censusing and DNA Barcoding Techniques

    Science.gov (United States)

    Hsu, Chia-Min; de Palmas, Stéphane; Kuo, Chao-Yang; Denis, Vianney; Chen, Chaolun Allen

    2014-01-01

    The identification of coral recruits has been problematic due to a lack of definitive morphological characters being available for higher taxonomic resolution. In this study, we tested whether fluorescent detection of coral recruits used in combinations of different DNA-barcoding markers (cytochrome oxidase I gene [COI], open reading frame [ORF], and nuclear Pax-C intron [PaxC]) could be useful for increasing the resolution of coral spat identification in ecological studies. One hundred and fifty settlement plates were emplaced at nine sites on the fringing reefs of Kenting National Park in southern Taiwan between April 2011 and September 2012. A total of 248 living coral spats and juveniles (with basal areas ranging from 0.21 to 134.57 mm2) were detected on the plates with the aid of fluorescent light and collected for molecular analyses. Using the COI DNA barcoding technique, 90.3% (224/248) of coral spats were successfully identified into six genera, including Acropora, Isopora, Montipora, Pocillopora, Porites, and Pavona. PaxC further separated I. cuneata and I. palifera of Isopora from Acropora, and ORF successfully identified the species of Pocillopora (except P. meandrina and P. eydouxi). Moreover, other cnidarian species such as actinarians, zoanthids, and Millepora species were visually found using fluorescence and identified by COI DNA barcoding. This combination of existing approaches greatly improved the taxonomic resolution of early coral life stages, which to date has been mainly limited to the family level based on skeletal identification. Overall, this study suggests important improvements for the identification of coral recruits in ecological studies. PMID:25211345

  3. Spatial heterogeneity in active chlorophyll fluorescence and PSII activity of coral tissues

    DEFF Research Database (Denmark)

    Ralph, P.J.; Gademann, R.; Larkum, A.W.D.

    2002-01-01

    Chlorophyll-a fluorescence was measured in six species of coral, using pulse-amplitude-modulated fluorometers employing fibre-optic probes with diameters of 8 mm, 1 mm and 140 µm. The 8-mm probe integrated responses over a large area, giving more weight to coenosarc than polyp tissue for Acropora...

  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. Temporal variability in chlorophyll fluorescence of back-reef corals in Ofu, American Samoa

    Science.gov (United States)

    Piniak, G.A.; Brown, E.K.

    2009-01-01

    Change in the yield of chlorophyll a fluorescence is a common indicator of thermal stress in corals. The present study reports temporal variability in quantum yield measurements for 10 coral species in Ofu, American Samoa - a place known to experience elevated and variable seawater temperatures. In winter, the zooxanthellae generally had higher dark-adapted maximum quantum yield (F v/Fm), higher light- adapted effective quantum yield (??F/F'm), and lower relative electron transport rates (rETR) than in the summer. Temporal changes appeared unrelated to the expected bleaching sensitivity of corals. All species surveyed, with the exception of Montipora grisea, demonstrated significant temporal changes in the three fluorescence parameters. Fluorescence responses were influenced by the microhabitat - temporal differences in fluorescence parameters were usually observed in the habitat with a more variable temperature regime (pool 300), while differences in Fv/Fm between species were observed only in the more environmentally stable habitat (pool 400). Such species-specific responses and microhabitat variability should be considered when attempting to determine whether observed in situ changes are normal seasonal changes or early signs of bleaching. ?? 2009 Marine Biological Laboratory.

  6. 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.

  7. Highlights of the optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, G H

    2011-07-01

    The development of super-resolution microscopy techniques using molecular localization, such as photoactivated localization microscopy, fluorescence photoactivated localization microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy with independent running acquisition and many others, has heightened interest in molecules that will be grouped here into a category referred to as 'optical highlighter' fluorescent proteins. This review will survey many of the advances in development of fluorescent proteins for optically highlighting sub-populations of fluorescently labelled molecules.

  8. Fluorescence dynamics of green fluorescent protein in AOT reversed micelles

    NARCIS (Netherlands)

    Uskova, M.A.; Borst, J.W.; Hink, M.A.; Hoek, van A.; Schots, A.; Klyachko, N.L.; Visser, A.J.W.G.

    2000-01-01

    We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the

  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. Fluorescence-based classification of Caribbean coral reef organisms and substrates

    Science.gov (United States)

    Zawada, David G.; Mazel, Charles H.

    2014-01-01

    A diverse group of coral reef organisms, representing several phyla, possess fluorescent pigments. We investigated the potential of using the characteristic fluorescence emission spectra of these pigments to enable unsupervised, optical classification of coral reef habitats. We compiled a library of characteristic fluorescence spectra through in situ and laboratory measurements from a variety of specimens throughout the Caribbean. Because fluorescent pigments are not species-specific, the spectral library is organized in terms of 15 functional groups. We investigated the spectral separability of the functional groups in terms of the number of wavebands required to distinguish between them, using the similarity measures Spectral Angle Mapper (SAM), Spectral Information Divergence (SID), SID-SAM mixed measure, and Mahalanobis distance. This set of measures represents geometric, stochastic, joint geometric-stochastic, and statistical approaches to classifying spectra. Our hyperspectral fluorescence data were used to generate sets of 4-, 6-, and 8-waveband spectra, including random variations in relative signal amplitude, spectral peak shifts, and water-column attenuation. Each set consisted of 2 different band definitions: ‘optimally-picked’ and ‘evenly-spaced.’ The optimally-picked wavebands were chosen to coincide with as many peaks as possible in the functional group spectra. Reference libraries were formed from half of the spectra in each set and used for training purposes. Average classification accuracies ranged from 76.3% for SAM with 4 evenly-spaced wavebands to 93.8% for Mahalanobis distance with 8 evenly-spaced wavebands. The Mahalanobis distance consistently outperformed the other measures. In a second test, empirically-measured spectra were classified using the same reference libraries and the Mahalanobis distance for just the 8 evenly-spaced waveband case. Average classification accuracies were 84% and 87%, corresponding to the extremes in modeled

  11. Correlation of coral fluorescence with nearshore rainfall and runoff in Hainan Island, South China Sea

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Under UV-laser excitation, the Porites coral from Longwan bay waters, Hainan Island, could emit yellow-green fluorescence. After fitting the data of the fluorescent intensity (FI) of the coral with the rainfall (RF) in nearshore area of Qionghai, a good relationship between FI and RF was found with a linear formula of RF =889-4.54 FI and a correlation coefficient of 0.78. Based on this formula, the rainfall sequence from 1982 to 1997 has been reconstructed. The peak annual rainfall is usually related to global El'nino events. By fitting the data of the fluorescent intensity and the runoff (RO), a quadric formula was obtained as RO=-0.0279FI2-18.59 FI-950.9 with a correlation coefficient of 0.74.The reconstructed runoff sequence from 1982 to 1997 coincided with the rainfall occurrence. The results indicate that flood and drought incidents in coastal areas of the south China are affected by global change to a great extent.

  12. X-ray diffraction analysis and molecular-replacement solution of the cyan fluorescent protein dsFP483

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Meitian; Patel, Hetal N.; Wachter, Rebekka M., E-mail: rwachter@asu.edu [Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona (United States)

    2005-10-01

    The coral-derived cyan fluorescent protein dsFP483 has been crystallized and diffraction data were collected to 2.1 Å. A molecular-replacement solution is presented for 83% of the protein contents of the asymmetric unit. A novel cyan fluorescent protein, dsFP483 from the coral Discosoma striata, has been crystallized. Diffraction data were collected to 2.1 Å and processed in space group C2. Molecular-replacement methods were applied using the closely related red fluorescent protein DsRed as a search model. The asymmetric unit appears to contain six protein molecules (1.5 tetramers), five of which (83%) could be located by the molecular-replacement searches.

  13. Fluorescent protein integrated white LEDs for displays

    Science.gov (United States)

    Press, Daniel Aaron; Melikov, Rustamzhon; Conkar, Deniz; Nur Firat-Karalar, Elif; Nizamoglu, Sedat

    2016-11-01

    The usage time of displays (e.g., TVs, mobile phones, etc) is in general shorter than their functional life time, which worsens the electronic waste (e-waste) problem around the world. The integration of biomaterials into electronics can help to reduce the e-waste problem. In this study, we demonstrate fluorescent protein integrated white LEDs to use as a backlight source for liquid crystal (LC) displays for the first time. We express and purify enhanced green fluorescent protein (eGFP) and monomeric Cherry protein (mCherry), and afterward we integrate these proteins as a wavelength-converter on a blue LED chip. The protein-integrated backlight exhibits a high luminous efficacy of 248 lm/Wopt and the area of the gamut covers 80% of the NTSC color gamut. The resultant colors and objects in the image on the display can be well observed and distinguished. Therefore, fluorescent proteins show promise for display applications.

  14. Rapid evolution of coral proteins responsible for interaction with the environment.

    KAUST Repository

    Voolstra, Christian R.

    2011-05-25

    Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably.

  15. 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.

  16. The UV Induced Fluorescence in Porites Coral from Hainan Island, China and Its Paleo-Environmental Implications

    Institute of Scientific and Technical Information of China (English)

    彭子成; 谢端; 何学贤; 张兆峰; 盛六四; 高辉; 聂宝符; 陈特固; 钟晋梁

    2001-01-01

    Porites coral in the nearshore waters off Hainan Island can give off yellowish-green fluorescence under UV laser induction. After fitting the data between rainfall and fluorescent intensity, it can be found that both appear in a simply linear equation with a correlation coefficient of 0.78. The model equation can be used to reconstruct the rainfall in the past time by means of the determined fluorescent intensity. The correlation diagram of the determined and calculated or hindcast rainfall shows a synchronous change from 1982 to 1997. The peak positions of the variant curve are usually related to the El'nino year. That means the drought and flood recycles are restrained to some extent by globally environmental changes. Therefore, the coral fluorescent intensity acts as a geochemical proxy for reconstructing the rainfall series in the past hundreds of years and predicting the drought-flood variant tendency in the future time.

  17. The UV Induced Fluorescence in Porites Coral from Hainan Island,China and Its Paleo—Environmental Implications

    Institute of Scientific and Technical Information of China (English)

    彭子成; 谢端; 等

    2001-01-01

    Porites coral in the nearshore waters off Hainan Island can give off yellowish-green fluorescence under UV laser induction.After fitting the data between rainfall and fluorescent intensity,it can be found that both appear in a simply linear equation with a correlation coefficeient of 0.78.The model equation can be used to reconstruct the rainfall in the past time by means of the determined fluorescent intensity.The correlation diagram of the determined and calculated or hindcast rainfall shows a synchronous change from 1982 to 1997.The peak positions of the variant curve are usually related to the El'nino year.That means the drought and flood recycles are restrained to some extent by globally environmental changes.Therefore,the coral fluorescent intensity acts as a geochemical proxy for reconstructing the rainfall series in the past hundreds of years and predicting the drought-flood variant tendency in the future time.

  18. Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.

    Directory of Open Access Journals (Sweden)

    Hsing-Hui Li

    Full Text Available The cellular and molecular-scale processes underlying the stability of coral-Symbiodinium endosymbioses remain unclear despite decades of investigation. As the coral gastroderm is the only tissue layer characterized by this unique symbiotic association, the membranes of these symbiotic gastrodermal cells (SGCs may play important roles in the initiation and maintenance of the endosymbiosis. In order to elucidate the interactions between the endosymbiotic dinoflagellates and their coral hosts, a thorough characterization of SGC membranes is therefore required. Cell surface proteins of isolated SGCs were biotinylated herein by a cell impermeant agent, biotin-XX sulfosuccinimidyl ester. The in situ distribution of these biotinylated proteins was uncovered by both fluorescence and transmission electron microscopic imaging of proteins bound to Alexa Fluor® 488-conjugated streptavidin. The identity of these proteins was then determined by two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. Nineteen (19 proteins were identified, and they are known to be involved in the molecular chaperone/stress response, cytoskeletal remodeling, and energy metabolism. These results not only reveal the molecular characters of the host SGC membrane, but also provide critical insight into understanding the possible role of host membranes in this ecologically important endosymbiotic association.

  19. Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.

    Science.gov (United States)

    Li, Hsing-Hui; Huang, Zi-Yu; Ye, Shih-Png; Lu, Chi-Yu; Cheng, Pai-Chiao; Chen, Shu-Hwa; Chen, Chii-Shiarng

    2014-01-01

    The cellular and molecular-scale processes underlying the stability of coral-Symbiodinium endosymbioses remain unclear despite decades of investigation. As the coral gastroderm is the only tissue layer characterized by this unique symbiotic association, the membranes of these symbiotic gastrodermal cells (SGCs) may play important roles in the initiation and maintenance of the endosymbiosis. In order to elucidate the interactions between the endosymbiotic dinoflagellates and their coral hosts, a thorough characterization of SGC membranes is therefore required. Cell surface proteins of isolated SGCs were biotinylated herein by a cell impermeant agent, biotin-XX sulfosuccinimidyl ester. The in situ distribution of these biotinylated proteins was uncovered by both fluorescence and transmission electron microscopic imaging of proteins bound to Alexa Fluor® 488-conjugated streptavidin. The identity of these proteins was then determined by two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. Nineteen (19) proteins were identified, and they are known to be involved in the molecular chaperone/stress response, cytoskeletal remodeling, and energy metabolism. These results not only reveal the molecular characters of the host SGC membrane, but also provide critical insight into understanding the possible role of host membranes in this ecologically important endosymbiotic association.

  20. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    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).

  1. Photochromicity and fluorescence lifetimes of green fluorescent protein

    OpenAIRE

    1999-01-01

    The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea and its mutants have gained widespread usage as an indicator of structure and function within cells. Proton transfer has been implicated in the complex photophysics of the wild-type molecule, exhibiting a protonated A species excited at 400 nm, and two deprotonated excited-state species I* and B* with red-shifted excitation similar to 475 nm. Photochromicity between the protonated and deprotonated species has been re...

  2. Conjugation of fluorescent proteins with DNA oligonucleotides.

    Science.gov (United States)

    Lapiene, Vidmantas; Kukolka, Florian; Kiko, Kathrin; Arndt, Andreas; Niemeyer, Christof M

    2010-05-19

    This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

  3. 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.; 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, respec...

  4. 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.

  5. Rapid Evolution of Coral Proteins Responsible for Interaction with the Environment

    Energy Technology Data Exchange (ETDEWEB)

    Voolstra, Christian R.; Sunagawa, Shinichi; Matz, Mikhail V.; Bayer, Till; Aranda, Manuel; Buschiazzo, Emmanuel; DeSalvo, Michael K.; Lindquist, Erika; Szmant, Alina M.; Coffroth, Mary Alice; Medina, Monica

    2011-01-31

    Background: Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably. Methodology/Principal Findings: We screened a set of 2,604 putative orthologs from EST-based sequence datasets of the coral species Acropora millepora and Acropora palmata to determine the fraction and identity of proteins that may experience adaptive evolution. 7percent of the orthologs show elevated rates of evolution. Taxonomically-restricted (i.e. lineagespecific) genes show a positive selection signature more frequently than genes that are found across many animal phyla. The class of proteins that displayed elevated evolutionary rates was significantly enriched for proteins involved in immunity and defense, reproduction, and sensory perception. We also found elevated rates of evolution in several other functional groups such as management of membrane vesicles, transmembrane transport of ions and organic molecules, cell adhesion, and oxidative stress response. Proteins in these processes might be related to the endosymbiotic relationship corals maintain with dinoflagellates in the genus Symbiodinium. Conclusion/Relevance: This study provides a birds-eye view of the processes potentially underlying coral adaptation, which will serve as a foundation for future work to elucidate the rates, patterns, and mechanisms of corals? evolutionary response to global climate change.

  6. Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Förster Resonance Energy Transfer (FRET) studies

    NARCIS (Netherlands)

    Hink, M.A.; Visser, N.V.; Borst, J.W.; Hoek, van A.; Visser, A.J.W.G.

    2003-01-01

    Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein (EYFP

  7. Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Förster Resonance Energy Transfer (FRET) studies

    NARCIS (Netherlands)

    Hink, M.A.; Visser, N.V.; Borst, J.W.; Hoek, van A.; Visser, A.J.W.G.

    2003-01-01

    Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein

  8. 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

  9. 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

    2016-03-18

    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.

  10. Generation of red fluorescent protein transgenic dogs.

    Science.gov (United States)

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.

  11. Genetically encoded biosensors based on engineered fluorescent proteins.

    Science.gov (United States)

    Frommer, Wolf B; Davidson, Michael W; Campbell, Robert E

    2009-10-01

    Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date.

  12. 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.

  13. Photoactivation and imaging of optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, George H

    2011-07-01

    A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins, which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be "turned on" by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks.

  14. Non-excitable fluorescent protein orthologs found in ctenophores.

    Science.gov (United States)

    Francis, Warren R; Christianson, Lynne M; Powers, Meghan L; Schnitzler, Christine E; D Haddock, Steven H

    2016-08-24

    Fluorescent proteins are optically active proteins found across many clades in metazoans. A fluorescent protein was recently identified in a ctenophore, but this has been suggested to derive from a cnidarian, raising again the question of origins of this group of proteins. Through analysis of transcriptome data from 30 ctenophores, we identified a member of an orthologous group of proteins similar to fluorescent proteins in each of them, as well as in the genome of Mnemiopsis leidyi. These orthologs lack canonical residues involved in chromophore formation, suggesting another function. The phylogenetic position of the ctenophore protein family among fluorescent proteins suggests that this gene was present in the common ancestor of all ctenophores and that the fluorescent protein previously found in a ctenophore actually derives from a siphonophore.

  15. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Al, Hui-wang; Henderson, J. Nathan; Remington, S. James; Campbell, Robert E. (Alberta); (Oregon)

    2008-05-07

    The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 {angstrom} crystal structure (1 {angstrom}=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

  16. Ultrafast excited state dynamics of the green fluorescent protein chromophore and its kindling fluorescent protein analogue.

    Science.gov (United States)

    Addison, Kiri; Heisler, Ismael A; Conyard, Jamie; Dixon, Tara; Page, Philip C Bulman; Meech, Stephen R

    2013-01-01

    Fluorescent proteins exhibit a very diverse range of photochemical behaviour, from efficient fluorescence through photochromism to photochemical reactivity. Remarkably this diverse behaviour arises from chromophores which have very similar structures. Here we describe measurements and modelling of the excited state dynamics in the chromophores of GFP (HBDI) and the kindling fluorescent protein, KFP (FHBMI). The methods are ultrafast fluorescence spectroscopy with sub 50 fs time resolution and the modelling is based on the Smoluchowski equation. The excited state decays of both chromophores are very fast, longer for their anions than for the neutral form and independent of wavelength. Detailed studies show the mean fluorescence wavelength to be independent of time. The excited state decay times are also observed to be a very weak function of solvent polarity and viscosity. These results are modelled utilising recently calculated potential energy surfaces for the ground and excited states as a function of the twist coordinates about the two bridging bonds of the chromophore. For FHBMI and the scarce data on the neutral HBDI the calculations are not successful suggesting the need for refinement of these potential energy surfaces. For HBDI in methanol the simulation is successful provided a strong dependence of the radiationless decay rate on the coordinate is assumed. Such dependence should be included in future calculations of excited state dynamics. When the simulations are extended to more viscous solvents they fail to reproduce the observed weak viscosity dependence. The implications of these results for the nature of the coordinate leading to radiationless decay in the chromophore and for the photodynamics of fluorescent proteins are discussed.

  17. A never ending race for new and improved fluorescent proteins.

    Science.gov (United States)

    Jones, Alexander M; Ehrhardt, David W; Frommer, Wolf B

    2012-05-03

    Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants.

  18. 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.

  19. Cell tracking using a photoconvertible fluorescent protein.

    Science.gov (United States)

    Hatta, Kohei; Tsujii, Hitomi; Omura, Tomomi

    2006-01-01

    The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.

  20. Protein conformation in solution by three-dimensional fluorescence spectrometry

    Institute of Scientific and Technical Information of China (English)

    鄢远; 许金钩; 陈国珍

    1996-01-01

    The conformations of bovine serum albumin (USA) and egg albumin (EA) in solution and their conformation changes under different conditions were studied by using three-dimensional fluorescence spectrometry (TDFS) such as three-dimensional fluorescence (TDF) spectra and three-dimensional fluorescence polarization (TDFP) spectra with tryptophan residues in protein molecules as an intrinsic fluorescent probe. The results show that the microenvironment of tryptophan residues of protein molecules in various solutions can be directly indicated and TDFS is an effective tool for studying protein conformation in solution. Meantime, some valuable results were obtained.

  1. Toward fluorescence detection of protein residues on surgical instruments

    Science.gov (United States)

    Richardson, Patricia R.; Jones, Anita C.; Baxter, Robert L.; Baxter, Helen C.; Whittaker, A. Gavin; Campbell, Gaynor A.

    2004-06-01

    Prion proteins are the infectious agents that cause Creutzfeldt-Jakob Disease (CJD) in humans. These proteins are particularly resistant to normal sterilization procedures, and the theoretical risk of prion transmission via surgical instruments is of current public and professional concern. We are currently investigating fluorescence methods for the detection of proteins on surfaces, with a view to developing an optical-fiber-based system for routine, online monitoring of residual protein contamination on surgical instruments, in hospital sterilization departments. This paper presents preliminary results on the detection of femtomole amounts of fluorescently labelled protein on surgical steel and discusses some of the problems involved in the detection of fluorescence from metal samples.

  2. Fluorescence of Alexa Fluor dye tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Borst, J.W.; Visser, A.J.W.G.; Mierlo, van C.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

  3. Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.

    Directory of Open Access Journals (Sweden)

    Bobin George Abraham

    Full Text Available Fluorescence Resonance Energy Transfer (FRET using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM.

  4. 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.

  5. Ratiometric fluorescent pH nanoprobes based on in situ assembling of fluorescence resonance energy transfer between fluorescent proteins.

    Science.gov (United States)

    Yu, Haijun; Chen, Chao; Cao, Xiaodan; Liu, Yueling; Zhou, Shengmin; Wang, Ping

    2017-08-01

    pH-dependent protein adsorption on mesoporous silica nanoparticle (MSN) was examined as a unique means for pH monitoring. Assuming that the degree of protein adsorption determines the distance separating protein molecules, we examined the feasibility of nanoscale pH probes based on fluorescence resonance energy transfer (FRET) between two fluorescent proteins (mTurquoise2 and mNeonGreen, as donor and acceptor, respectively). Since protein adsorption on MSN is pH-sensitive, both fluorescent proteins were modified to make their isoelectric points (pIs) identical, thus achieving comparable adsorption between the proteins and enhancing FRET signals. The adsorption behaviors of such modified fluorescent proteins were examined along with ratiometric FRET signal generation. Results demonstrated that the pH probes could be manipulated to show feasible sensitivity and selectivity for pH changes in hosting solutions, with a good linearity observed in the pH range of 5.5-8.0. In a demonstration test, the pH probes were successfully applied to monitor progress of enzymatic reactions. Such an "in situ-assembling" pH sensor demonstrates a promising strategy in developing nanoscale fluorescent protein probes. Graphical abstract Working principle of the developed pH sensor TNS; and FRET Ratio (I528/I460) as a function of pH under different protein feed ratios (mNeonGreen to mTurquoise2).

  6. Quenching of fluorescence in membrane protein by hypocrellin B.

    Science.gov (United States)

    Yue, J; Pang, S

    1997-04-01

    The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical charactcristics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quenchtr between membrane and water, and the fluorescence quenching constant of protein (K(sv); K(q),). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was observed in detail by using the ESR technique. The signal of HB- was found to arise from an electron transfer from excited trytophan to HB.

  7. Quenching of fluorescence in membrane protein by hypocrellin B

    Institute of Scientific and Technical Information of China (English)

    乐加昌; 庞素珍

    1997-01-01

    The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical characteris-tics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quencher between membrane and water, and the fluorescence quenching constant of protein (Ksv; Kq). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was ob-served in detail by using the ESR technique. The signal of HB" was found to arise from an electron transfer from ex-cited trytophan to HB.

  8. Immunity through early development of coral larvae.

    Science.gov (United States)

    Palmer, C V; Graham, E; Baird, A H

    2012-10-01

    As a determinant of survival, immunity is likely to be significant in enabling coral larvae to disperse and successfully recruit, however, whether reef-building coral larvae have immune defenses is unknown. We investigated the potential presence and variation in immunity in the lecithotrophic larvae of Acropora tenuis through larval development. Enzymes indicative of tyrosinase and laccase-type melanin-synthesis were quantified, and the concentration of three coral fluorescent proteins was measured over six developmental stages; egg, embryo, motile planula, planula post-exposure to crustose coralline algae (CCA; settlement cue), settled, settled post-exposure to Symbiodinium (endosymbiont). Both types of melanin-synthesis pathways and the three fluorescent proteins were present in A. tenuis throughout development. Laccase-type activity and red fluorescence increased following exposure of planula to CCA, whereas tyrosinase-type activity and cyan fluorescence increased following settlement. No change was detected in the measured parameters following exposure to Symbiodinium. This study is the first to document coral larval immune responses and suggests the melanin-synthesis pathways have disparate roles-the laccase-type potentially non-immunological and the tyrosinase-type in cytotoxic defense. Our results indicate that corals have the potential to resist infection from the earliest life history phase. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Fluorescence of Alexa fluor dye tracks protein folding.

    Science.gov (United States)

    Lindhoud, Simon; Westphal, Adrie H; Visser, Antonie J W G; Borst, Jan Willem; 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 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.

  10. 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.

  11. Exposure to elevated sea-surface temperatures below the bleaching threshold impairs coral recovery and regeneration following injury.

    Science.gov (United States)

    Bonesso, Joshua Louis; Leggat, William; Ainsworth, Tracy Danielle

    2017-01-01

    Elevated sea surface temperatures (SSTs) are linked to an increase in the frequency and severity of bleaching events due to temperatures exceeding corals' upper thermal limits. The temperatures at which a breakdown of the coral-Symbiodinium endosymbiosis (coral bleaching) occurs are referred to as the upper thermal limits for the coral species. This breakdown of the endosymbiosis results in a reduction of corals' nutritional uptake, growth, and tissue integrity. Periods of elevated sea surface temperature, thermal stress and coral bleaching are also linked to increased disease susceptibility and an increased frequency of storms which cause injury and physical damage to corals. Herein we aimed to determine the capacity of corals to regenerate and recover from injuries (removal of apical tips) sustained during periods of elevated sea surface temperatures which result in coral stress responses, but which do not result in coral bleaching (i.e., sub-bleaching thermal stress events). In this study, exposure of the species Acropora aspera to an elevated SST of 32 °C (2 °C below the bleaching threshold, 34 °C) was found to result in reduced fluorescence of green fluorescent protein (GFP), reduced skeletal calcification and a lack of branch regrowth at the site of injury, compared to corals maintained under ambient SST conditions (26 °C). Corals maintained under normal, ambient, sea surface temperatures expressed high GFP fluorescence at the injury site, underwent a rapid regeneration of the coral branch apical tip within 12 days of sustaining injury, and showed extensive regrowth of the coral skeleton. Taken together, our results have demonstrated that periods of sustained increased sea surface temperatures, below the corals' bleaching threshold but above long-term summertime averages, impair coral recovery from damage, regardless of the onset or occurrence of coral bleaching.

  12. 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…

  13. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; Berg, van den Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase

  14. A never ending race for new and improved fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Jones Alexander M

    2012-05-01

    Full Text Available Abstract Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants. See research article http://www.biomedcentral.com/1472-6750/12/17

  15. A never ending race for new and improved fluorescent proteins

    Science.gov (United States)

    2012-01-01

    Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants. See research article http://www.biomedcentral.com/1472-6750/12/17 PMID:22554191

  16. 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.

  17. 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.

  18. 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,…

  19. 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,…

  20. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET

    NARCIS (Netherlands)

    Subach, F.V.; Zhang, L.; Gadella, T.W.J.; Gurskaya, N.G.; Lukyanov, K.A.; Verkhusha, V.V.

    2010-01-01

    We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF stat

  1. 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.

  2. 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.

  3. Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis.

    Science.gov (United States)

    Mass, Tali; Putnam, Hollie M; Drake, Jeana L; Zelzion, Ehud; Gates, Ruth D; Bhattacharya, Debashish; Falkowski, Paul G

    2016-04-27

    Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization 'toolkit' in the early life history of a stony coral. © 2016 The Author(s).

  4. Fluorescence properties of porcine odorant binding protein Trp 16 residue

    Energy Technology Data Exchange (ETDEWEB)

    Albani, Jihad Rene, E-mail: Jihad-Rene.Albani@univ-lille1.f [Laboratoire de Biophysique Moleculaire, Universite des Sciences et Technologies de Lille, F-59655 Villeneuve d' Ascq Cedex (France)

    2010-11-15

    Summary: The present work deals with fluorescence studies of adult porcine odorant binding protein at pH=7.5. At this pH, the protein is a dimer, each monomer contains one tryptophan residue. Our results show that tryptophan residue displays significant motions and emits with three fluorescence lifetimes. Decay associated spectra showed that the three lifetime's components emanate from sub-structures surrounded by the same microenvironment.

  5. Origin of tryptophan fluorescence lifetimes. Part 2: fluorescence lifetimes origin of tryptophan in proteins.

    Science.gov (United States)

    Albani, J R

    2014-01-01

    Fluorescence intensity decays of L-tryptophan in proteins dissolved in pH 7 buffer, in ethanol and in 6 M guanidine pH 7.8 and in lyophilized proteins were measured. In all protein conditions, three lifetimes were obtained along the emission spectrum (310-410 nm). The two shortest lifetimes are in the same range of those obtained for L-Trp in water or in ethanol. Thus, these two lifetimes originate from specific two sub-structures existing in the excited state and are inherent to the tryptophan structure independently of the surrounding environment (amino acids residues, solvent, etc.) In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Populations of these lifetimes are independent of the excitation wavelength and thus originate from pre-defined sub structures existing in the excited state and put into evidence after tryptophan excitation. Fluorescence decay studies of different tripeptides having a tryptophan residue in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetimes. Indole dissolved in water and/or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.

  6. Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models

    Directory of Open Access Journals (Sweden)

    Natalia V. Povarova

    2017-07-01

    Full Text Available Despite great advances in practical applications of fluorescent proteins (FPs, their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

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

    Science.gov (United States)

    Kanki, Hiroaki; Shimabukuro, Marilia Kimie; Miyawaki, Atsushi; Okano, Hideyuki

    2010-02-02

    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 green. This two-color initial

  8. 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

  9. TIR-domain-containing protein repertoire of nine anthozoan species reveals coral-specific expansions and uncharacterized proteins.

    Science.gov (United States)

    Poole, Angela Z; Weis, Virginia M

    2014-10-01

    The intracellular toll/interleukin-1 receptor (TIR) domain plays an important role in vertebrate immunity, but the evolution and function of invertebrate TIR-domain-containing proteins is not fully understood. This study characterized and compared the TIR-domain-containing protein repertoire of nine cnidarians in class Anthozoa. A diverse set of proteins, including MyD88 (myeloid differentiation primary response protein 88), toll-like receptor (TLR)-like, interleukin-1 receptor (IL-1R)-like, and TIR-only proteins are present in the species surveyed. Increased numbers of TIR-only proteins were observed in corals compared to anemones, especially in the Acroporid and Pocilloporid coral families. This expansion could be linked to diversity of the microbial community on or in hosts and managing both positive and negative associations. Phylogenetic analysis indicates there are two groups of proteins with IL-1R-like domain architecture in anthozoans that potentially evolved independently of the vertebrate family. Bacterial-like TIR_2 domain proteins are also present, including one sequence with novel domain architecture. Overall this work promotes a better understanding of the anthozoan immune repertoire, which is important in the context learning about ancestral immune pathways and host-microbe interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. [Fluorescent fusion proteins with 10th human fibronectin domain].

    Science.gov (United States)

    Petrovskaia, L E; Gapizov, S Sh; Shingarova, L N; Kriukova, E A; Boldyreva, E F; Iakimov, S A; Svirshchevskaia, E V; Lukashev, E P; Dolgikh, D A; Kirpichnikov, M P

    2014-01-01

    In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.

  11. 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.

  12. Effect of refractive index on the fluorescence lifetime of green fluorescent protein.

    Science.gov (United States)

    Tregidgo, Carolyn; Levitt, James A; Suhling, Klaus

    2008-01-01

    The average fluorescence lifetime of the green fluorescent protein (GFP) in solution is a function of the refractive index of its environment. We report that this is also the case for GFP-tagged proteins in cells. Using time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging (FLIM) with a confocal scanning microscope, images of GFP-tagged proteins in cells suspended in different refractive index media are obtained. It is found that the average fluorescence lifetime of GFP decreases on addition of glycerol or sucrose to the media in which the fixed cells are suspended. The inverse GFP lifetime is proportional to the refractive index squared. This is the case for GFP-tagged major histocompatibility complex (MHC) proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP that is located outside the cell membrane. The implications of these findings are discussed with regard to total internal reflection fluorescence (TIRF) techniques where the change in refractive index is crucial in producing an evanescent wave to excite fluorophores near a glass interface. Our findings show that the GFP fluorescence lifetime is shortened in TIRF microscopy in comparison to confocal microscopy.

  13. On the purported "backbone fluorescence" in protein three-dimensional fluorescence spectra

    DEFF Research Database (Denmark)

    Bortolotti, Annalisa; Wong, Yin How; Korsholm, Stine S.

    2016-01-01

    as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence......In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220-230 nm. In the last 10 years we have observed a worrying increase in the number of articles...... claiming that this fluorescence originates from the protein backbone, contrary to the established knowledge that UV protein emission is due to aromatic amino acids only. Overall, our data clearly demonstrate that the observed emission upon excitation at 220-230 nm is due to the excitation of Tyr and/or Trp...

  14. Fast repetition rate (FRR) fluorometry: variability of chlorophyll a fluorescence yields in colonies of the corals, Montastraea faveolata (w.) and Diploria labyrinthiformes (h.) recovering from bleaching.

    Science.gov (United States)

    Lombardi; Lesser; Gorbunov

    2000-09-05

    Recently, an underwater version of a fast repetition rate fluorometer (FRRF) was developed for the non-destructive study of fluorescence yields in benthic photoautotrophs. We used an FRRF to study bleached colonies of the corals, Montastraea faveolata and Diploria labyrinthiformes at sites surrounding Lee Stocking Island, Exuma, Bahamas, to assess their recovery from bleaching ( approximately 1 year after the initial bleaching event) induced by elevated temperatures. The steady state quantum yields of chlorophyll a fluorescence (DeltaF'/F'(m)) from photosystem II (PSII) within coral colonies were separated into three categories representing visibly distinct degrees of bleaching ranging from no bleaching to completely bleached areas. Differences in DeltaF'/F'(m) were significantly different from bleached to unbleached regions within colonies. Dark, unbleached regions within colonies exhibited significantly higher DeltaF'/F'(m) values (0.438+/-0.019; mean+/-S.D.) when compared to lighter regions, and occupied a majority of the colonies' surface area (46-73%). Bleached regions exhibited significantly lower DeltaF'/F'(m) (0.337+/-0.014) and covered only 7-25% of the colonies' surface area. The observations from this study suggest that zooxanthellae in bleached regions of a colony exhibit reduced photosynthetic activity as long as one year after a bleaching event and that in situ fluorescence techniques such as FRRF are an effective means of studying coral responses and recovery from natural or anthropogenic stress in a non-destructive manner.

  15. 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 absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum.

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

    Science.gov (United States)

    Duan, Chenxi; Byrdin, Martin; El Khatib, Mariam; Henry, Xavier; Adam, Virgile; Bourgeois, Dominique

    2015-03-01

    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.

  17. 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.

  18. Are G-protein-coupled receptors involved in mediating larval settlement and metamorphosis of coral planulae?

    Science.gov (United States)

    Tran, Cawa; Hadfield, Michael G

    2012-04-01

    Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae.

  19. Fluorescence resonance energy transfer between fluorescent proteins as powerful toolkits for in vivo studies

    Science.gov (United States)

    Rusanov, A. L.; Savitsky, A. P.

    2011-02-01

    To expand the field of research in biological systems development of extra-sensitive analytical methods is highly desirable. In this review, the latest advances in technologies relying on the fluorescence resonance energy transfer between fluorescent proteins (FP's) to visualize numerous molecular processes in living cells are discussed. Variety of FP's as well as of novel experimental techniques allows one to choose the most appropriate tools to attack concrete problems.

  20. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins.

    OpenAIRE

    2000-01-01

    We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotr...

  1. 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.

  2. Fluobodies : green fluorescent single-chain Fv fusion proteins

    NARCIS (Netherlands)

    Griep, R.A.; Twisk, van C.; Wolf, van der J.M.; Schots, A.

    1999-01-01

    An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selec

  3. 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.

  4. Effects of drilling fluids (muds) and turbidity on the metabolic state of the coral Acropora cervicornis: calcification rate and protein concentration

    Energy Technology Data Exchange (ETDEWEB)

    Kendall, J.J. Jr.

    1983-01-01

    The effects of ten used drilling muds on coral health have been examined by monitoring changes in calcification rates and soluble tissue protein in the coral Acropora cervicornis. Exposure to 25-ppm (v/v) of one mud for 24 h reduced calcification rate in the growing tips by as much as 63%. Soluble tissue protein concentration dropped sig

  5. A PCR-Based Assay Targeting the Major Capsid Protein Gene of a Dinorna-Like ssRNA Virus That Infects Coral Photosymbionts

    Directory of Open Access Journals (Sweden)

    Jose Montalvo-Proaño

    2017-09-01

    Full Text Available The coral-Symbiodinium association is a critical component of coral reefs as it is the main primary producer and builds the reef's 3-dimensional structure. A breakdown of this endosymbiosis causes a loss of the dinoflagellate photosymbiont, Symbiodinium, and/or its photosynthetic pigments from the coral tissues (i.e., coral bleaching, and can lead to coral mortality. Coral bleaching has mostly been attributed to environmental stressors, and in some cases to bacterial infection. Viral lysis of Symbiodinium has been proposed as another possible cause of some instances of coral bleaching, but this hypothesis has not yet been experimentally confirmed. In this study, we used coral virome data to develop a novel PCR-based assay for examining the presence and diversity of a single-stranded RNA (ssRNA virus by targeting its major capsid protein (MCP gene. Illumina sequence analysis of amplicons obtained with novel primers showed 99.8% of the reads had the closest taxonomic affinity with the MCP gene of the virus, Heterocapsa circularisquama RNA virus (HcRNAV known to infect dinoflagellates, indicating that dinorna-like viruses are commonly associated with corals on the Great Barrier Reef. A phylogenetic analysis of MCP gene sequences revealed strong coral species specificity of viral operational taxon units (OTUs. This assay allows a relatively easy and rapid evaluation of the presence and diversity of this particular viral group and will assist in enhancing our understanding of the role of viral lysis in coral bleaching.

  6. Microarray analysis reveals transcriptional plasticity in the reef building coral Acropora millepora

    DEFF Research Database (Denmark)

    Bay, L. K.; Ulstrup, K. E.; Nielsen, H. B.

    2009-01-01

    We investigated variation in transcript abundance in the scleractinian coral, Acropora millepora, within and between populations characteristically exposed to different turbidity regimes and hence different levels of light and suspended particulate matter. We examined phenotypic plasticity...... exclusively downregulated; however, green fluorescent protein levels remained unchanged following translocation. Photophysiological responses of corals from both locations were characterized by a decline when introduced to the common laboratory environment but remained healthy (F-v/F-m > 0.6). Declines...... in total lipid content following translocation were the greatest for inshore corals, suggesting that turbid water corals have a strong reliance on heterotrophic feeding....

  7. A fluorescent probe for imaging p53-MDM2 protein-protein interaction.

    Science.gov (United States)

    Liu, Zhenzhen; Miao, Zhenyuan; Li, Jin; Fang, Kun; Zhuang, Chunlin; Du, Lupei; Sheng, Chunquan; Li, Minyong

    2015-04-01

    In this article, we describe a no-wash small-molecule fluorescent probe for detecting and imaging p53-MDM2 protein-protein interaction based on an environment-sensitive fluorescent turn-on mechanism. After extensive biological examination, this probe L1 exhibited practical activity and selectivity in vitro and in cellulo.

  8. 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.

  9. Fluorescent IgG fusion proteins made in E. coli.

    Science.gov (United States)

    Luria, Yael; Raichlin, Dina; Benhar, Itai

    2012-01-01

    Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called "Inclonals." By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the "Inclonals" technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.

  10. Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications

    Science.gov (United States)

    Hendy, Erica J.; Tomiak, Peter J.; Collins, Matthew J.; Hellstrom, John; Tudhope, Alexander W.; Lough, Janice M.; Penkman, Kirsty E. H.

    2012-06-01

    Over 500 Free Amino Acid (FAA) and corresponding Total Hydrolysed Amino Acid (THAA) analyses were completed from eight independently-dated, multi-century coral cores of massive Porites sp. colonies. This dataset allows us to re-evaluate the application of amino acid racemization (AAR) for dating late Holocene coral material, 20 years after Goodfriend et al. (GCA56 (1992), 3847) first showed AAR had promise for developing chronologies in coral cores. This re-assessment incorporates recent method improvements, including measurement by RP-HPLC, new quality control approaches (e.g. sampling and sub-sampling protocols, statistically-based data screening criteria), and cleaning steps to isolate the intra-crystalline skeletal protein. We show that the removal of the extra-crystalline contaminants and matrix protein is the most critical step for reproducible results and recommend a protocol of bleaching samples in NaOCl for 48 h to maximise removal of open system proteins while minimising the induced racemization. We demonstrate that AAR follows closed system behaviour in the intra-crystalline fraction of the coral skeletal proteins. Our study is the first to assess the natural variability in intra-crystalline AAR between colonies, and we use coral cores taken from the Great Barrier Reef, Australia, and Jarvis Island in the equatorial Pacific to explore variability associated with different environmental conditions and thermal histories. Chronologies were developed from THAA Asx D/L, Ala D/L, Glx D/L and FAA Asx D/L for each core and least squares Monte Carlo modelling applied in order to quantify uncertainty of AAR age determinations and assess the level of dating resolution possible over the last 5 centuries. AAR within colonies follow consistent stratigraphic aging. However, there are systematic differences in rates between the colonies, which would preclude direct comparison from one colony to another for accurate age estimation. When AAR age models are developed from

  11. 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.

  12. Photosystem II recovery in the presence and absence of chloroplast protein repair in the symbionts of corals exposed to bleaching conditions

    Science.gov (United States)

    Hill, R.; Takahashi, S.

    2014-12-01

    Increased seawater temperature causes photoinhibition due to accumulation of photodamaged photosystem II (PSII) in symbiotic algae (genus Symbiodinium) within corals, and it is assumed to be associated with coral bleaching. To avoid photoinhibition, photosynthetic organisms repair the photodamaged PSII through replacing the PSII proteins, primarily the D1 protein, with newly synthesised proteins. However, in experiments using cultured Symbiodinium strains, the PSII repair of Symbiodinium has been suggested not to be related to the synthesis of the D1 protein. In this study, we examined the relationship between the recovery of PSII photochemical efficiency ( F V/ F M) and the content of D1 protein after high-light and high-temperature treatments using the bleaching-sensitive coral species, Pocillopora damicornis and Acropora millepora, and the bleaching-tolerant coral species, Montipora digitata and Pavona decussata. When corals were exposed to strong light (600 µmol photons m-2 s-1) at elevated temperature (32 °C) for 8 h, significant bleaching occurred in bleaching-sensitive coral species although an almost similar extent of reduced PSII function was found across all coral species tested. During a subsequent 15-h recovery under low light (10 µmol photons m-2 s-1) at optimal temperature (22 °C), the reduced F V/ F M recovered close to initial levels in all coral species, but the reduced D1 content recovered only in one coral species ( Pavona decussata). D1 content was therefore not strongly linked to chloroplast protein synthesis-dependent PSII repair. These results demonstrate that the recovery of photodamaged PSII does not always correspond with the recovery of D1 protein content in Symbiodinium within corals, suggesting that photodamaged PSII can be repaired by a unique mechanism in Symbiodinium within corals.

  13. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  14. "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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wielgus-Kutrowska, Beata [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Narczyk, Marta [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Buszko, Anna [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Bzowska, Agnieszka [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Clark, Patricia L [Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556-5670 (United States)

    2007-07-18

    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.

  16. Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure.

    Directory of Open Access Journals (Sweden)

    Tomonobu M Watanabe

    Full Text Available Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.

  17. 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.

  18. 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 is pro...

  19. Detection of protein-protein interactions in plants using bimolecular fluorescence complementation.

    Science.gov (United States)

    Bracha-Drori, Keren; Shichrur, Keren; Katz, Aviva; Oliva, Moran; Angelovici, Ruthie; Yalovsky, Shaul; Ohad, Nir

    2004-11-01

    Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.

  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. Colorful protein-based fluorescent probes for collagen imaging.

    Directory of Open Access Journals (Sweden)

    Stijn J A Aper

    Full Text Available Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488. The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

  2. 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.

  3. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-04-15

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.

  4. Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

    Science.gov (United States)

    Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-01-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  5. Calcite formation in soft coral sclerites is determined by a single reactive extracellular protein.

    Science.gov (United States)

    Rahman, M Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-09-09

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called "calcite and aragonite seas." Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that -OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature.

  6. Colloidal quantum dots for fluorescent labels of proteins

    Science.gov (United States)

    Gladyshev, P.; Kouznetsov, V.; Martinez Bonilla, C.; Dezhurov, S.; Krilsky, D.; Vasiliev, A.; Morenkov, O.; Vrublevskaya, V.; Tsygankov, P.; Ibragimova, S.; Rybakova, A.

    2016-10-01

    The work is devoted to the synthesis of colloidal quantum dots (QDs) and their bioconjugates with proteins. Various QDs were obtained as well with synthesis method in an organic solvent followed by hydrophilization and functionalization or synthesis in aqueous phase provides obtaining hydrophilic QDs directly. Particular attention is paid to the synthesis of QDs as fluorescent tags in the near infrared where minimum absorption occurs and the fluorescence of biological tissue and synthetic materials used in analytical systems. A method for the QDs synthesis of type fluorescent core/shell CdTeSe/CdS/CdZnS-PolyT with mixed telluride, selenide cadmium core with a high quantum yield and high resistance to photoaging. It is shown that these quantum dots may be effectively used in the immunoassay.

  7. 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…

  8. Quantitative Analysis of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    Science.gov (United States)

    Wu, Yong; Liu, Yi-Kuang; Eghbali, Mansoureh; Stefani, Enrico

    2009-02-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component.

  9. Fluorescent biosensors for high throughput screening of protein kinase inhibitors.

    Science.gov (United States)

    Prével, Camille; Pellerano, Morgan; Van, Thi Nhu Ngoc; Morris, May C

    2014-02-01

    High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.

  10. 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).

  11. The fluorescent protein palette: tools for cellular imaging†

    Science.gov (United States)

    Davidson, Michael W.

    2010-01-01

    This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems. “If wood is rubbed with the Pulmo marinus, it will have all the appearance of being on fire; so much so, indeed, that a walking-stick, thus treated, will light the way like a torch” (translation of Pliny the Elder from John Bostock, 1855). PMID:19771335

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

    Science.gov (United States)

    Cao, Hop S Tran; Kimura, Hiroaki; Kaushal, Sharmeela; Snyder, Cynthia S; Reynoso, Jose; Hoffman, Robert M; Bouvet, Michael

    2015-01-01

    Context 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. PMID:19287108

  13. 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.

  14. Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

    Science.gov (United States)

    Robinson, Lisbeth C; Marchant, Jonathan S

    2005-02-01

    The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.

  15. Protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves.

    Science.gov (United States)

    Schweiger, Regina; Schwenkert, Serena

    2014-03-09

    Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

  16. Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

    Science.gov (United States)

    2016-01-13

    and β-sheet structure present in the stabilizing protein. These data support the hypothesis that the pressure -induced fluorescence enhancement...ARL-TR-7572 ● JAN 2016 US Army Research Laboratory Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold...JAN 2016 US Army Research Laboratory Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters by Abby

  17. 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

  18. Protein expression and genetic structure of the coral Porites lobata in an environmentally extreme Samoan back reef: Does host genotype limit phenotypic plasticity?

    Science.gov (United States)

    Barshis, D.J.; Stillman, J.H.; Gates, R.D.; Toonen, R.J.; Smith, L.W.; Birkeland, C.

    2010-01-01

    The degree to which coral reef ecosystems will be impacted by global climate change depends on regional and local differences in corals' susceptibility and resilience to environmental stressors. Here, we present data from a reciprocal transplant experiment using the common reef building coral Porites lobata between a highly fluctuating back reef environment that reaches stressful daily extremes, and a more stable, neighbouring forereef. Protein biomarker analyses assessing physiological contributions to stress resistance showed evidence for both fixed and environmental influence on biomarker response. Fixed influences were strongest for ubiquitin-conjugated proteins with consistently higher levels found in back reef source colonies both pre and post-transplant when compared with their forereef conspecifics. Additionally, genetic comparisons of back reef and forereef populations revealed significant population structure of both the nuclear ribosomal and mitochondrial genomes of the coral host (FST = 0.146 P < 0.0001, FST = 0.335 P < 0.0001 for rDNA and mtDNA, respectively), whereas algal endosymbiont populations were genetically indistinguishable between the two sites. We propose that the genotype of the coral host may drive limitations to the physiological responses of these corals when faced with new environmental conditions. This result is important in understanding genotypic and environmental interactions in the coral algal symbiosis and how corals may respond to future environmental changes. ?? 2010 Blackwell Publishing Ltd.

  19. 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.

  20. Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

    Science.gov (United States)

    Chen, Shengxi; Fahmi, Nour Eddine; Bhattacharya, Chandrabali; Wang, Lin; Jin, Yuguang; Benkovic, Stephen J; Hecht, Sidney M

    2013-11-26

    In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational

  1. Simultaneous time-lamination imaging of protein association using a split fluorescent timer protein.

    Science.gov (United States)

    Takamura, Ayari; Hattori, Mitsuru; Yoshimura, Hideaki; Ozawa, Takeaki

    2015-03-17

    Studies of temporal behaviors of protein association in living cells are crucially important for elucidating the fundamental roles and the mechanism of interactive coordination for cell activities. We developed a method for investigating the temporal alternation of a particular protein assembly using monomeric fluorescent proteins, fluorescent timers (FTs), of which the fluorescent color changes from blue to red over time. We identified a dissection site of the FTs, which allows complementation of the split FT fragments. The split fragments of each FT variant recovered their fluorescence and maintained inherent rates of the color changes upon the reassembly of the fragments in vitro. We applied this method to visualize the aggregation process of α-synuclein in living cells. The size of the aggregates with the temporal information was analyzed from ratio values of the blue and red fluorescence of the reconstituted FTs, from which the aggregation rates were evaluated. This method using the split FT fragments enables tracing and visualizing temporal alternations of various protein associations by single fluorescence measurements at a given time point.

  2. 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…

  3. Intracellular distribution of cowpea mosaic virus movement protein as visualised by green fluorescent protein fusions

    NARCIS (Netherlands)

    Gopinath, K.; Bertens, P.; Pouwels, J.; Marks, H.; Lent, van J.W.M.; Wellink, J.E.; Kammen, van A.

    2003-01-01

    Cowpea mosaic virus (CPMV) derivatives expressing movement protein (MP) green fluorescent protein (GFP) fusions (MP:GFP) were used to study the intracellular targeting and localization of the MP in cowpea protoplasts and plants. In protoplasts, a virus coding for a wild type MP:GFP (MPfGFP) induced

  4. Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

    Science.gov (United States)

    Dang, Dung Thanh; Phan, Anh Tuân

    2016-01-01

    We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.

  5. Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table

    Science.gov (United States)

    Einstein, Gnanatheepam; Udayakumar, Kanniyappan; Aruna, Prakasarao; Ganesan, Singaravelu

    2017-03-01

    Fluorescence of Protein has been widely used in diagnostic oncology for characterizing cellular metabolism. However, the intensity of fluorescence emission is affected due to the absorbers and scatterers in tissue, which may lead to error in estimating exact protein content in tissue. Extraction of intrinsic fluorescence from measured fluorescence has been achieved by different methods. Among them, Monte Carlo based method yields the highest accuracy for extracting intrinsic fluorescence. In this work, we have attempted to generate a lookup table for Monte Carlo simulation of fluorescence emission by protein. Furthermore, we fitted the generated lookup table using an empirical relation. The empirical relation between measured and intrinsic fluorescence is validated using tissue phantom experiments. The proposed relation can be used for estimating intrinsic fluorescence of protein for real-time diagnostic applications and thereby improving the clinical interpretation of fluorescence spectroscopic data.

  6. 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.

  7. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  8. P-glycoprotein (multi-xenobiotic resistance) and heat shock protein gene expression in the reef coral Montastraea franksi in response to environmental toxicants.

    Science.gov (United States)

    Venn, Alexander A; Quinn, Jennifer; Jones, Ross; Bodnar, Andrea

    2009-07-26

    The deleterious impacts of marine pollutants on reef corals and their symbiotic algae are an important element of global coral reef decline. In the current study we examined the impacts of toxicants on the reef coral Montastraea franksi by analysing the expression of three stress-related genes belonging to the coral host, using Taqman real-time quantitative reverse transcription-PCR. Gene expression profiles of P-glycoprotein (or multi-xenobiotic resistance protein) (P-gp); heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) were examined following 4 and 8h exposures to the heavy metal copper (3, 10, 30 and 100 microgL(-1)) or the third generation oil dispersant Corexit9527 (1, 5, 10 and 50 ppm). Additionally, the expression of P-gp was examined following exposure to 0.5 and 5 microM concentrations of the chemotherapeutic drug vinblastine, a classic substrate of P-gp. The expression of P-gp increased significantly in corals treated with vinblastine and also increased following exposure to copper and Corexit9527. Hsp70, and to a lesser extent Hsp90 expression increased following exposure to copper and Corexit9527 indicating a general cellular stress response. Densities of symbiotic algae in the tissues of the corals did not change significantly during the experiments, nor was any loss or paling of coral tissues observed. These findings provide important insight into how corals defend themselves against pollution and complement ongoing initiatives developing molecular biomarkers of stress in reef-building corals.

  9. Binding phenomena and fluorescence quenching. II: Photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation

    Science.gov (United States)

    Callis, Patrik R.

    2014-12-01

    The three amino acids with aromatic ring side chains-phenylalanine (Phe), tyrosine (Tyr), and especially tryptophan (Trp) have played a long and productive role in helping unlock the secrets of protein behavior by optical spectroscopy (absorption, fluorescence, circular dichroism, etc.) In principle, an appropriately placed Trp will undergo fluorescence wavelength and/or intensity changes upon whatever functional process a protein performs. Although perceived to be enigmatic and not well understood, Trp is arguably now better understood than many of the extrinsic probes currently in use. Basic principles of intrinsic tryptophan fluorescence quenching and wavelength shifts in proteins are presented, with strong emphasis on the importance of electrostatics. The condensed description of findings from recent experiments and simulations of tryptophan fluorescence and intrinsic quenching in proteins is designed to help authors in planning and interpreting experimental results of ligand binding studies.

  10. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis, streptavidin-binding peptide (SBP, and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM. These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

  11. Quantitative Determination of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    CERN Document Server

    Wu, Yong; Ou, Jimmy; Li, Min; Toro, Ligia; Stefani, Enrico

    2009-01-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component. This new method is illustrated by analyzing colocalization in both simulated and biological images.

  12. Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity.

    Science.gov (United States)

    Zeng, Yan-Syun; Gao, Ruo-Cing; Wu, Ting-Wei; Cho, Chien; Tan, Kui-Thong

    2016-08-17

    Despite the promising improvements made recently on fluorescence probes for the detection of enzymes and reactive small molecules, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag protein and three environment-sensitive fluorescence probes to overcome these two problems. The probes were constructed by following the current probe design for enzymes and reactive small molecules but with an additional benzylguanine moiety for selective SNAP-tag conjugation. The SNAP-tag conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma, whereas analyte concentrations were overestimated up to 700-fold when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically, as our probes displayed 390-fold fluorescence enhancement upon SNAP-tag conjugation, in stark contrast to the weak fluorescence of the free probes in aqueous solutions. Compared with the conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcame many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

  13. Plasmon-enhanced emission from single fluorescent proteins

    Science.gov (United States)

    Donehue, Jessica E.; Haas, Beth L.; Wertz, Esther; Talicska, Courtney N.; Biteen, Julie S.

    2013-02-01

    In this work, we use evaporated gold nanoparticle films (GNPFs) as substrates for plasmon-enhanced imaging of two fluorescent proteins (FPs): mCherry and YFP. Through single-molecule epifluorescence microscopy, we show enhancement of single FP emission in the presence of GNPFs. The gold-coupled FPs demonstrate emission up to four times brighter and seven times longer lived, yielding order-of-magnitude enhancements in total photons detected. Ultimately, this results in increased localization accuracies for single-molecule imaging. Furthermore, we introduce preliminary results for enhancement of mCherry-labeled TcpP membrane proteins inside live Vibrio cholerae cells coupled to GNPFs. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

  14. 叶绿素荧光技术在珊瑚礁研究中的应用%Application of Chlorophyll Fluorescence Technique in the Study of Coral Reefs

    Institute of Scientific and Technical Information of China (English)

    周洁; 施祺; 余克服

    2011-01-01

    Mutualism between corals and their zooxanthellae is the essential feature of reef formation. Through performing photosynthesis, zooxanthellae nourish the host coral as well as help it deposit its skeleton. Thus, the chlorophyll fluorescence technique, previously being used in the study of photosynthesis of plants, including algae, can shed light on the research of coral reefs, and has been widely spread in the field. Its application contains several aspects: revealing of photophysiology principles of symbiont dinoflagelates, explanation of the machanism of coral bleaching, early warning of coral bleaching, monitoring the responses of coral to pollutions, monitoring the effects of turbidity to coral growth, and exploring the ecological pattern of coral reef. PAM fluorometry has good potential for such applications, as fluorescence yield is a sensitive, non-invasive indicator of sublethal stress. Therefore, the study of physiological ecology of scleractinian corals may be more effective in the aid of chlorophyll fluorescence technique.%珊瑚礁生态系统最基本的生态特征是虫黄藻与珊瑚虫的共生,虫黄藻的光合作用在珊瑚礁生态系统中发挥着重要作用,因此用于测定植物光合作用的叶绿素荧光技术在认识珊瑚礁生态系统中得到越来越广泛的应用.应用方面主要包括:①揭示珊瑚共生藻光生理学原理;②探索珊瑚白化的机制;③监测及预警珊瑚白化事件;④研究珊瑚对污染的响应;⑤监测珊瑚对水体浑浊的响应;⑥探寻珊瑚礁生态模式.叶绿素荧光技术具有快速、灵敏和非破坏性测量等优点,在造礁珊瑚生理生态研究方面将有广阔的应用前景.

  15. Levels of immunity parameters underpin bleaching and disease susceptibility of reef corals.

    Science.gov (United States)

    Palmer, Caroline V; Bythell, John C; Willis, Bette L

    2010-06-01

    Immunity is a key life history trait that may explain hierarchies in the susceptibility of corals to disease and thermal bleaching, two of the greatest current threats to coral health and the persistence of tropical reefs. Despite their ongoing and rapid global decline, there have been few investigations into the immunity mechanisms of reef-building corals. Variables commonly associated with invertebrate immunity, including the presence of melanin, size of melanin-containing granular cells, and phenoloxidase (PO) activity, as well as concentrations of fluorescent proteins (FPs), were investigated in hard (Scleractinia) and soft (Alcyonacea) corals spanning 10 families from the Great Barrier Reef. Detectable levels of these indicators were present in all corals investigated, although relative investment differed among coral taxa. Overall levels of investment were inversely correlated to thermal bleaching and disease susceptibility. In addition, PO activity, melanin-containing granular cell size, and FP concentration were each found to be significant predictors of susceptibility and thus may play key roles in coral immunity. Correlative evidence that taxonomic (family-level) variation in the levels of these constituent immunity parameters underpins susceptibility to both thermal bleaching and disease indicates that baseline immunity underlies the vulnerability of corals to these two threats. This reinforces the necessity of a holistic approach to understanding bleaching and disease in order to accurately determine the resilience of coral reefs.

  16. Fluorescence energy transfer in the bi-fluorescent S-layer tandem fusion protein ECFP-SgsE-YFP.

    Science.gov (United States)

    Kainz, Birgit; Steiner, Kerstin; Sleytr, Uwe B; Pum, Dietmar; Toca-Herrera, José L

    2010-12-01

    This work reports for the first time on the fabrication of a bi-functional S-layer tandem fusion protein which is able to self-assemble on solid supports without losing its functionality. Two variants of the green fluorescent protein (GFP) were genetically combined with a self-assembly system having the remarkable opportunity to interact with each other and act as functional nanopatterning biocoating. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the cyan ECFP donor protein at the SgsE N-terminus and with the yellow YFP acceptor protein at the C-terminus. The fluorescence energy transfer was studied with spectrofluorimetry, confocal microscopy and flow cytometry, whilst protein self-assembly (on silicon dioxide particles) and structural investigations were carried out with atomic force microscopy (AFM). The fluorescence resonance energy transfer efficiency of reassembled SgsE tandem protein was 20.0 ± 6.1% which is almost the same transfer efficiency shown in solution (19.6 ± 0.1%). This work shows that bi-fluorescent S-layer fusion proteins self-assemble on silica particles retaining their fluorescent properties.

  17. Fluorescent detection of C-reactive protein using polyamide beads

    Science.gov (United States)

    Jagadeesh, Shreesha; Chen, Lu; Aitchison, Stewart

    2016-03-01

    Bacterial infection causes Sepsis which is one of the leading cause of mortality in hospitals. This infection can be quantified from blood plasma using C - reactive protein (CRP). A quick diagnosis at the patient's location through Point-of- Care (POC) testing could give doctors the confidence to prescribe antibiotics. In this paper, the development and testing of a bead-based procedure for CRP quantification is described. The size of the beads enable them to be trapped in wells without the need for magnetic methods of immobilization. Large (1.5 mm diameter) Polyamide nylon beads were used as the substrate for capturing CRP from pure analyte samples. The beads captured CRP either directly through adsorption or indirectly by having specific capture antibodies on their surface. Both methods used fluorescent imaging techniques to quantify the protein. The amount of CRP needed to give a sufficient fluorescent signal through direct capture method was found suitable for identifying bacterial causes of infection. Similarly, viral infections could be quantified by the more sensitive indirect capture method. This bead-based assay can be potentially integrated as a disposable cartridge in a POC device due to its passive nature and the small quantities needed.

  18. Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection.

    Science.gov (United States)

    Benz, Christian; Retzbach, Heiko; Nagl, Stefan; Belder, Detlev

    2013-07-21

    Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.

  19. 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.

  20. 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.

  1. Highly Fluorescent Green Fluorescent Protein Chromophore Analogues Made by Decorating the Imidazolone Ring.

    Science.gov (United States)

    Gutiérrez, Sara; Martínez-López, David; Morón, María; Sucunza, David; Sampedro, Diego; Domingo, Alberto; Salgado, Antonio; Vaquero, Juan J

    2015-12-14

    The synthesis and photophysical behavior of an unexplored family of green fluorescent protein (GFP)-like chromophore analogues is reported. The compound (Z)-4-(4-hydroxybenzylidene)-1-propyl-2-(propylamino)-1H-imidazol-5(4 H)-one (p-HBDNI, 2 a) exhibits significantly enhanced fluorescence properties relative to the parent compound (Z)-5-(4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one (p-HBDI, 1). p-HBDNI was considered as a model system and the photophysical properties of other novel 2-amino-3,5-dihydro-4H-imidazol-4-one derivatives were evaluated. Time-dependent DFT calculations were carried out to rationalize the results. The analogue AIDNI (2 c), in which the 4-hydroxybenzyl group of p-HBDNI was replaced by an azaindole group, showed improved photophysical properties and potential for cell staining. The uptake and intracellular distribution of 2 c in living cells was investigated by confocal microscopy imaging.

  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. 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

  4. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

    Science.gov (United States)

    Caldwell, C R

    1987-07-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

  5. 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.

  6. Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

    Science.gov (United States)

    Talukder, Poulami; Chen, Shengxi; Roy, Basab; Yakovchuk, Petro; Spiering, Michelle M; Alam, Mohammad P; Madathil, Manikandadas M; Bhattacharya, Chandrabali; Benkovic, Stephen J; Hecht, Sidney M

    2015-12-29

    Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

  7. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Science.gov (United States)

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.

    2015-01-01

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  8. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Directory of Open Access Journals (Sweden)

    Bernhard Hochreiter

    2015-10-01

    Full Text Available Fluorescence- or Förster resonance energy transfer (FRET is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a cleavage; (b conformational-change; (c mechanical force and (d changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  9. Fluorescent proteins as genetically encoded FRET biosensors in life sciences.

    Science.gov (United States)

    Hochreiter, Bernhard; Garcia, Alan Pardo; Schmid, Johannes A

    2015-10-16

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  10. The electronic excited states of green fluorescent protein chromophore models

    Science.gov (United States)

    Olsen, Seth Carlton

    We explore the properties of quantum chemical approximations to the excited states of model chromophores of the green fluorescent protein of A. victoria. We calculate several low-lying states by several methods of quantum chemical calculation, including state-averaged complete active space SCF (CASSCF) methods, time dependent density functional theory (TDDFT), equation-of motion coupled cluster (EOM-CCSD) and multireference perturbation theory (MRPT). Amongst the low-lying states we identify the optically bright pipi* state of the molecules and examine its properties. We demonstrate that the state is dominated by a single configuration function. We calculate zero-time approximations to the resonance Raman spectrum of GFP chromophore models, and assign published spectra based upon these.

  11. Enhanced green fluorescent protein-mediated synthesis of biocompatible graphene.

    Science.gov (United States)

    Gurunathan, Sangiliyandi; Woong Han, Jae; Kim, Eunsu; Kwon, Deug-Nam; Park, Jin-Ki; Kim, Jin-Hoi

    2014-10-03

    Graphene is the 2D form of carbon that exists as a single layer of atoms arranged in a honeycomb lattice and has attracted great interest in the last decade in view of its physical, chemical, electrical, elastic, thermal, and biocompatible properties. The objective of this study was to synthesize an environmentally friendly and simple methodology for the preparation of graphene using a recombinant enhanced green fluorescent protein (EGFP). The successful reduction of GO to graphene was confirmed using UV-vis spectroscopy, and FT-IR. DLS and SEM were employed to demonstrate the particle size and surface morphology of GO and EGFP-rGO. The results from Raman spectroscopy suggest the removal of oxygen-containing functional groups from the surface of GO and formation of graphene with defects. The biocompatibility analysis of GO and EGFP-rGO in human embryonic kidney (HEK) 293 cells suggests that GO induces significant concentration-dependent cell toxicity in HEK cells, whereas graphene exerts no adverse effects on HEK cells even at a higher concentration (100 μg/mL). Altogether, our findings suggest that recombinant EGFP can be used as a reducing and stabilizing agent for the preparation of biocompatible graphene. The novelty and originality of this work is that it describes a safe, simple, and environmentally friendly method for the production of graphene using recombinant enhanced green fluorescent protein. Furthermore, the synthesized graphene shows excellent biocompatibility with HEK cells; therefore, biologically synthesized graphene can be used for biomedical applications. To the best of our knowledge, this is the first and novel report describing the synthesis of graphene using recombinant EGFP.

  12. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

    Science.gov (United States)

    Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

    2015-07-01

    In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.

  13. A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range.

    Science.gov (United States)

    Xue, Lin; Prifti, Efthymia; Johnsson, Kai

    2016-04-27

    We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.

  14. Coral microbiology

    Science.gov (United States)

    Rosenberg, Eugene; Kellogg, Christina A.; Rohwer, Forest

    2007-01-01

    In the last 30 years, there has been approximately a 30% loss of corals worldwide, largely due to emerging diseases (Harvell et al., 2002, 2007; Hughes et al., 2003). Coral microbiology is a new field, driven largely by a desire to understand the interactions between corals and their symbiotic microorganisms and to use this knowledge to eventually prevent the spread of coral diseases.

  15. Bond selection in the photoisomerization reaction of anionic green fluorescent protein and kindling fluorescent protein chromophore models.

    Science.gov (United States)

    Olsen, Seth; Smith, Sean C

    2008-07-09

    The chromophores of the most widely known fluorescent proteins (FPs) are derivatives of a core p-hydroxybenzylidene-imidazolinon-5-one (HBI) motif, which usually occurs as a phenolate anion. Double bond photoisomerization of the exocyclic bridge of HBI is widely held to be an important internal conversion mechanism for FP chromophores. Herein we describe the ground and excited-state electronic structures and potential energy surfaces of two model chromophores: 4- p-hydroxybenzylidiene-1,2-dimethyl-imidazolin-5-one anion (HBDI), representing green FPs (GFPs), and 2-acetyl-4-hydroxybenylidene-1-methyl-imidazolin-5-one anion (AHBMI), representing kindling FPs (KFPs). These chromophores differ by a single substitution, but we observe qualitative differences in the potential energy surfaces which indicate inversion of bond selection in the photoisomerization reaction. Bond selection is also modulated by whether the reaction proceeds from a Z or an E conformation. These configurations correspond to fluorescent and nonfluorescent states of structurally characterized FPs, including some which can be reversibly switched by specific illumination regimes. We explain the difference in bond selectivity via substituent stabilization effects on a common set of charge-localized chemical structures. Different combinations of these structures give rise to both optically active (planar) and twisted intramolecular charge-transfer (TICT) states of the molecules. We offer a prediction of the gas-phase absorption of AHBMI, which has not yet been measured. We offer a hypothesis to explain the unusual fluorescence of AHBMI in DMF solution, as well as an experimental proposal to test our hypothesis.

  16. 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.

  17. Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

    Science.gov (United States)

    Pattanaik, Sitakanta; Werkman, Joshua R; Yuan, Ling

    2011-01-01

    Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

  18. Recent progress in design of protein-based fluorescent biosensors and their cellular applications.

    Science.gov (United States)

    Tamura, Tomonori; Hamachi, Itaru

    2014-12-19

    Protein-based fluorescent biosensors have emerged as key bioanalytical tools to visualize and quantify a wide range of biological substances and events in vitro, in cells, and even in vivo. On the basis of the construction method, the protein-based fluorescent biosensors can be principally classified into two classes: (1) genetically encoded fluorescent biosensors harnessing fluorescent proteins (FPs) and (2) semisynthetic biosensors comprised of protein scaffolds and synthetic fluorophores. Recent advances in protein engineering and chemical biology not only allowed the further optimization of conventional biosensors but also facilitated the creation of novel biosensors based on unique strategies. In this review, we survey the recent studies in the development and improvement of protein-based fluorescent biosensors and highlight the successful applications to live cell and in vivo imaging. Furthermore, we provide perspectives on possible future directions of the technique.

  19. Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

    Science.gov (United States)

    Young, W S; Iacangelo, A; Luo, X Z; King, C; Duncan, K; Ginns, E I

    1999-12-01

    Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

  20. A fluorescence spectroscopic study of a coagulating protein extracted from Moringa oleifera seeds.

    Science.gov (United States)

    Kwaambwa, H M; Maikokera, R

    2007-11-15

    The fluorescence studies of coagulating protein extracted from Moringa oleifera seeds have been studied using steady-state intrinsic fluorescence. The fluorescence spectra are dominated by tryptophan emission and the emission peak maximum (lambda(max)=343+ or -2nm) indicated that the tryptophan residue is not located in the hydrophobic core of the protein. Changes in solution pH affected the protein conformation as indicated by changes in the tryptophan fluorescence above pH 9 whereas the ionic strength had minimal effect. The exposure and environments of the tryptophan residue were determined using collisional quenchers.

  1. A pH-sensitive red fluorescent protein compatible with hydrophobic resin embedding

    Science.gov (United States)

    Guo, Wenyan; Gang, Yadong; Liu, Xiuli; Zhou, Hongfu; Zeng, Shaoqun

    2017-02-01

    pH sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EYFP or EGFP improved from GFP in jellyfish are good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is of urgent need. Here a pH sensitive red fluorescent protein, pHuji, is selected and verified to be compatible with hydrophobic resin embedding and thus may be promising for dual-colour chemical reactivation imaging in conjunction with EGFP or EYFP.

  2. Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin : Slow Water-Protein Relaxation Unmasked

    NARCIS (Netherlands)

    Xu, Jianhua; Chen, Binbin; Callis, Patrik Robert; Muiño, Pedro L; Rozeboom, Henriette J; Broos, Jaap; Toptygin, Dmitri; Brand, Ludwig; Knutson, Jay R

    2015-01-01

    Time Dependent Fluorescence Stokes (emission wavelength) Shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many fluor

  3. Picosecond Fluorescence Dynamics of Tryptophan and 5-Fluorotryptophan in Monellin : Slow Water-Protein Relaxation Unmasked

    NARCIS (Netherlands)

    Xu, Jianhua; Chen, Binbin; Callis, Patrik Robert; Muiño, Pedro L; Rozeboom, Henriette J; Broos, Jaap; Toptygin, Dmitri; Brand, Ludwig; Knutson, Jay R

    2015-01-01

    Time Dependent Fluorescence Stokes (emission wavelength) Shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many

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

    OpenAIRE

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

    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 corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based F...

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

    Science.gov (United States)

    Nguyen, Hau B; Hung, Li-Wei; Yeates, Todd O; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-12-01

    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.

  6. Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

    Directory of Open Access Journals (Sweden)

    S.S.G. Ferguson

    1998-11-01

    Full Text Available G protein-coupled receptor (GPCR activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs. Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.

  7. 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...

  8. 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 con...

  9. 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...

  10. FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

    OpenAIRE

    Raina, Shweta A.; Jeffrey Tsai; Montserrat Samsó; Fessenden, James D.

    2012-01-01

    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) inse...

  11. In vitro regulation of CaCO(3) crystal growth by the highly acidic proteins of calcitic sclerites in soft coral, Sinularia Polydactyla.

    Science.gov (United States)

    Rahman, M Azizur; Oomori, Tamotsu

    2009-01-01

    Acidic proteins are generally thought to control mineral formation and growth in biocalcification. Analysis of proteinaceous components in the soluble and insoluble matrix fractions of sclerites in Sinularia polydactyla indicates that aspartic acid composes about 60% of the insoluble and 29% of the soluble matrix fractions. We previously analyzed aspartic acids in the matrix fractions (insoluble = 17 mol%; soluble = 38 mol%) of sclerites from a different type of soft coral, Lobophytum crassum, which showed comparatively lower aspartic acid-rich proteins than S. polydactyla. Thus, characterization of highly acidic proteins in the organic matrix of present species is an important first step toward linking function to individual proteins in soft coral. Here, we show that aspartic-acid rich proteins can control the CaCO(3) polymorph in vitro. The CaCO(3) precipitates in vitro in the presence of aspartic acid-rich proteins and 50 mM Mg(2+) was verified by Raman microprobe analysis. The matrix proteins of sclerites demonstrated that the aspartic-acid rich domain is crucial for the calcite precipitation in soft corals. The crystalline form of CaCO(3) in the presence of aspartic acid-rich proteins in vitro was identified by X-ray diffraction and, revealed calcitic polymorphisms with a strong (104) reflection. The structure of soft coral organic matrices containing aspartate-rich proteins and polysaccharides was assessed by Fourier transform infrared spectroscopy. These results strongly suggest that the aspartic acid-rich proteins within the organic matrix of soft corals play a key role in biomineralization regulation.

  12. Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

    Science.gov (United States)

    Nienhaus, Karin; Nienhaus, G. Ulrich

    2016-11-01

    Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

  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. Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

    Science.gov (United States)

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

    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 corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

  15. Fluorescence lifetime measurements of intrinsically unstructured proteins: application to α-synuclein.

    Science.gov (United States)

    Schreurs, Sarah; Kluba, Malgorzata; Meuvis, Jessika; Engelborghs, Yves

    2012-01-01

    Lifetimes of fluorescent states and their fluorescence intensities are strictly coupled and very sensitive to the environment of the fluorophores. The advantage of measuring lifetimes, next to intensities, comes from the fact that it can reveal heterogeneity and dynamic properties of this environment. In this way lifetime analysis can be used to characterize static and dynamic conformational properties and heterogeneity of fluorescent groups in different areas of a protein and as a function of time for an evolving protein. The phenomena that determine the lifetime of a label are its intrinsic properties, dynamic quenching by neighboring groups, exposure to the solvent, as well as Förster resonance energy transfer (FRET) between different groups. The basic principles of these fluorescence phenomena can be found extensively described in the excellent book of Lakowicz (Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, 2006). The fluorescent groups involved are either natural amino acid side chains like tryptophan (Trp) or tyrosine (Tyr), or fluorescent labels covalently engineered into the protein. Even a single fluorescent group can show indications of heterogeneity in the local environment. If several natural fluorescent groups are present, the properties of the individual groups can be separated using site-directed mutagenesis, and additivity of their contributions can be analyzed (Engelborghs, Spectrochim Acta A Mol Biomol Spectrosc 57(11):2255-2270, 2001). If no fluorescent group is naturally present, site-directed mutagenesis can be used to introduce either a fluorescent amino acid or a cysteine allowing chemical labeling.

  16. Mapping fast protein folding with multiple-site fluorescent probes.

    Science.gov (United States)

    Prigozhin, Maxim B; Chao, Shu-Han; Sukenik, Shahar; Pogorelov, Taras V; Gruebele, Martin

    2015-06-30

    Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

  17. 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 i......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...

  18. To understand coral disease, look at coral cells

    Science.gov (United States)

    Work, Thierry M.; Meteyer, Carol

    2014-01-01

    Diseases threaten corals globally, but 40 years on their causes remain mostly unknown. We hypothesize that inconsistent application of a complete diagnostic approach to coral disease has contributed to this slow progress. We quantified methods used to investigate coral disease in 492 papers published between 1965 and 2013. Field surveys were used in 65% of the papers, followed by biodetection (43%), laboratory trials (20%), microscopic pathology (21%), and field trials (9%). Of the microscopic pathology efforts, 57% involved standard histopathology at the light microscopic level (12% of the total investigations), with the remainder dedicated to electron or fluorescence microscopy. Most (74%) biodetection efforts focused on culture or molecular characterization of bacteria or fungi from corals. Molecular and immunological tools have been used to incriminate infectious agents (mainly bacteria) as the cause of coral diseases without relating the agent to specific changes in cell and tissue pathology. Of 19 papers that declared an infectious agent as a cause of disease in corals, only one (5%) used microscopic pathology, and none fulfilled all of the criteria required to satisfy Koch’s postulates as applied to animal diseases currently. Vertebrate diseases of skin and mucosal surfaces present challenges similar to corals when trying to identify a pathogen from a vast array of environmental microbes, and diagnostic approaches regularly used in these cases might provide a model for investigating coral diseases. We hope this review will encourage specialists of disease in domestic animals, wildlife, fish, shellfish, and humans to contribute to the emerging field of coral disease.

  19. Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants.

    Science.gov (United States)

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

    2011-09-01

    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.

  20. 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.

  1. New insights in the interpretation of tryptophan fluorescence : origin of the fluorescence lifetime and characterization of a new fluorescence parameter in proteins: the emission to excitation ratio.

    Science.gov (United States)

    Albani, J R

    2007-07-01

    Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore-environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were

  2. 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.

  3. New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

    OpenAIRE

    Kuznetsova, Alexandra A.; Kuznetsov, Nikita A.; Vorobjev, Yuri N.; Barthes, Nicolas P. F.; Benoît Y Michel; Alain Burger; 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...

  4. In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichiacoli expressing infrared fluorescent protein in mice

    OpenAIRE

    Berlec, Aleš; Štrukelj, Borut; Završnik, Janja; Turk, Boris; Butinar, Miha

    2016-01-01

    Background In vivo imaging of orally administered lactic acid bacteria (LAB) and commensal bacteria in mice is shown to provide information on the spatial and temporal distribution of bacteria in the gastrointestinal tract. The bacteria can be detected and monitored using bioluminescence or near-infrared fluorescence. Results Fluorescence imaging of bacteria was established by expressing the infrared fluorescent protein IRFP713 in Lactococcus lactis, Lactobacillus plantarum and Escherichia co...

  5. Nitric oxide and heat shock protein 90 co-regulate temperature-induced bleaching in the soft coral Eunicea fusca

    Science.gov (United States)

    Ross, Cliff

    2014-06-01

    Coral bleaching represents a complex physiological process that is affected not only by environmental conditions but by the dynamic internal cellular biology of symbiotic dinoflagellates ( Symbiodinium spp.) and their cnidarian hosts. Recently, nitric oxide (NO) has emerged as a key molecule involved with the expulsion of Symbiodinium from host cnidarian cells. However, the site of production remains under debate, and the corresponding signaling pathways within and between host and endosymbiont remain elusive. In this study, using freshly isolated Symbiodinium from the soft coral Eunicea fusca, I demonstrate that thermally induced stress causes an upregulation in Symbiodinium heat shock protein 90 (Hsp90). In turn, Hsp90 shows a concomitant ability to enhance the activity of a constitutively expressed isoform of NO synthase. The resulting production of NO constitutes a signaling molecule capable of inducing Symbiodinium expulsion. Using nitric oxide synthase (NOS) and Hsp90 polyclonal antibodies, thermal stress-induced Hsp90 was shown to co-immunoprecipitate with a constitutive isoform of NOS. The specific blocking of Hsp90 activity, with the Hsp90 inhibitor geldanamycin, was capable of inhibiting NO production implicating the involvement of a coordinated regulatory system. These results have strong evolutionary implications for Hsp90-NOS chaperone complexes among biological kingdoms and provide evidence for a new functional role in symbiotic associations.

  6. Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.

    Science.gov (United States)

    Smith, Elizabeth M; Hennen, Jared; Chen, Yan; Mueller, Joachim D

    2015-07-01

    This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The importance of monitoring metabolic recovery in the coral Acropora cervicornis after short-term exposure to drilling muds: Calcification rate and protein concentration

    Science.gov (United States)

    Kendall, J. J.; Powell, E. N.; Connor, S. J.; Bright, T. J.; Zastrow, C. E.

    1984-04-01

    The effect of used drilling muds on coral health was examined by monitoring changes in calcification rate and soluble tissue protein concentration in the coral Acropora cervicornis. Exposure to 25 ppm (v/v) of one mud for 24 h reduced calcification rate in the growing tips by as much as 62%. In recovery experiments, corals were exposed to drilling muds for 24 h; some of them were allowed to recover in clean seawater for 48 h. After the 24-hour exposure, calcification rates were significantly less than those of the controls. After a 48-hour recovery period, calcification rates returned to control levels for one mud but were still significantly below control levels for another. The results indicate that the capacity for recovery after exposure cannot be predicted from the results of experiments on exposure only. Recovery capacity must be independently verified for all studies on the effects of short-term exposure to drilling muds.

  8. Absorption, steady-state fluorescence, fluorescence lifetime, and 2D self-assembly properties of engineered fluorescent S-layer fusion proteins of Geobacillus stearothermophilus NRS 2004/3a.

    Science.gov (United States)

    Kainz, Birgit; Steiner, Kerstin; Möller, Marco; Pum, Dietmar; Schäffer, Christina; Sleytr, Uwe B; Toca-Herrera, José L

    2010-01-11

    S-layer fusion protein technology was used to design four different fluorescent fusion proteins with three different GFP mutants and the red fluorescent protein mRFP1. Their absorption spectra, steady-state fluorescence, and fluorescence lifetime were investigated as a function of pH. It was found that fluorescence intensities and lifetime of the GFP mutant S-layer fusion proteins decreased about 50% between pH 6 and pH 5. The spectral properties of the red S-layer fusion protein were minimally affected by pH variations. These results were compared with His-tagged reference fluorescent proteins, demonstrating that the S-layer protein did not change the general spectral properties of the whole fusion protein. In addition, the pK(a) values of the fluorescent S-layer fusion proteins were calculated. Finally, it was shown that the S-layer fusion proteins were able to self-assemble forming 2D nanostructures of oblique p2 symmetry with lattice parameters of about a = 11 nm, b = 14 nm, and gamma = 80 degrees . The fluorescence tag did not hinder the natural self-assembly process of the S-layer protein. The combination of the fluorescence properties and the self-assembly ability of the engineered fusion proteins make them a promising tool to generate biomimetic surfaces for future applications in nanobiotechnology at a wide range of pH.

  9. Microspectroscopic analysis of green fluorescent proteins infiltrated into mesoporous silica nanochannels.

    NARCIS (Netherlands)

    Ma, Y.; Rajendran, P.; Blum, C.; Cesa, Y.; Gartmann, N.; Bruhwiler, D.; Subramaniam, V.

    2011-01-01

    The infiltration of enhanced green fluorescent protein (EGFP) into nanochannels of different diameters in mesoporous silica particles was studied in detail by fluorescence microspectroscopy at room temperature. Silica particles from the MCM-41, ASNCs and SBA-15 families possessing nanometer-sized (3

  10. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

    Science.gov (United States)

    Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

    2014-05-01

    Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

  11. Photo-initiated dynamics and spectroscopy of the deprotonated Green Fluorescent Protein chromophore

    DEFF Research Database (Denmark)

    Bochenkova, Anastasia; Andersen, Lars Henrik

    2013-01-01

    This chapter combines recent advances in understanding the photophysics of the chromophore anion of the Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria. GFP and its homologues are widely used for in vivo labeling in biology through their remarkable fluorescent properties...

  12. Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli

    NARCIS (Netherlands)

    García-Cayuela, T.; Cadiñanos, de L.P.; Mohedano, M.L.; Palencia, de P.F.; Boden, D.; Wells, J.; Peláez, C.; López, P.; Requena, T.

    2012-01-01

    Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked,

  13. New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

    Institute of Scientific and Technical Information of China (English)

    Christian Gehl; Rainer Waadt; J(o)rg Kudla; Ralf-R. Mendel; Robert Hansch

    2009-01-01

    Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluores-cence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the locali-zation of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of can-didate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the com-bination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cn×6/Cn×6 and Cn×6/Cn×7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

  14. 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.

  15. Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

    Science.gov (United States)

    Ah-Fong, Audrey M V; Judelson, Howard S

    2011-09-01

    Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  16. Very bright green fluorescent proteins from the Pontellid copepod Pontella mimocerami.

    Directory of Open Access Journals (Sweden)

    Marguerite E Hunt

    Full Text Available BACKGROUND: Fluorescent proteins (FP homologous to the green fluorescent protein (GFP from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. RESULTS: Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae, which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M(-1 cm(- and quantum yields (0.92, which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. CONCLUSIONS: The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.

  17. Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen for human G-protein-coupled receptor signaling in microbial yeast cells.

    Directory of Open Access Journals (Sweden)

    Yasuyuki Nakamura

    Full Text Available G-protein-coupled receptors (GPCRs are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer's disease and Parkinson's disease, respectively were chosen as human GPCR(s. The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s.

  18. Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

    National Research Council Canada - National Science Library

    Danielle E. Johnson; Hui-wang Ai; Peter Wong; James D. Young; Robert E. Campbell; Joseph R. Casey

    2009-01-01

    .... 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...

  19. POLA EKSPRESI GEN ENHANCED GREEN FLUORESCENT PROTEIN PADA EMBRIO DAN LARVA IKAN PATIN SIAM (Pangasianodon hypophthalmus)

    OpenAIRE

    Raden Roro Sri Pudji Sinarni Dewi; Alimuddin Alimuddin; Agus Oman Sudrajat; Komar Sumantadinata; Erma Primanita Hayuningtyas

    2016-01-01

    Penelitian ekspresi sementara (transient expression) dari transgen secara in vivo menggunakan gen reporter berguna untuk mendesain konstruksi gen yang akan digunakan pada penelitian transgenesis. Gen reporter yang umum digunakan dalam penelitian ekspresi sementara transgen adalah gen GFP (green fluorescent protein). Pengamatan gen EGFP (enhanced green fluorescent protein) pada embrio dan larva ikan patin siam (Pangasianodon hypophthalmus) ditujukan untuk mendapatkan informasi mengenai kema...

  20. Fiber-optic system for monitoring fast photoactivation dynamics of optical highlighter fluorescent proteins.

    Science.gov (United States)

    Pei, Zhiguo; Qin, Lingsong; Zhang, Zhihong; Zeng, Shaoqun; Huang, Zhen-Li

    2011-08-01

    Characterizing the photoactivation performance of optical highlighter fluorescent proteins is crucial to the realization of photoactivation localization microscopy. In contrast to those fluorescence-based approaches that require complex data processing and calibration procedures, here we report a simple and quantitative alternative, which relies on the measurement of small absorption spectra changes over time with a fiber-optic system. Using Dronpa as a representative highlighter protein, we have investigated the capacity of this system in monitoring the fast photoactivation process.

  1. Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation

    NARCIS (Netherlands)

    de Kort, B.J.

    2012-01-01

    Proteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive prot

  2. [Spectral diversity among the members of the family of Green Fluorescent Protein in hydroid jellyfish (Cnidaria, Hydrozoa)].

    Science.gov (United States)

    Ianushevich, Iu G; Shagin, D A; Fradkov, A F; Shakhbazov, K S; Barsova, E V; Gurskaia, N G; Labas, Iu A; Matts, M V; Luk'ianov, k A; Lul'ianov, S A

    2005-01-01

    The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.

  3. Biology of corals and coral reefs

    Digital Repository Service at National Institute of Oceanography (India)

    Rajkumar, R.; Parulekar, A.H.

    This chapter deals with biology of corals, coral reefs (in general) and coral reefs of the Indian Ocean. Biology of corals is lucidly dealt with, beginning from the clarification on hermatypic and ahermatypic forms. A complete account...

  4. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  5. 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.

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

    DEFF Research Database (Denmark)

    Hojman, Pernille; Eriksen, Jens; Gehl, Julie

    2009-01-01

    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...... 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. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

    Directory of Open Access Journals (Sweden)

    Michele L Markwardt

    Full Text Available Cyan fluorescent proteins (CFPs, such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

  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.

  9. Fluorescent Pressure Response of Protein-Nanocluster Polymer Composites

    Science.gov (United States)

    2016-05-01

    fluorescence spectra of BSA:AuNCs found in literature.10 Notably, the BSA:AuNCs experience peak broadening and blue shifting upon incorporation into the...semiconductor quantum dots. Nature Biotechnology . 2004;22:969–976. 9. Medintz IL, Uyeda TH, Goldman ER, Mattoussi H. Quantum dot bioconjugates for imaging

  10. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    Administrator

    times faster in E. coli and exhibits eighteen-fold greater fluorescence .... The generation time (g, min) necessary to double the .... Fitted models for µg GFPuv/ mL adjusted from the polynomial model (µg GFPuv/ mL = 10.28 - 1.13 x1 - 5.65 x3 +.

  11. Fluorescent Ensemble Based on Bispyrene Fluorophore and Surfactant Assemblies: Sensing and Discriminating Proteins in Aqueous Solution.

    Science.gov (United States)

    Fan, Junmei; Ding, Liping; Bo, Yu; Fang, Yu

    2015-10-14

    A particular bispyrene fluorophore (1) with two pyrene moieties covalently linked via a hydrophilic spacer was synthesized. Fluorescence measurements reveal that the fluorescence emission of 1 could be well modulated by a cationic surfactant, dodecyltrimethylammonium bromide (DTAB). Protein sensing studies illustrate that the selected ensemble based on 1/DTAB assemblies exhibits ratiometric responses to nonmetalloproteins and turn-off responses to metalloproteins, which can be used to differentiate the two types of proteins. Moreover, negatively charged nonmetalloproteins can be discriminated from the positively charged ones according to the difference in ratiometric responses. Fluorescence sensing studies with control bispyrenes indicate that the polarity of the spacer connecting two pyrene moieties plays an important role in locating bispyrene fluorophore in DTAB assemblies, which further influences its sensing behaviors to noncovalent interacting proteins. This study sheds light on the influence of the probe structure on the sensing performance of a fluorescent ensemble based on probe and surfactant assemblies.

  12. 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,…

  13. 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,…

  14. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

    Science.gov (United States)

    Ast, Cindy; Foret, Jessica; Oltrogge, Luke M; De Michele, Roberto; Kleist, Thomas J; Ho, Cheng-Hsun; Frommer, Wolf B

    2017-09-05

    Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

  15. Coral choreography

    Science.gov (United States)

    Showstack, Randy

    Viewers clicking onto the Waikiki Aquarium's “Coral Research Cam” any time during daylight hours in Hawaii can catch the latest action of three species of living corals (Acropora sp., Acropora elseyi,and Montipora digitata) and the yellow tang and blue tang fish swimming amongst them in an outdoor aquarium.Waikiki Aquarium Director Bruce Carlson says the camera is part of a new exhibit, “Corals Are Alive!,” which encourages people to view living corals close-up at the aquarium or via the Internet, in order to gain a better appreciation of the corals. “Hopefully through education and awareness, people will be more interested and willing to help with conservation efforts to preserve coral reefs,” says Carlson.

  16. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    Science.gov (United States)

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  17. 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

  18. Improved Fluorescent Protein Contrast and Discrimination by Optically Controlling Dark State Lifetimes.

    Science.gov (United States)

    Chen, Yen-Cheng; Dickson, Robert M

    2017-02-16

    Modulation and optical control of photoswitchable fluorescent protein (PS-FP) dark state lifetimes drastically improves sensitivity and selectivity in fluorescence imaging. The dark state population of PS-FPs generates an out-of-phase fluorescence component relative to the sinusoidally modulated 488 nm laser excitation. Because this apparent phase advanced emission results from slow recovery to the fluorescent manifold, we hasten recovery and, therefore, modulation frequency by varying coillumination intensity at 405 nm. As 405 nm illumination regenerates the fluorescent ground state more rapidly than via thermal recovery, we experimentally demonstrate that secondary illumination can control PS-FPs dark state lifetime to act as an additional dimension for discriminating spatially and spectrally overlapping emitters. This experimental combination of out of phase imaging after optical modulation (OPIOM) and synchronously amplified fluorescence image recovery (SAFIRe) optically controls the fluorescent protein dark state lifetimes for improved time resolution, with the resulting modulation-based selective signal recovery being quantitatively modeled. The combined experimental results and quantitative numerical simulations further demonstrate the potential of SAFIRe-OPIOM for wide-field biological imaging with improved speed, sensitivity, and optical resolution over other modulation-based fluorescence microscopies.

  19. Illuminating plant biology: using fluorescent proteins for high-throughput analysis of protein localization and function in plants.

    Science.gov (United States)

    DeBlasio, Stacy L; Sylvester, Anne W; Jackson, David

    2010-03-01

    First discovered in jellyfish, fluorescent proteins (FPs) have been successfully optimized for use as effective biomarkers within living plant cells. When exposed to light, FPs fused to a protein or regulatory element will fluoresce, and non-invasively mark expression and protein localization, which allows for the in vivo monitoring of diverse cellular processes. In this review, we discuss how FP technology has evolved from small-scale analysis of individual genes to more high-throughput techniques for global expression and functional profiling in plants.

  20. Lumazine Protein from the Bioluminescent Bacterium Photobacterium Phosphoreum: A Fluorescence Study of the Protein-Ligand Equilibrium

    NARCIS (Netherlands)

    Visser, A.J.W.G.; Lee, J.

    1980-01-01

    The changes of fluorescence spectral distribution, polarization, and lifetime of the lumazine protein from Photobacterium phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1′-D-ribityl)lumazine. The equilibrium is

  1. Lumazine Protein from the Bioluminescent Bacterium Photobacterium Phosphoreum: A Fluorescence Study of the Protein-Ligand Equilibrium

    NARCIS (Netherlands)

    Visser, A.J.W.G.; Lee, J.

    1980-01-01

    The changes of fluorescence spectral distribution, polarization, and lifetime of the lumazine protein from Photobacterium phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1′-D-ribityl)lumazine. The equilibrium is rapid

  2. 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

    . Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation......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...

  3. Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

    OpenAIRE

    2015-01-01

    Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as...

  4. PNA-induced assembly of fluorescent proteins using DNA as a framework

    OpenAIRE

    2013-01-01

    Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein−PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)−peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation ...

  5. 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.

  6. 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

  7. Measurement of protein-like fluorescence in river and waste water using a handheld spectrophotometer.

    Science.gov (United States)

    Baker, Andy; Ward, David; Lieten, Shakti H; Periera, Ryan; Simpson, Ellie C; Slater, Malcolm

    2004-07-01

    Protein-like fluorescence intensity in rivers increases with increasing anthropogenic DOM inputs from sewerage and farm wastes. Here, a portable luminescence spectrophotometer was used to investigate if this technology could be used to provide both field scientists with a rapid pollution monitoring tool and process control engineers with a portable waste water monitoring device, through the measurement of river and waste water tryptophan-like fluorescence from a range of rivers in NE England and from effluents from within two waste water treatment plants. The portable spectrophotometer determined that waste waters and sewerage effluents had the highest tryptophan-like fluorescence intensity, urban streams had an intermediate tryptophan-like fluorescence intensity, and the upstream river samples of good water quality the lowest tryptophan-like fluorescence intensity. Replicate samples demonstrated that fluorescence intensity is reproducible to +/- 20% for low fluorescence, 'clean' river water samples and +/- 5% for urban water and waste waters. Correlations between fluorescence measured by the portable spectrophotometer with a conventional bench machine were 0.91; (Spearman's rho, n = 143), demonstrating that the portable spectrophotometer does correlate with tryptophan-like fluorescence intensity measured using the bench spectrophotometer.

  8. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    Science.gov (United States)

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  9. 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

  10. Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells.

    Science.gov (United States)

    Kremers, Gert-Jan; Piston, David

    2010-06-26

    Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

  11. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

    Science.gov (United States)

    Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

    2013-11-01

    Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

  12. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding

    Science.gov (United States)

    Hagelueken, Gregor; Naismith, James H

    2013-01-01

    Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins. PMID:24091556

  13. Comparative immune responses of corals to stressors associated with offshore reef-based tourist platforms.

    Science.gov (United States)

    van de Water, Jeroen A J M; Lamb, Joleah B; van Oppen, Madeleine J H; Willis, Bette L; Bourne, David G

    2015-01-01

    Unravelling the contributions of local anthropogenic and seasonal environmental factors in suppressing the coral immune system is important for prioritizing management actions at reefs exposed to high levels of human activities. Here, we monitor health of the model coral Acropora millepora adjacent to a high-use and an unused reef-based tourist platform, plus a nearby control site without a platform, over 7 months spanning a typical austral summer. Comparisons of temporal patterns in a range of biochemical and genetic immune parameters (Toll-like receptor signalling pathway, lectin-complement system, prophenoloxidase-activating system and green fluorescent protein-like proteins) among healthy, injured and diseased corals revealed that corals exhibit a diverse array of immune responses to environmental and anthropogenic stressors. In healthy corals at the control site, expression of genes involved in the Toll-like receptor signalling pathway (MAPK p38, MEKK1, cFos and ATF4/5) and complement system (C3 and Bf) was modulated by seasonal environmental factors in summer months. Corals at reef platform sites experienced additional stressors over the summer, as evidenced by increased expression of various immune genes, including MAPK p38 and MEKK1. Despite increased expression of immune genes, signs of white syndromes were detected in 31% of study corals near tourist platforms in the warmest summer month. Evidence that colonies developing disease showed reduced expression of genes involved in the complement pathway prior to disease onset suggests that their immune systems may have been compromised. Responses to disease and physical damage primarily involved the melanization cascade and GFP-like proteins, and appeared to be sufficient for recovery when summer heat stress subsided. Overall, seasonal and anthropogenic factors may have interacted synergistically to overwhelm the immune systems of corals near reef platforms, leading to increased disease prevalence in summer at

  14. ZnO nanoparticles assist the refolding of denatured green fluorescent protein.

    Science.gov (United States)

    Pandurangan, Muthuraman; Zamany, Ahmad Jawid; Kim, Doo Hwan

    2016-04-01

    Proteins are essential for cellular and biological processes. Proteins are synthesized and fold into the native structure to become active. The inability of a protein molecule to remain in its native conformation is called as protein misfolding, and this is due to several environmental factors. Protein misfolding and aggregation handle several human diseases. Protein misfolding is believed to be one of the causes of several disorders such as cancer, degenerative diseases, and metabolic pathologies. The zinc oxide (ZnO) nanoparticle was significantly promoted refolding of thermally denatured green fluorescent protein (GFP). In the present study, ZnO nanoparticles interaction with GFP was investigated by ultraviolet-visible spectrophotometer, fluorescence spectrophotometer, and dynamic light scattering. Results suggest that the ZnO nanoparticles significantly assist the refolding of denatured GFP. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Valero Francisco

    2007-05-01

    Full Text Available Abstract Background Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL in Pichia pastoris by means of 2D-fluorimetric techniques Results In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity Conclusion P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.

  16. 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

  17. 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.

  18. Drug/protein interactions studied by time-resolved fluorescence spectroscopy

    Science.gov (United States)

    Gustavsson, Thomas; Markovitsi, Dimitra; Vayá, Ignacio; Bonancía, Paula; Jiménez, M. C.; Miranda, Miguel A.

    2014-09-01

    We report here on a recent time-resolved fluorescence study [1] of the interaction between flurbiprofen (FBP), a chiral non-steroidal anti-inflammatory drug, and human serum albumin (HSA), the main transport protein in the human body. We compare the results obtained for the drug-protein complex with those of various covalently linked flurbiprofentryptophan dyads having well-defined geometries. In all cases stereoselective dynamic fluorescence quenching is observed, varying greatly from one system to another. In addition, the fluorescence anisotropy decays also display a clear stereoselectivity. For the drug-protein complexes, this can be interpreted in terms of the protein microenvironment playing a significant role in the conformational relaxation of FBP, which is more restricted in the case of the (R)- enantiomer.

  19. 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.

  20. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein

    Directory of Open Access Journals (Sweden)

    Hadjantonakis Anna-Katerina

    2005-07-01

    Full Text Available Abstract Background DsRed the red fluorescent protein (RFP isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems. Results We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing. Conclusion Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they

  1. Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopy

    Science.gov (United States)

    Sun, Yuansheng; Booker, Cynthia F.; Day, Richard N.; Periasamy, Ammasi

    2009-02-01

    Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However, the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy. Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor photobleaching FRET microscopy.

  2. 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.

  3. Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins

    Directory of Open Access Journals (Sweden)

    Anna Rosati

    2004-01-01

    Full Text Available Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV, have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR or MRP (multidrug resistance‐related protein (PANC‐1 and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine or MRP (MK571 and probenecid has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

  4. Fluorescence Lifetime Imaging of Free and Protein-Bound NADH

    Science.gov (United States)

    Lakowicz, Joseph R.; Szmacinski, Henryk; Nowaczyk, Kazimierz; Johnson, Michael L.

    1992-02-01

    We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challenging case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images, each obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe an imaging procedure that allows specific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.

  5. Mass Spectrometric Imaging of Red Fluorescent Protein in Breast Tumor Xenografts

    Science.gov (United States)

    Chughtai, Kamila; Jiang, Lu; Post, Harm; Winnard, Paul T.; Greenwood, Tiffany R.; Raman, Venu; Bhujwalla, Zaver M.; Heeren, Ron M. A.; Glunde, Kristine

    2013-05-01

    Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

  6. Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

    Science.gov (United States)

    Köhler, Stephan; Ouahrani-Bettache, Safia; Layssac, Marion; Teyssier, Jacques; Liautard, Jean-Pierre

    1999-01-01

    A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized. PMID:10569794

  7. Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

    OpenAIRE

    Köhler, Stephan; Ouahrani-Bettache, Safia; Layssac, Marion; Teyssier, Jacques; Liautard, Jean-Pierre

    1999-01-01

    A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

  8. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Jae Eun Lee

    2015-06-01

    Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

  9. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Science.gov (United States)

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  10. Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.

    Science.gov (United States)

    Iijima, Issei; Hohsaka, Takahiro

    2009-04-17

    Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in fluorescence upon binding of maltose. Tryptophan-to-phenylalanine substitutions suggest that the increase in fluorescence was the result of a decrease in the quenching of BODIPY-FL by tryptophan located around the binding site. MBP containing a BODIPY-558 moiety also showed a maltose-dependent increase in fluorescence. BODIPY-FL was then additionally incorporated in place of Lys1 of the BODIPY-558-containing MBP as a response to the amber codon. Fluorescence measurements with excitation of BODIPY-FL showed a large change in fluorescence intensity ratio (0.13 to 1.25) upon binding of maltose; this change can be attributed to fluorescence resonance energy transfer (FRET) and maltose-dependent quenching of BODIPY-558. These results demonstrate the usefulness of the position-specific incorporation of fluorescent amino acids in the fluorescence-based detection of protein functions.

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

    Science.gov (United States)

    Straschewski, Sarah; Warmer, Martin; Frascaroli, Giada; Hohenberg, Heinrich; Mertens, Thomas; Winkler, Michael

    2010-02-11

    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.

  12. 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.

  13. A tunable fluorescent timer method for imaging spatial-temporal protein dynamics using light-driven photoconvertible protein.

    Science.gov (United States)

    Zhu, Xinxin; Zhang, Luyuan; Kao, Ya-Ting; Xu, Fang; Min, Wei

    2015-03-01

    Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial-temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation. Herein we propose a light-driven timer concept that could report relative protein ages at specific sub-cellular locations, by weakly but chronically illuminating photoconvertible fluorescent proteins inside cells. This new method exploits light, instead of oxygen, as the driving force. Therefore its timing speed is optically tunable by adjusting the photoconverting laser intensity. We characterized this light-driven timer method both in vitro and in vivo and applied it to image spatiotemporal distributions of several proteins with different lifetimes. This novel timer method thus offers a flexible "ruler" for studying temporal hierarchy of spatially ordered processes with exquisite spatial-temporal resolution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi.

    Science.gov (United States)

    dos Santos, W G; Buck, G A

    2000-12-01

    The ribosomal RNA (rRNA) gene promoter was used to construct plasmid vectors that simultaneously express multiple exogenous genes in Trypanosoma cruzi. Vector pBSPANEO expresses neomycin phosphotransferase, and pPAGFPAN expresses both green fluorescent protein and neomycin phosphotransferase from a single promoter. Both vectors require the presence of the rRNA promoter for stable transfection; epimastigotes transfected with pPAGFPAN strongly fluoresced due to green fluorescent protein expression. Intact plasmids were rescued from the T. cruzi-transfected population after >8 mo of culture, indicating stable replication of these vectors. Vectors were integrated into the rRNA locus by homologous recombination and into other loci, presumably by illegitimate recombination. Parasites bearing tandem concatamers of plasmids were also found among the transfectants. Transfectants expressing green fluorescent protein showed a bright green fluorescence distributed throughout the cell. Fluorescence was also detected in amastigotes after infection of mammalian cells with transfected parasites, indicating that the rRNA promoter can drive efficient expression of these reporter genes in multiple life-cycle stages of the parasite. Expression of the heterologous genes was detected after passage in mice or in the insect vector. These vectors will be useful for the genetic dissection of T. cruzi biology and pathogenesis.

  15. Single fluorescent protein-based Ca2+ sensors with increased dynamic range

    Directory of Open Access Journals (Sweden)

    Labas Yulii A

    2007-06-01

    Full Text Available Abstract Background Genetically encoded sensors developed on the basis of green fluorescent protein (GFP-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP. Results Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. Conclusion We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

  16. Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

    Science.gov (United States)

    Inagaki, Yoshinori; Matsumoto, Yasuhiko; Ishii, Masaki; Uchino, Keiro; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2015-06-10

    In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

  17. Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation.

    Science.gov (United States)

    Kerppola, Tom K

    2009-10-01

    Investigations of the molecular processes that sustain life must include studies of these processes in their normal cellular environment. The bimolecular fluorescence complementation (BiFC) assay provides an approach for the visualization of protein interactions and modifications in living cells. This assay is based on the facilitated association of complementary fragments of a fluorescent protein that are fused to interaction partners. Complex formation by the interaction partners tethers the fluorescent protein fragments in proximity to each other, which can facilitate their association. The BiFC assay enables sensitive visualization of protein complexes with high spatial resolution. The temporal resolution of BiFC analysis is limited by the time required for fluorophore formation, as well as the stabilization of complexes by association of the fluorescent protein fragments. Many modifications and enhancements to the BiFC assay have been developed. The multicolor BiFC assay enables simultaneous visualization of multiple protein complexes in the same cell, and can be used to investigate competition among mutually exclusive interaction partners for complex formation in cells. The ubiquitin-mediated fluorescence complementation (UbFC) assay enables visualization of covalent ubiquitin family peptide conjugation to substrate proteins in cells. The BiFC assay can also be used to visualize protein binding to specific chromatin domains, as well as other molecular scaffolds in cells. BiFC analysis therefore provides a powerful approach for the visualization of a variety of processes that affect molecular proximity in living cells. The visualization of macromolecular interactions and modifications is of great importance owing to the central roles of proteins, nucleic acids and other macromolecular complexes in the regulation of cellular functions. This tutorial review describes the BiFC assay, and discusses the advantages and disadvantages of this experimental approach

  18. 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.

  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. Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein

    Directory of Open Access Journals (Sweden)

    Bewley J Derek

    2008-09-01

    Full Text Available Abstract Background The actin cytoskeleton responds quickly to diverse stimuli and plays numerous roles in cellular signalling, organelle motility and subcellular compartmentation during plant growth and development. Molecular and cell biological tools that can facilitate visualization of actin organization and dynamics in a minimally invasive manner are essential for understanding this fundamental component of the living cell. Results A novel, monomeric (m Eos-fluorescent protein derived from the coral Lobophyllia hemprichii was assessed for its green to red photo-convertibility in plant cells by creating mEosFP-cytosolic. mEosFP was fused to the F-(filamentous-Actin Binding Domain of the mammalian Talin gene to create mEosFP::FABDmTalin. Photo-conversion, visualization and colour quantification protocols were developed for EosFP targeted to the F-actin cytoskeleton. Rapid photo-conversion in the entire cell or in a region of interest was easily achieved upon illumination with an approximately 400 nm wavelength light beam using an epi-fluorescent microscope. Dual color imaging after photo-conversion was carried out using a confocal laser-scanning microscope. Time-lapse imaging revealed that although photo-conversion of single mEosFP molecules can be rapid in terms of live-cell imaging it involves a progressive enrichment of red fluorescent molecules over green species. The fluorescence of photo-converted cells thus progresses through intermediate shades ranging from green to red. The time taken for complete conversion to red fluorescence depends on protein expression level within a cell and the quality of the focusing lens used to deliver the illuminating beam. Three easily applicable methods for obtaining information on fluorescent intensity and colour are provided as a means of ensuring experimental repeatability and data quantification, when using mEosFP and similar photo-convertible proteins. Conclusion The mEosFP::FABD-mTn probe retains

  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.

  2. Fluorescence turn-on responses of anionic and cationic conjugated polymers toward proteins: effect of electrostatic and hydrophobic interactions.

    Science.gov (United States)

    Pu, Kan-Yi; Liu, Bin

    2010-03-11

    Cationic and anionic poly(fluorenyleneethynylene-alt-benzothiadiazole)s (PFEBTs) are designed and synthesized via Sonagashira coupling reaction to show light-up signatures toward proteins. Due to the charge transfer character of the excited states, the fluorescence of PFEBTs is very weak in aqueous solution, while their yellow fluorescence can be enhanced by polymer aggregation. PFEBTs show fluorescence turn-on rather than fluorescence quenching upon complexation with proteins. Both electrostatic and hydrophobic interactions between PFEBTs and proteins are found to improve the polymer fluorescence, the extent of which is dependent on the nature of the polymer and the protein. Changes in solution pH adjust the net charges of proteins, providing an effective way to manipulate electrostatic interactions and in turn the increment in the polymer fluorescence. In addition, the effect of protein digestion on the fluorescence of polymer/protein complexes is probed. The results indicate that electrostatic interaction induced polymer fluorescence increase cannot be substantially reduced through cleaving protein into peptide fragments. In contrast, hydrophobic interactions, mainly determined by the hydrophobicity of proteins, can be minimized by digestion, imparting a light-off signature for the polymer/protein complexes. This study thus not only highlights the opportunities of exerting nonspecific interactions for protein sensing but also reveals significant implications for biosensor design.

  3. 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 estab......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 establishing the ability of HPLC-LIF protein profiling technique for discrimination, using hard clustering and Fuzzy clustering methods. The clustering algorithms have quite successfully classified the profiles as belonging to normal, cancer of cervix, and oral cancer conditions....

  4. Coral reefs in crisis.

    Science.gov (United States)

    Hinrichsen, D

    1997-01-01

    This article reports on the crisis facing reefs throughout the world and the struggle to save them. Coral reefs, one of the biological wonders of the world, are among the largest and oldest living communities of plants and animals on earth, having been evolved between 200 and 450 million years ago. Located mostly in the Pacific region, most established coral reefs are now dead and only the upper layer is covered by a thin changeable skin of living coral. Reefs, over the years, have been the main source of animal protein for over 1 billion people in Asia. Countries near the coastlines, which relied on the seas, have resorted to dynamite fishing, poisoning and other illegal and dangerous techniques. Overpopulation and pollution has caused the deteriorating conditions of the 600,000 sq. km of coral reefs worldwide. Despite these conditions, the government has ignored this problem as they struggle to develop their economies at the expense of common resources. In addition, this article narrates the efforts that are exerted by governments in promoting coral reef protection and management of these coastal resources, setting the Apo Island in the Philippines as an example of good management and sustainability.

  5. DBD dyes as fluorescence lifetime probes to study conformational changes in proteins.

    Science.gov (United States)

    Wawrzinek, Robert; Ziomkowska, Joanna; Heuveling, Johanna; Mertens, Monique; Herrmann, Andreas; Schneider, Erwin; Wessig, Pablo

    2013-12-16

    Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

  6. Quantitative single-molecule detection of protein based on DNA tetrahedron fluorescent nanolabels.

    Science.gov (United States)

    Ding, Yongshun; Liu, Xingti; Zhu, Jing; Wang, Lei; Jiang, Wei

    2014-07-01

    A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10(-14) to 1.0×10(-12)mol L(-1). What is more, this method showed an excellent specificity and a low matrix effect.

  7. 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.

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

    Science.gov (United States)

    Raina, Shweta A; Tsai, Jeffrey; Samsó, Montserrat; Fessenden, James D

    2012-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Changhua; Mao, Mao [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Yuan, Hang [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Shen, Huaibin [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Wu, Feng; Ma, Lan, E-mail: malan@sz.tsinghua.edu.cn [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Li, Lin Song, E-mail: lsli@henu.edu.cn [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China)

    2013-09-15

    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 Degree-Sign 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.

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

    Science.gov (United States)

    Zhou, Changhua; Mao, Mao; Yuan, Hang; Shen, Huaibin; Wu, Feng; Ma, Lan; Li, Lin Song

    2013-09-01

    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.

  11. 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.

  12. Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins.

    Science.gov (United States)

    Pires, S F; DaRocha, W D; Freitas, J M; Oliveira, L A; Kitten, G T; Machado, C R; Pena, S D J; Chiari, E; Macedo, A M; Teixeira, S M R

    2008-03-01

    Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

  13. 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.

  14. Early transcriptional changes in the reef-building coral Acropora aspera in response to thermal and nutrient stress.

    Science.gov (United States)

    Rosic, Nedeljka; Kaniewska, Paulina; Chan, Chon-Kit Kenneth; Ling, Edmund Yew Siang; Edwards, David; Dove, Sophie; Hoegh-Guldberg, Ove

    2014-12-02

    Changes to the environment as a result of human activities can result in a range of impacts on reef building corals that include coral bleaching (reduced concentrations of algal symbionts), decreased coral growth and calcification, and increased incidence of diseases and mortality. Understanding how elevated temperatures and nutrient concentration affect early transcriptional changes in corals and their algal endosymbionts is critically important for evaluating the responses of coral reefs to global changes happening in the environment. Here, we investigated the expression of genes in colonies of the reef-building coral Acropora aspera exposed to short-term sub-lethal levels of thermal (+6°C) and nutrient stress (ammonium-enrichment: 20 μM). The RNA-Seq data provided hundreds of differentially expressed genes (DEGs) corresponding to various stress regimes, with 115 up- and 78 down-regulated genes common to all stress regimes. A list of DEGs included up-regulated coral genes like cytochrome c oxidase and NADH-ubiquinone oxidoreductase and up-regulated photosynthetic genes of algal origin, whereas coral GFP-like fluorescent chromoprotein and sodium/potassium-transporting ATPase showed reduced transcript levels. Taxonomic analyses of the coral holobiont disclosed the dominant presence of transcripts from coral (~70%) and Symbiodinium (~10-12%), as well as ~15-20% of unknown sequences which lacked sequence identity to known genes. Gene ontology analyses revealed enriched pathways, which led to changes in the dynamics of protein networks affecting growth, cellular processes, and energy requirement. In corals with preserved symbiont physiological performance (based on Fv/Fm, photo-pigment and symbiont density), transcriptomic changes and DEGs provided important insight into early stages of the stress response in the coral holobiont. Although there were no signs of coral bleaching after exposure to short-term thermal and nutrient stress conditions, we managed to detect

  15. Chromophore Structure of Photochromic Fluorescent Protein Dronpa: Acid-Base Equilibrium of Two Cis Configurations.

    Science.gov (United States)

    Higashino, Asuka; Mizuno, Misao; Mizutani, Yasuhisa

    2016-04-07

    Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid-base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pKa of 5.2 ± 0.3 and maintains the cis configuration. The observed resonance Raman bands showed that the other photochromic state of the chromophore is in a trans configuration. The results demonstrate that Raman bands selectively enhanced for the chromophore yield valuable information on the molecular structure of the chromophore in photochromic fluorescent proteins after careful elimination of the fluorescence background.

  16. Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

    Science.gov (United States)

    Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, Rainer

    2016-01-01

    Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles. PMID:27783656

  17. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

    Directory of Open Access Journals (Sweden)

    Mazahir T Hasan

    2004-06-01

    Full Text Available Genetically encoded fluorescent calcium indicator proteins (FCIPs are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs and synaptic and sensory stimulation can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

  18. 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%.

  19. 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.

  20. “Turn-On” Protein Fluorescence: In Situ Formation of Cyanine Dyes

    Science.gov (United States)

    2015-01-01

    Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2–11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues. PMID:25534273

  1. Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

    Science.gov (United States)

    Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

    1999-02-01

    Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

  2. 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.

  3. Crowning corals

    Digital Repository Service at National Institute of Oceanography (India)

    Raghukumar, C.

    and oil transport, thermal pollution and freshwater inflow are the major threats to corals growing along the urban and industrialised centres. Therefore, a concerted effort from academicians, governmental and non-governmental bodies to educate the public...

  4. Cytoplasmic illuminations: in planta targeting of fluorescent proteins to cellular organelles.

    Science.gov (United States)

    Hawes, C; Saint-Jore, C M; Brandizzi, F; Zheng, H; Andreeva, A V; Boevink, P

    2001-01-01

    Use of the jellyfish green-fluorescent protein as an in vivo reporter is in the process of revolutionising plant cell biology. By fusing the protein to specific targeting peptides or to sequences of complete proteins, it is now possible to observe the location, structure, and dynamics of a number of intracellular organelles over extended periods of time. In this review we discuss the most recent developments and unexpected results originating from the targeting of this unique protein and its derivatives to elements of the cytoskeleton and to membrane-bounded organelles in a range of plant cell types.

  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. Gene discovery in the threatened elkhorn coral: 454 sequencing of the Acropora palmata transcriptome.

    Directory of Open Access Journals (Sweden)

    Nicholas R Polato

    Full Text Available BACKGROUND: Cnidarians, including corals and anemones, offer unique insights into metazoan evolution because they harbor genetic similarities with vertebrates beyond that found in model invertebrates and retain genes known only from non-metazoans. Cataloging genes expressed in Acropora palmata, a foundation-species of reefs in the Caribbean and western Atlantic, will advance our understanding of the genetic basis of ecologically important traits in corals and comes at a time when sequencing efforts in other cnidarians allow for multi-species comparisons. RESULTS: A cDNA library from a sample enriched for symbiont free larval tissue was sequenced on the 454 GS-FLX platform. Over 960,000 reads were obtained and assembled into 42,630 contigs. Annotation data was acquired for 57% of the assembled sequences. Analysis of the assembled sequences indicated that 83-100% of all A. palmata transcripts were tagged, and provided a rough estimate of the total number genes expressed in our samples (~18,000-20,000. The coral annotation data contained many of the same molecular components as in the Bilateria, particularly in pathways associated with oxidative stress and DNA damage repair, and provided evidence that homologs of p53, a key player in DNA repair pathways, has experienced selection along the branch separating Cnidaria and Bilateria. Transcriptome wide screens of paralog groups and transition/transversion ratios highlighted genes including: green fluorescent proteins, carbonic anhydrase, and oxidative stress proteins; and functional groups involved in protein and nucleic acid metabolism, and the formation of structural molecules. These results provide a starting point for study of adaptive evolution in corals. CONCLUSIONS: Currently available transcriptome data now make comparative studies of the mechanisms underlying coral's evolutionary success possible. Here we identified candidate genes that enable corals to maintain genomic integrity despite

  7. Fluorescent-protein-based biosensors: modulation of energy transfer as a design principle.

    Science.gov (United States)

    Campbell, Robert E

    2009-08-01

    Genetically-encoded biosensors based on FRET between fluorescent proteins of different hues enable quantitative measurement of intracellular enzyme activities and small molecule concentrations. (To listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.acs.org/journal/ancham.).

  8. 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...

  9. Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins

    DEFF Research Database (Denmark)

    Lambertsen, L.; Sternberg, Claus; Molin, Søren

    2004-01-01

    The mini-Tn7 transposon system is a convenient tool for site-specific tagging of bacteria in which the tagging DNA is inserted at a unique and neutral chromosomal site. We have expanded the panel of mini-Tn7 delivery plasmids expressing different fluorescent proteins (stable and unstable) from th...

  10. Functional Expression of Aquaporin-2 Tagged with Photoconvertible Fluorescent Protein in mpkCCD Cells

    Directory of Open Access Journals (Sweden)

    Kay-Pong Yip

    2015-05-01

    Full Text Available Background: Vasopressin induced trafficking of aquaporin-2 (AQP2 containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. Methods: A spacer was used to link a photoconvertible fluorescent protein (mEos2 to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. Results: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. Conclusion: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated.

  11. Fluorescent protein aided insights on plastids and their extensions: A critical appraisal.

    Directory of Open Access Journals (Sweden)

    Kathleen eDelfosse

    2016-01-01

    Full Text Available Multi-coloured fluorescent proteins targeted to plastids have provided new insights on the dynamic behaviour of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signalling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes.

  12. 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.

  13. 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

    2016-08-09

    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.

  14. 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.

  15. 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.

  16. Modeling structure and spectra of the kindling fluorescent protein asFP595

    Science.gov (United States)

    Collins, Jack R.; Topol, Igor A.; Savitsky, Alexander P.; Nemukhin, Alexander V.

    2011-03-01

    Modern computational approaches based on quantum mechanical methods to characterize structures and optical spectra of biological chromophores in proteins are intensively used to gain knowledge of events occurring upon of their photoexcitation. Primary attention is paid to the species from the family of the green fluorescent protein applied as biomarkers in living cells. We apply quantum chemical approaches for accurate calculations of the structures of the chromophore binding pockets and to estimate spectral bands corresponding to the S0-S1 optical transitions of the intriguing kindling protein asFP595. Its precursor, the chromoprotein asCP from the sea anemony Anemonia sulcata is characterized by distinctive spectral properties: at low light intensities the wild-type protein is weakly fluorescent with the very low quantum yield, however, high intensity irradiation with green light leads to a drastic increase of quantum yield. This phenomenon is now termed "kindling". In simulations, the model system is designed as a molecular cluster constructed on the basis of available crystal structures of the related protein. The equilibrium geometry of the cluster is optimized using density functional theory approximations. The vertical excitation energies corresponding to the S0-S1 transitions are computed by using the semiempirical ZINDO technique. A special attention is paid to evaluate effects of point mutations in the vicinity of the chromophore group. Theoretical data provide important information on the chromophore properties aiming to interpret the results of experimental studies and applications of this fluorescent protein.

  17. 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 en...

  18. Dynamic measurement of fluorescent proteins spectral distribution on virus infected cells

    Science.gov (United States)

    Lee, Ja-Yun; Wu, Ming-Xiu; Kao, Chia-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

    2006-09-01

    We constructed a dynamic spectroscopy system that can simultaneously measure the intensity and spectral distributions of samples with multi-fluorophores in a single scan. The system was used to monitor the fluorescence distribution of cells infected by the virus, which is constructed by a recombinant baculoviruses, vAcD-Rhir-E, containing the red and green fluorescent protein gene that can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. The system was composed of an excitation light source, a scanning system and a spectrometer. We also developed an algorithm and fitting process to analyze the pattern of fluorescence distribution of the dual fluorescence produced in the recombinant virus-infected cells. All the algorithm and calculation are automatically processed in a visualized scanning program and can monitor the specific region of sample by calculating its intensity distribution. The spectral measurement of each pixel was performed at millisecond range and the two dimensional distribution of full spectrum was recorded within several seconds. We have constructed a dynamic spectroscopy system to monitor the process of virus-infection of cells. The distributions of the dual fluorescence were simultaneously measured at micrometer resolution.

  19. 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.

  20. Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-12-01

    Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP- and Dreiklang-labeled plasma membrane protein complexes.

  1. Fluorescence property of photosystem II protein complexes bound to a gold nanoparticle.

    Science.gov (United States)

    Tahara, Kazuki; Mohamed, Ahmed; Kawahara, Kousuke; Nagao, Ryo; Kato, Yuki; Fukumura, Hiroshi; Shibata, Yutaka; Noguchi, Takumi

    2017-03-08

    Development of an efficient photo-anode system for water oxidation is key to the success of artificial photosynthesis. We previously assembled photosystem II (PSII) proteins, which are an efficient natural photocatalyst for water oxidation, on a gold nanoparticle (GNP) to prepare a PSII-GNP conjugate as an anode system in a light-driven water-splitting nano-device (Noji et al., J. Phys. Chem. Lett., 2011, 2, 2448-2452). In the current study, we characterized the fluorescence property of the PSII-GNP conjugate by static and time-resolved fluorescence measurements, and compared with that of free PSII proteins. It was shown that in a static fluorescence spectrum measured at 77 K, the amplitude of a major peak at 683 nm was significantly reduced and a red shoulder at 693 nm disappeared in PSII-GNP. Time-resolved fluorescence measurements showed that picosecond components at 683 nm decayed faster by factors of 1.4-2.1 in PSII-GNP than in free PSII, explaining the observed quenching of the major fluorescence peak. In addition, a nanosecond-decay component arising from a 'red chlorophyll' at 693 nm was lost in time-resolved fluorescence of PSII-GNP, probably due to a structural perturbation of this chlorophyll by interaction with GNP. Consistently with these fluorescence properties, degradation of PSII during strong-light illumination was two times slower in PSII-GNP than in free PSII. The enhanced durability of PSII is an advantageous property of the PSII-GNP conjugate in the development of an artificial photosynthesis device.

  2. Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

    Science.gov (United States)

    Katoh, A; Shoguchi, K; Matsuoka, H; Yoshimura, M; Ohkubo, J-I; Matsuura, T; Maruyama, T; Ishikura, T; Aritomi, T; Fujihara, H; Hashimoto, H; Suzuki, H; Murphy, D; Ueta, Y

    2014-05-01

    The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

  3. In vivo cell biology of cancer cells visualized with fluorescent proteins.

    Science.gov (United States)

    Hoffman, Robert M

    2005-01-01

    This chapter describes a new cell biology where the behavior of individual cells can be visualized in the living animal. Previously it has been demonstrated that fluorescent proteins can be used for whole-body imaging of metastatic tumor growth, bacterial infection, and gene expression. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of tumor-stroma interactions and especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts, and macrophages. Another example is the color coding of cells with RFP or GFP such that both cell types can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem cell marker nestin drive GFP expression enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Highly elongated cancer cells in capillaries in living mice were observed within skin flaps. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells in the capillaries elongated to fit the width of these vessels. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

  4. A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins

    Science.gov (United States)

    Liu, Jun; Pei, Zhiguo; Wang, Liang; Zhang, Zhihong; Zeng, Shaoqun; Huang, Zhen-Li

    2010-11-01

    Characterizing the photoactivation performance of highlighter fluorescence proteins (FPs) is crucial for screening better highlighter FPs and optimizing the photoactivation efficiency of a certain highlighter FP. Currently, photoactivation contrast and half-time values of photoactivation and photobleaching processes are used for such purpose. However, the relations among these parameters are not clear, and little guidance for practical experiments could be obtained from the half-time values. Here, we show that light dose dependent fluorescence curve, which is calculated from activation-intensity-dependent photoactivation and photobleaching rates, is capable of quantifying the photoactivation performance straightforwardly. Moreover, the photoactivation contrast is easily obtained from the curve.

  5. 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. 

  6. Fluorescent proteins as singlet oxygen photosensitizers: mechanistic studies in photodynamic inactivation of bacteria

    Science.gov (United States)

    Ruiz-González, Rubén.; White, John H.; Cortajarena, Aitziber L.; Agut, Montserrat; Nonell, Santi; Flors, Cristina

    2013-02-01

    Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer, light and oxygen to produce reactive oxygen species (ROS), mainly singlet oxygen (1O2), to photo-oxidize important biomolecules and induce cell death. aPDT is a promising alternative to standard antimicrobial strategies, but its mechanisms of action are not well understood. One of the reasons for that is the lack of control of the photosensitizing drugs location. Here we report the use of geneticallyencoded fluorescent proteins that are also 1O2 photosensitizers to address the latter issue. First, we have chosen the red fluorescent protein TagRFP as a photosensitizer, which unlike other fluorescent proteins such as KillerRed, is able to produce 1O2 but not other ROS. TagRFP photosensitizes 1O2 with a small, but not negligible, quantum yield. In addition, we have used miniSOG, a more efficient 1O2 photosensitizing fluorescent flavoprotein that has been recently engineered from phototropin 2. We have genetically incorporated these two photosensitizers into the cytosol of E. coli and demonstrated that intracellular 1O2 is sufficient to kill bacteria. Additional assays have provided further insight into the mechanism of cell death. Photodamage seems to occur primarily in the inner membrane, and extends to the outer membrane if the photosensitizer's efficiency is high enough. These observations are markedly different to those reported for external photosensitizers, suggesting that the site where 1O2 is primarily generated proves crucial for inflicting different types of cell damage.

  7. 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.

  8. 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.

  9. 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

    2016-03-03

    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.

  10. TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

    Science.gov (United States)

    Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

    2014-01-01

    In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. PMID:25287913

  11. 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.

  12. 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

    2016-04-14

    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.

  13. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    Science.gov (United States)

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-01-01

    Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore. PMID:23008753

  14. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    Science.gov (United States)

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-09-01

    Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore.

  15. 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.

  16. Toxicity detection using lysosomal enzymes, glycoamylase and thioredoxin fused with fluorescent protein in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Ngoc-Tu; Shin, Hwa-Yoon; Kim, Yang-Hoon; Min, Jiho

    2015-11-20

    Saccharomyces cerevisiae is the simplest and a favorite eukaryotic system that contains lysosome and thus, is a suitable organism for monitoring some toxic effects in environmental pollution. In this study, S. cerevisiae was transformed with two recombinant plasmids. Sporulation-specific glycoamylase (SGA1), which was upregulated in response to arsenic, was fused with the blue fluorescent protein (BFP) for the construction of an oxidative stress-causing chemicals sensor. Additionally, thioredoxin (TRX2), a protein overexpressed exclusively under tetracycline's influence, fused with the cyan fluorescent protein (CFP) to create a detector for this kind of chemical. In summary, we developed two recombinant S. cerevisiae that facilitate the detection of both kinds of toxic chemicals, specifically visualized by different color indicators.

  17. Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

    Science.gov (United States)

    Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

    2013-03-18

    Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. PNA-induced assembly of fluorescent proteins using DNA as a framework.

    Science.gov (United States)

    Gholami, Zahra; Brunsveld, Luc; Hanley, Quentin

    2013-08-21

    Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein-PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)-peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation (EPL). A DNA beacon, with 6-Fam and Dabcyl at its ends, acts as a framework to create an assembled hetero-FRET system with the mTFP-PNA conjugate. Using fluorescence intensity, frequency domain lifetime measurements, and anisotropy measurements, the system was shown to produce FRET as indicated by decreased donor intensity, decreased donor lifetime, and increased donor anisotropy. Extension of the DNA scaffold allowed for the assembly of multiple mTFP-PNA constructs. Efficient formation of protein dimers and oligomers on the DNA-PNA frameworks could be shown, as visualized via size exclusion chromatography (SEC) and electrophoresis (SDS-PAGE). Assembly of multiple proteins in a row induced homo-FRET for the mTFP-PNA's assembled on the DNA scaffolds. The oligonucleotide framework allows an induced and controllable assembly of proteins by fusing them to PNAs directed to align on DNA scaffolds.

  19. Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

    Science.gov (United States)

    Chen, L Y; Chen, X; Tian, X L; Yu, X H

    2000-10-01

    To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

  20. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

    Directory of Open Access Journals (Sweden)

    Stefanie Besser

    Full Text Available GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65. TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

  1. Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo.

    Science.gov (United States)

    Song, Lina; Bachert, Collin; Linstedt, Adam D

    2016-01-01

    Mucin-type O-glycosylation occurring in the Golgi apparatus is an important protein posttranslational modification initiated by up to 20 GalNAc-transferase isozymes with largely distinct substrate specificities. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and misregulation causes human diseases. Here we describe the use of protein-based fluorescence sensors that traffic in the secretory pathway to monitor GalNAc-transferase activity in living cells. The sensors can either be "pan" or isozyme specific.

  2. Steady-state tryptophan fluorescence spectroscopy study to probe tertiary structure of proteins in solid powders.

    Science.gov (United States)

    Sharma, Vikas K; Kalonia, Devendra S

    2003-04-01

    The purpose of this work was to obtain information about protein tertiary structure in solid state by using steady state tryptophan (Trp) fluorescence emission spectroscopy on protein powders. Beta-lactoglobulin (betaLg) and interferon alpha-2a (IFN) powder samples were studied by fluorescence spectroscopy using a front surface sample holder. Two different sets of dried betaLg samples were prepared by vacuum drying of solutions: one containing betaLg, and the other containing a mixture of betaLg and guanidine hydrochloride. Dried IFN samples were prepared by vacuum drying of IFN solutions and by vacuum drying of polyethylene glycol precipitated IFN. The results obtained from solid samples were compared with the emission scans of these proteins in solutions. The emission scans obtained from protein powders were slightly blue-shifted compared to the solution spectra due to the absence of water. The emission scans were red-shifted for betaLg samples dried from solutions containing GuHCl. The magnitude of the shifts in lambda(max) depended on the extent of drying of the samples, which was attributed to the crystallization of GuHCl during the drying process. The shifts in the lambda(max) of the Trp emission spectrum are associated with the changes in the tertiary structure of betaLg. In the case of IFN, the emission scans obtained from PEG-precipitated and dried sample were different compared to the emission scans obtained from IFN in solution and from vacuum dried IFN. The double peaks observed in this sample were attributed to the unfolding of the protein. In the presence of trehalose, the two peaks converged to form a single peak, which was similar to solution emission spectra, whereas no change was observed in the presence of mannitol. We conclude that Trp fluorescence spectroscopy provides a simple and reliable means to characterize Trp microenvironment in protein powders that is related to the tertiary conformation of proteins in the solid state. This study shows

  3. Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.

    Science.gov (United States)

    Richards, Ryan; Dempski, Robert E

    2011-05-29

    Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels, ion pumps, and transporters. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells. We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling. Furthermore, this method provides an approach to determine distance constraints between specific residues. This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest. In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein. Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%) upon a conformational change of the protein. Second, these changes in fluorescence

  4. How do corals make rocks?

    Science.gov (United States)

    Falkowski, P. G.; Mass, T.; Drake, J.; Schaller, M. F.; Rosenthal, Y.; Schofield, O.; Sherrell, R. M.

    2014-12-01

    We have developed a three pronged approach to understanding how corals precipitate aragonite crystals and contain proxy biogeochemical information. Using proteomic and genomic approaches, we have identified 35 proteins in coral skeletons. Among these are a series of coral acidic proteins (CARPs). Based on their gene sequences, we cloned a subset of these proteins and purified them. Each of the proteins precipitate aragonite in vitro in unamended seawater. Antibodies raised against these proteins react with individual crystals of the native coral, clearly revealing that they are part of a biomineral structure. Based on the primary structure of the proteins we have developed a model of the precipitation reaction that focuses on a Lewis acid displacement of protons from bicarbonate anions by calcium ligated to the carboxyl groups on the CARPs. The reactions are highly acidic and are not manifestly influenced by pH above ca. 6. These results suggest that corals will maintain the ability to calcify in the coming centuries, despite acidification of the oceans.

  5. Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein.

    Science.gov (United States)

    Brient-Litzler, Elodie; Plückthun, Andreas; Bedouelle, Hugues

    2010-04-01

    Designed ankyrin repeat proteins (DARPins) can be selected from combinatorial libraries to bind any target antigen. They show high levels of recombinant expression, solubility and stability, and contain no cysteine residue. The possibility of obtaining, from any DARPin and at high yields, fluorescent conjugates which respond to the binding of the antigen by a variation of fluorescence, would have numerous applications in micro- and nano-analytical sciences. This possibility was explored with Off7, a DARPin directed against the maltose binding protein (MalE) from Escherichia coli, with known crystal structure of the complex. Eight residues of Off7, whose solvent accessible surface area varies on association with the antigen but which are not in direct contact with the antigen, were individually mutated into cysteine and then chemically coupled with a fluorophore. The conjugates were ranked according to their relative sensitivities. All of them showed an increase in their fluorescence intensity on antigen binding by >1.7-fold. The best conjugate retained the same affinity as the parental DARPin. Its signal increased linearly and specifically with the concentration of antigen, up to 15-fold in buffer and 3-fold in serum when fully saturated, the difference being mainly due to the absorption of light by serum. Its lower limit of detection was equal to 0.3 nM with a standard spectrofluorometer. Titrations with potassium iodide indicated that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the antigen. These results suggest rules for the design of reagentless fluorescent biosensors from any DARPin.

  6. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits.

    Science.gov (United States)

    Isailovic, Dragan; Sultana, Ishrat; Phillips, Gregory J; Yeung, Edward S

    2006-11-01

    Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

  7. Chapter 3: A fluorescent window into protein folding and aggregation in cells.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2008-01-01

    Evolutionary selective pressures have tuned the efficiency of the protein-folding reaction in the crowded complex environment in the cell. Nevertheless, the fidelity of folding is imperfect, leading to off-pathway intermolecular interactions that compete with proper folding and to consequent formation of thermodynamically stable aggregates. Such aggregates constitute the histopathological hallmarks of many neurodegenerative pathologies. Yet, most of the approaches to characterize protein folding and/or misfolding are limited to in vitro conditions. Here, we describe a strategy to directly monitor the behavior of a protein in prokaryotic and eukaryotic cells. The method is based on incorporation of structurally non-perturbing, specific binding motifs for a bis-arsenical fluoroscein dye, FlAsH, in sites that result in distinct dye fluorescence signals for the folded and unfolded states of the protein under study. Our approach has been developed using as a case study the predominantly beta-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein, alone or as a chimera fused to the exon 1-encoded fragment of huntingtin, which harbors a polyglutamine repeat tract. We have designed protocols to label this protein in vivo and to monitor the resulting fluorescence signal, which reports on any misfolding transition and formation of aggregates, yielding quantitatively interpretable data.

  8. Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

    Science.gov (United States)

    Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

    2016-06-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

  9. The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Watkins, Jennifer L.; Kim, Hanseong [Arizona State University, Tempe, AZ 85287-1604 (United States); Markwardt, Michele L. [University of Maryland School of Medicine, Baltimore, MD 21201-1559 (United States); Chen, Liqing; Fromme, Raimund [Arizona State University, Tempe, AZ 85287-1604 (United States); Rizzo, Mark A. [University of Maryland School of Medicine, Baltimore, MD 21201-1559 (United States); Wachter, Rebekka M., E-mail: rwachter@asu.edu [Arizona State University, Tempe, AZ 85287-1604 (United States)

    2013-05-01

    The high resolution X-ray structure of the cyan fluorescent protein mCerulean3 demonstrates that different combinations of correlated residue substitutions can provide near optimum quantum yield values for fluorescence. Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

  10. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pletneva, Nadya V.; Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Souslova, Ekaterina; Chudakov, Dmitry M. [Russian Academy of Sciences, Moscow (Russian Federation); Lukyanov, Sergey [Russian Academy of Sciences, Moscow (Russian Federation); Nizhny Novgorod State Medical Academy, Nizhny Novgorod (Russian Federation); Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor [Russian Academy of Sciences, Moscow (Russian Federation); Wlodawer, Alexander [National Cancer Institute, Frederick, MD 21702 (United States); Dauter, Zbigniew [National Cancer Institute, Argonne, IL 60439 (United States); Pletnev, Sergei [National Cancer Institute, Argonne, IL 60439 (United States); SAIC-Frederick, 9700 South Cass Avenue, Argonne, IL 60439 (United States); Russian Academy of Sciences, Moscow (Russian Federation)

    2013-06-01

    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 (λ{sub em}{sup max} ≃ 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.

  11. Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

    Science.gov (United States)

    Hoff, Signe; Larsen, Flemming H; Andersen, Mogens L; Lund, Marianne N

    2013-04-07

    A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 μM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

  12. 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.

  13. Probing DNA-Protein Interactions on Surfaces Using Spectral Self-interference Fluorescence Microscopy

    Science.gov (United States)

    Dogan, Mehmet; Droge, Peter; Swan, Anna K.; Unlu, Selim; Goldberg, Bennett B.

    2007-03-01

    We are probing the interactions between double-stranded DNA and integration host factor (IHF) proteins [1] on surfaces using Spectral Self-interference Fluorescence Microscopy (SSFM) [2].The probing technique utilizes the spectral fringes produced by interference of direct and reflected emission from fluorescent molecules. The modified spectrum provides a unique signature of the axial position of the fluorophores. Using the SSFM technique, we probe the average location of the fluorescent markers attached to the DNA molecules to study the conformational changes in double-stranded DNA tethered to SiO2 surfaces. In the presence of IHF, a DNA bending protein, we observe reduction in the vertical position of fluorescent molecules suggesting the formation of IHF-DNA complex and IHF-induced DNA bending. We also discuss the results with different IHF strains and different binding conditions. [1] Q. Bao et. al., Gene, Vol.343 pp.99-106 (2004) [2] L.A. Moiseev et. al., Journal of Applied Physics, Vol.96, pp. 5311-5315 (2004)

  14. Emission shaping in fluorescent proteins: role of electrostatics and π-stacking.

    Science.gov (United States)

    Park, Jae Woo; Rhee, Young Min

    2016-02-07

    For many decades, simulating the excited state properties of complex systems has been an intriguing but daunting task due to its high computational cost. Here, we apply molecular dynamics based techniques with interpolated potential energy surfaces toward calculating fluorescence spectra of the green fluorescent protein (GFP) and its variants in a statistically meaningful manner. With the GFP, we show that the diverse electrostatic tuning can shape the emission features in many different ways. By computationally modulating the electrostatic interactions between the chromophore phenoxy oxygen and its nearby residues, we demonstrate that we indeed can shift the emission to the blue or to the red side in a predictable manner. We rationalize the shifting effects of individual residues in the GFP based on the responses of both the adiabatic and the diabatic electronic states of the chromophore. We next exhibit that the yellow emitting variant, the Thr203Tyr mutant, generates changes in the electrostatic interactions and an additional π-stacking interaction. These combined effects indeed induce a red shift to emit the fluorescence into the yellow region. With the series of demonstrations, we suggest that our approach can provide sound rationales and useful insights in understanding different responses of various fluorescent complexes, which may be helpful in designing new light emitting proteins and other related systems in future studies.

  15. 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.

  16. 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

  17. Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe.

    Science.gov (United States)

    Ma, Changbei; Lv, Xiaoyuan; Wang, Kemin; Jin, Shunxin; Liu, Haisheng; Wu, Kefeng; Zeng, Weimin

    2017-06-08

    Protein kinase A was detected by quantifying the amount of ATP used after a protein kinase reaction. The ATP assay was performed using the T4 DNA ligase and a molecular beacon (MB). In the presence of ATP, DNA ligase catalyzed the ligation of short DNA. The ligation product then hybridized to MB, resulting in a fluorescence enhancement of the MB. This assay was capable of determining protein kinase A in the range of 12.5∼150 nM, with a detection limit of 1.25 nM. Furthermore, this assay could also be used to investigate the effect of genistein on protein kinase A. It was a universal, non-radioisotopic, and homogeneous method for assaying protein kinase A.

  18. An intrinsically fluorescent recognition ligand scaffold based on chaperonin protein and semiconductor quantum-dot conjugates.

    Science.gov (United States)

    Xie, Hongzhi; Li, Yi-Fen; Kagawa, Hiromi K; Trent, Jonathan D; Mudalige, Kumara; Cotlet, Mircea; Swanson, Basil I

    2009-05-01

    Genetic engineering of a novel protein-nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18-subunit double ring, which self-assembles in the presence of Mg ions and ATP. Described here is a mutant chaperonin (His-beta-loopless, HBLL) with increased access to the central cavity and His-tags on each subunit extending into the central cavity. This mutant binds water-soluble semiconductor quantum dots, creating a protein-encapsulated fluorescent nanoparticle. The new bioconjugate has high affinity, in the order of strong antibody-antigen interactions, a one-to-one protein-nanoparticle stoichiometry, and high stability. By adding selective binding sites to the solvent-exposed regions of the chaperonin, this protein-nanoparticle bioconjugate becomes a sensor for specific targets.

  19. 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.

  20. Use of green fluorescent protein to monitor Lactobacillus plantarum in the gastrointestinal tract of goats

    Directory of Open Access Journals (Sweden)

    Xufeng Han

    2015-09-01

    Full Text Available The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum isolated from goat. Green fluorescent protein (GFP was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

  1. Simulations on the kindling mechanism of the asFP595 fluorescent protein

    Science.gov (United States)

    Grigorenko, Bella L.; Nemukhin, Alexander V.; Savitsky, Alexander P.

    2008-02-01

    We report the results of quantum mechanical - molecular mechanical (QM/MM) simulations aiming to elucidate the mechanism of kindling of the initially non-fluorescent protein asFP595, which is a mutated variant of the chromoprotein asCP from the sea anemone Anemonia sulcata. asFP595 becomes brightly fluorescent (kindles) with emission at 595 nm in response to intense light irradiation at 568 nm. In simulations, we use the flexible effective fragment QM/MM method with the complete active space self-consistent field (CASSCF) wavefunctions in the quantum part and the AMBER force field parameters in the molecular mechanical part. We analyze the computed scans over potential energy surfaces of the ground and excited electronic states and consider details of the working hypothesis that the trans-cis isomerization of the chromophore group inside the protein is responsible for kindling.

  2. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces 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...... 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...... 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....

  3. Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage.

    Science.gov (United States)

    Sokolenko, Stanislav; Nicastro, Jessica; Slavcev, Roderick; Aucoin, Marc G

    2012-12-01

    As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.

  4. Enhanced green fluorescent protein expression in Pleurotus ostreatus for in vivo analysis of fungal laccase promoters.

    Science.gov (United States)

    Amore, Antonella; Honda, Yoichi; Faraco, Vincenza

    2012-10-01

    The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

  5. Use of green fluorescent protein to monitor Lactobacillus plantarum in the gastrointestinal tract of goats.

    Science.gov (United States)

    Han, Xufeng; Wang, Lei; Li, Wei; Li, Bibo; Yang, Yuxin; Yan, Hailong; Qu, Lei; Chen, Yulin

    2015-01-01

    The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

  6. Generation and characterization of transgenic mice expressing mitochondrial targeted red fluorescent protein selectively in neurons: modeling mitochondriopathy in excitotoxicity and amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Wang Yi

    2011-11-01

    Full Text Available Abstract Background Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need. Results We created transgenic mice that express the coral (Discosoma sp. red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS mice expressing human mutant superoxide dismutase-1. Conclusions These novel transgenic mice will be a useful tool for better understanding

  7. Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins.

    Science.gov (United States)

    Steiner, Sebastian; Pfannschmidt, Thomas

    2009-01-01

    Changes in gene expression mediated by DNA-binding protein factors are a crucial part of many signal transduction pathways. Generally, these regulatory proteins are low abundant and thus their purification and characterisation is labour- and time-intensive. Here we describe a workflow for purification, characterisation and identification of DNA-binding proteins. We show the use of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and describe its advantages for a rapid and convenient screening for regulatory cis-elements. This involves a crude enrichment of nucleic acid binding proteins by heparin-Sepharose chromatography and the characterisation of fractions using overlapping fluorescence-labelled DNA probes spanning the promoter region of interest. The determined protein-binding sites can then be used for sequence-specific DNA-affinity chromatography to purify specifically interacting proteins. Finally, the DNA-binding complexes can be characterised and identified using two-dimensional EMSA, UV-cross-linking and mass spectrometry.

  8. Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

    Science.gov (United States)

    Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

    2009-02-01

    Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

  9. Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.

    Science.gov (United States)

    Errampalli, D; Leung, K; Cassidy, M B; Kostrzynska, M; Blears, M; Lee, H; Trevors, J T

    1999-04-01

    In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.

  10. Optimization of native fluorescence detection of proteins using a pulsed nano laser excitation source

    OpenAIRE

    Heywood, Matthew S.; Farnsworth, Paul B.

    2010-01-01

    We present a mathematical description of the S/N ratio in a fluorescence-based protein detector for capillary electrophoresis that uses a pulsed UV laser at 266 nm as an excitation source. The model accounts for photobleaching, detector volume, laser repetition rate, and analyte flow rate. We have experimentally characterized such a system, and present a comparison of the experimental data with the predictions of the model. Using the model, the system was optimized for test analytes tryptopha...

  11. 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...

  12. Lasing from Escherichia coli bacteria genetically programmed to express green fluorescent protein

    Science.gov (United States)

    Gather, Malte C.; Yun, Seok Hyun

    2011-08-01

    We report on lasing action from colonies of Escherichia coli bacteria that are genetically programmed to synthesize the green fluorescent protein (GFP). When embedded in a Fabry--Perot type cavity and excited by ns-pulses of blue light (465nm), the bacteria generate green laser emission (˜520nm). Broad illumination of pump light yields simultaneous lasing over a large area in bacterial colonies.

  13. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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.

  15. Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control.

    Directory of Open Access Journals (Sweden)

    Adam C Fisher

    Full Text Available Green fluorescent protein (GFP has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2, superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar 'superfolded' proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues.

  16. Protein mediated synthesis of fluorescent Au-nanoclusters for metal sensory coatings

    Energy Technology Data Exchange (ETDEWEB)

    Vogel, Manja; Raff, Johannes [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biogeochemistry

    2017-06-01

    Fluorescent Au-nanocluster were successfully synthesized and used for the selective detection of Cu{sup 2} {sup +}. The synthesized Au-BSA-nanoclusters remain functional also after immobilization and show high thermal stability. Additionally, the transfer of the protein mediated Au-nanocluster synthesis route to S-layer proteins was achieved. (The presented work is part of the project BIONEWS dealing with long-term stable cells for the set-up and regeneration of sensor and actor materials for strategic relevant metals, in particular rare earth elements).

  17. Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

    Science.gov (United States)

    Tsuji, Frederick I

    2010-08-01

    The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. (c) 2010 Wiley-Liss, Inc.

  18. Carotenoid-chlorophyll coupling and fluorescence quenching correlate with protein packing density in grana-thylakoids.

    Science.gov (United States)

    Holleboom, Christoph-Peter; Yoo, Sunny; Liao, Pen-Nan; Compton, Ian; Haase, Winfried; Kirchhoff, Helmut; Walla, Peter Jomo

    2013-09-26

    The regulation of light-harvesting in photosynthesis under conditions of varying solar light irradiation is essential for the survival and fitness of plants and algae. It has been proposed that rearrangements of protein distribution in the stacked grana region of thylakoid membranes connected to changes in the electronic pigment-interaction play a key role for this regulation. In particular, carotenoid-chlorophyll interactions seem to be crucial for the down-regulation of photosynthetic light-harvesting. So far, it has been difficult to determine the influence of the dense protein packing found in native photosynthetic membrane on these interactions. We investigated the changes of the electronic couplings between carotenoids and chlorophylls and the quenching in grana thylakoids of varying protein packing density by two-photon spectroscopy, conventional chlorophyll fluorometry, low-temperature fluorescence spectroscopy, and electron micrographs of freeze-fracture membranes. We observed an increasing carotenoid-chlorophyll coupling and fluorescence quenching with increasing packing density. Simultaneously, the antennas size and excitonic connectivity of Photosystem II increased with increasing quenching and carotenoid-chlorophyll coupling whereas isolated, decoupled LHCII trimers decreased. Two distinct quenching data regimes could be identified that show up at different protein packing densities. In the regime corresponding to higher protein packing densities, quenching is strongly correlated to carotenoid-chlorophyll interactions whereas in the second regime, a weak correlation is apparent with low protein packing densities. Native membranes are in the strong-coupling data regime. Consequently, PSII and LHCII in grana membranes of plants are already quenched by protein crowding. We concluded that this ensures efficient electronic connection of all pigment-protein complexes for intermolecular energy transfer to the reaction centers and allows simultaneously

  19. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  20. Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

    Science.gov (United States)

    Shrestha, Suresh; Salins, Lyndon L E.; Mark Ensor, C.; Daunert, Sylvia

    2002-01-01

    Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the

  1. 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.

  2. Scanning protein analysis of electrofocusing gels using X-ray fluorescence.

    Science.gov (United States)

    Matsuyama, Satoshi; Matsunaga, Akihiro; Sakamoto, Shinichi; Iida, Yutaka; Suzuki, Yoshinari; Ishizaka, Yukihito; Yamauchi, Kazuto; Ishikawa, Tetsuya; Shimura, Mari

    2013-05-01

    Recently, "metallomics," in addition to genomics and proteomics, has become a focus as a novel approach to identify sensitive fluctuations in homeostasis that accompany metabolic processes, such as stress responses, differentiation, and proliferation. Cellular elements and associated protein behavior provide important clues for understanding cellular and disease mechanism(s). It is important to develop a system for measuring the native status of the protein. In this study, we developed an original freeze-dried electrofocusing native gel over polyimide film (native-gel film) for scanning protein analysis using synchrotron radiation excited X-ray fluorescence (SPAX). To our knowledge, this is the first report detailing the successful mapping of metal-associated proteins of electrofocusing gels using X-ray fluorescence. SPAX can provide detection sensitivity equivalent to that of LA-ICP-MS. In addition to this increased sensitivity, SPAX has the potential to be combined with other X-ray spectroscopies. Our system is useful for further applications in proteomics investigating cellular element-associated protein behaviors and disease mechanisms.

  3. 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.

  4. LanFP10-A, first functional fluorescent protein whose chromophore contains the elusive mutation G67A.

    Science.gov (United States)

    Roldán-Salgado, Abigail; Sánchez-Barreto, Celidee; Gaytán, Paul

    2016-11-01

    Since Green Fluorescent Protein (GFP) was first successfully expressed in heterologous systems in 1994, many genes encoding other natural autofluorescent proteins (AFPs) have been cloned and subsequently modified by protein engineering to improve their physicochemical properties. Throughout this twenty-two-year period, glycine 67 (Gly67) has been regarded as the only amino acid in the entire protein family that is essential for the formation of the different reported chromophores. In this work, we demonstrate that a synthetic gene encoding LanFP10-A, a natural protein encoded in the genome of the lancelet Branchiostoma floridae containing the G67A mutation, produces a heterologous, functional yellow fluorescent protein when expressed in E. coli. In contrast to LanFP10-A, LanFP6-A, a second GFP-like protein found in the lancelet genome that also contains the natural G67A mutation, was non-fluorescent.

  5. 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.

  6. Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment

    NARCIS (Netherlands)

    Snippert, H.J.G.; Schepers, A.G.; Delconte, G.; Siersema, P.D.; Clevers, H.

    2011-01-01

    In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a

  7. 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.

  8. Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein.

    Science.gov (United States)

    Pirchi, Menahem; Ziv, Guy; Riven, Inbal; Cohen, Sharona Sedghani; Zohar, Nir; Barak, Yoav; Haran, Gilad

    2011-10-11

    Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinase's folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.

  9. 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.

  10. High-performance fluorescence-encoded magnetic microbeads as microfluidic protein chip supports for AFP detection.

    Science.gov (United States)

    Gong, Xiaoqun; Yan, Huan; Yang, Jiumin; Wu, Yudong; Zhang, Jian; Yao, Yingyi; Liu, Ping; Wang, Huiquan; Hu, Zhidong; Chang, Jin

    2016-10-01

    Fluorescence-encoded magnetic microbeads (FEMMs), with the fluorescence encoding ability of quantum dots (QDs) and magnetic enrichment and separation functions of Fe3O4 nanoparticles, have been widely used for multiple biomolecular detection as microfluidic protein chip supports. However, the preparation of FEMMs with long-term fluorescent encoding and immunodetection stability is still a challenge. In this work, we designed a novel high-temperature chemical swelling strategy. The QDs and Fe3O4 nanoparticles were effectively packaged into microbeads via the thermal motion of the polymer chains and the hydrophobic interaction between the nanoparticles and microbeads. The FEMMs obtained a highly uniform fluorescent property and long-term encoding and immunodetection stability and could be quickly magnetically separated and enriched. Then, the QD-encoded magnetic microbeads were applied to alpha fetoprotein (AFP) detection via sandwich immunoreaction. The properties of the encoded microspheres were characterized using a self-designed detecting apparatus, and the target molecular concentration in the sample was also quantified. The results suggested that the high-performance FEMMs have great potential in the field of biomolecular detection.

  11. 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.

  12. A fluorescence anisotropy method for measuring protein concentration in complex cell culture media.

    Science.gov (United States)

    Groza, Radu Constantin; Calvet, Amandine; Ryder, Alan G

    2014-04-22

    The rapid, quantitative analysis of the complex cell culture media used in biopharmaceutical manufacturing is of critical importance. Requirements for cell culture media composition profiling, or changes in specific analyte concentrations (e.g. amino acids in the media or product protein in the bioprocess broth) often necessitate the use of complicated analytical methods and extensive sample handling. Rapid spectroscopic methods like multi-dimensional fluorescence (MDF) spectroscopy have been successfully applied for the routine determination of compositional changes in cell culture media and bioprocess broths. Quantifying macromolecules in cell culture media is a specific challenge as there is a need to implement measurements rapidly on the prepared media. However, the use of standard fluorescence spectroscopy is complicated by the emission overlap from many media components. Here, we demonstrate how combining anisotropy measurements with standard total synchronous fluorescence spectroscopy (TSFS) provides a rapid, accurate quantitation method for cell culture media. Anisotropy provides emission resolution between large and small fluorophores while TSFS provides a robust measurement space. Model cell culture media was prepared using yeastolate (2.5 mg mL(-1)) spiked with bovine serum albumin (0 to 5 mg mL(-1)). Using this method, protein emission is clearly discriminated from background yeastolate emission, allowing for accurate bovine serum albumin (BSA) quantification over a 0.1 to 4.0 mg mL(-1) range with a limit of detection (LOD) of 13.8 μg mL(-1). Copyright © 2014. Published by Elsevier B.V.

  13. The preparation and use of fluorescent-protein conjugates for microvascular research.

    Science.gov (United States)

    McDonagh, P F; Williams, S K

    1984-01-01

    A procedure is described for making large quantities (100 ml) of fluorochrome-labeled albumin. Chromatographic techniques are described for the purification of commercial albumin (BSA) and the purification of albumin from serum. We report experimentally determined optimal conditions for the covalent attachment of fluorescent dyes (rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC] to albumin. Subsequent removal of all unreacted fluorescent material (UFM) was achieved using charcoal adsorption. We observed no loss of protein following charcoal treatment. The final protein conjugate was analyzed by polyacrylamide gel electrophoresis, gel chromatography, and isoelectric focusing. The conjugates were determined to be free of UFM and homogeneous with respect to molecular weight. However, FITC conjugation lowered the average isoelectric point of albumin by 0.1 to 0.3 pH units. Illustrations of combining fluorescence microscopy with FITC-BSA and RITC-BSA to view microvascular phenomena in skeletal muscle and the heart are given. Knowledge of the biochemical characteristics of the fluorochrome employed is important for proper interpretation of experimental results using this technique.

  14. 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.

  15. Fluorescence quenching as a tool to investigate quinolone antibiotic interactions with bacterial protein OmpF.

    Science.gov (United States)

    Neves, Patrícia; Sousa, Isabel; Winterhalter, Mathias; Gameiro, Paula

    2009-02-01

    The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp(214), Trp(61)), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp(61) and iodide Trp(214). Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp(214) and moxifloxacin near Trp(61). These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.

  16. 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.

  17. Green fluorescent protein labeling of food pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis.

    Science.gov (United States)

    Gensberger, Eva Theres; Kostić, Tanja

    2017-01-01

    Labeling of bacteria with marker genes, such as green fluorescent protein, is a useful and practicable tool for tracking and enumerating bacterial cells in a complex environment e.g. discrimination from the indigenous background population. In this study, novel TurboGFP prokaryotic expression vector was utilized for labeling of Yersinia species. Y. enterocolitica biovar 1A, biovar 2, biovar 4 and Y. pseudotuberculosis were successfully transformed with the vector and expressed bright green fluorescence that was even detectable visually by eye. No adverse effects were observed in growth behavior of the labeled strains compared to wild type (parental) strains and vector maintenance for longer time periods could be achieved for Y. enterocolitica biovar 1A, Y. enterocolitica biovar 2 and Y. pseudotuberculosis.

  18. Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage.

    Science.gov (United States)

    Hanazono, Y; Yu, J M; Dunbar, C E; Emmons, R V

    1997-07-20

    Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. Inclusion of this gene in a vector could allow rapid, nontoxic selection of successfully transduced cells. However, many attempts by our laboratory to isolate stable retroviral producer cell clones secreting biologically active vectors containing either the highly fluorescent S65T-GFP mutant or humanized GFP have failed. Vector plasmids containing various forms of GFP and the neomycin resistance gene were transfected into three different packaging cell lines and fluorescence was observed for several days, but stable clones selected with G418 no longer fluoresced. Using confocal microscopy, the brightest cells were observed to contract and die within a matter of days. RNA slot-blot analysis of retroviral producer supernatants showed no viral production from the GFP plasmid-transfected clones, although all clones derived after transfection with an identical retroviral construct not containing GFP produced virus. Genomic Southern analysis of the GFP-transduced clones showed a much higher probability of rearrangement of the priviral sequences than in the control non-GFP clones. Overall, 18/34 S65T-GFP clones and 17/33 humanized-GFP clones had rearrangements, whereas 2/15 control non-GFP clones had rearrangements. Hence, producer cells expressing high levels of these GFP genes seem to be selected against, with stable clones undergoing major rearrangements or other mutations that both abrogate GFP expression and prevent vector production. These observations indicate that GFP may not be an appropriate reporter gene for gene transfer applications in our vector/packaging system.

  19. Interfacial properties and fluorescence of a coagulating protein extracted from Moringa oleifera seeds and its interaction with sodium dodecyl sulphate.

    Science.gov (United States)

    Maikokera, R; Kwaambwa, H M

    2007-04-01

    The surfactant behaviour of aqueous coagulating protein extracted from Moringa oleifera seeds has been investigated by surface tension measurements. The interaction of the coagulant protein with an anionic surfactant sodium dodecyl sulphate (SDS) has been monitored by surface tension and intrinsic protein fluorescence measurements. The extracted protein shows some weak surface activity at low concentrations. To achieve maximum surface activity (i.e. maximum reduction in surface tension of water), substantially higher concentrations of protein are required. The coagulant protein-SDS interaction scheme did not exhibit the behaviour of weakly interacting polymer-surfactant systems and the SDS interacts in a monomeric form with the protein. The association process of SDS with the coagulant protein is supported by protein fluorescence measurements. SDS has an effect on the fluorescence of the coagulant protein indicating that the local environment of tryptophan in the protein changes as SDS concentration below its critical micelle concentration is increased. These results have led us to the conclusions that: (1) the protein extracted from M. oleifera seeds has significant surfactant behaviour; (2) the coagulant protein interacts strongly with SDS and the protein might have specific binding sites for SDS; (3) there is formation of protein-SDS complex.

  20. Analysis of Biophysical, Optical and Genetic Diversity of DoD Coral Reef Communities Using Advanced Fluorescence and Molecular Biology Techniques (Addendum)

    Science.gov (United States)

    2011-08-01

    Symbiodinium sp. as a bioindicator . 20 ii List of Figures 1. An example of the FIRe fluorescence transients. 6 2. Bench-top versions of the...dynoflagellates, Symbiodinium sp. as a bioindicator . Petroleum product Fv/Fm protocol Extended FIRe protocol Gasoline 87 AKI 300 ppm 30 ppm Kerosene...Gorbunov, and V. Fadeev (2010) - Phytoplankton as a fluorescent bioindicator of ecotoxicants in natural waters. - «SPIE Photonics Europe», 2010

  1. In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

    Science.gov (United States)

    Oliveira, Janaina Correia; da Silva, Aline Caroline; Oliveira, Renato Antonio Dos Santos; Pereira, Valéria Rêgo Alves; Gil, Laura Helena Vega Gonzales

    2016-11-01

    The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

  2. A subnanomolar fluorescent probe for protein kinase CK2 interaction studies.

    Science.gov (United States)

    Enkvist, Erki; Viht, Kaido; Bischoff, Nils; Vahter, Jürgen; Saaver, Siiri; Raidaru, Gerda; Issinger, Olaf-Georg; Niefind, Karsten; Uri, Asko

    2012-11-21

    Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.

  3. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

    Science.gov (United States)

    Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

  4. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    Science.gov (United States)

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  5. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

    2004-02-09

    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 ({lambda}{sub max}=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 -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-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{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -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 {beta}-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.

  6. Bacterial acquisition in juveniles of several broadcast spawning coral species.

    Directory of Open Access Journals (Sweden)

    Koty H Sharp

    Full Text Available Coral animals harbor diverse microorganisms in their tissues, including archaea, bacteria, viruses, and zooxanthellae. The extent to which coral-bacterial associations are specific and the mechanisms for their maintenance across generations in the environment are unknown. The high diversity of bacteria in adult coral colonies has made it challenging to identify species-specific patterns. Localization of bacteria in gametes and larvae of corals presents an opportunity for determining when bacterial-coral associations are initiated and whether they are dynamic throughout early development. This study focuses on the early onset of bacterial associations in the mass spawning corals Montastraea annularis, M. franksi, M. faveolata, Acropora palmata, A. cervicornis, Diploria strigosa, and A. humilis. The presence of bacteria and timing of bacterial colonization was evaluated in gametes, swimming planulae, and newly settled polyps by fluorescence in situ hybridization (FISH using general eubacterial probes and laser-scanning confocal microscopy. The coral species investigated in this study do not appear to transmit bacteria via their gametes, and bacteria are not detectable in or on the corals until after settlement and metamorphosis. This study suggests that mass-spawning corals do not acquire, or are not colonized by, detectable numbers of bacteria until after larval settlement and development of the juvenile polyp. This timing lays the groundwork for developing and testing new hypotheses regarding general regulatory mechanisms that control bacterial colonization and infection of corals, and how interactions among bacteria and juvenile polyps influence the structure of bacterial assemblages in corals.

  7. Bacterial acquisition in juveniles of several broadcast spawning coral species.

    Science.gov (United States)

    Sharp, Koty H; Ritchie, Kim B; Schupp, Peter J; Ritson-Williams, Raphael; Paul, Valerie J

    2010-05-28

    Coral animals harbor diverse microorganisms in their tissues, including archaea, bacteria, viruses, and zooxanthellae. The extent to which coral-bacterial associations are specific and the mechanisms for their maintenance across generations in the environment are unknown. The high diversity of bacteria in adult coral colonies has made it challenging to identify species-specific patterns. Localization of bacteria in gametes and larvae of corals presents an opportunity for determining when bacterial-coral associations are initiated and whether they are dynamic throughout early development. This study focuses on the early onset of bacterial associations in the mass spawning corals Montastraea annularis, M. franksi, M. faveolata, Acropora palmata, A. cervicornis, Diploria strigosa, and A. humilis. The presence of bacteria and timing of bacterial colonization was evaluated in gametes, swimming planulae, and newly settled polyps by fluorescence in situ hybridization (FISH) using general eubacterial probes and laser-scanning confocal microscopy. The coral species investigated in this study do not appear to transmit bacteria via their gametes, and bacteria are not detectable in or on the corals until after settlement and metamorphosis. This study suggests that mass-spawning corals do not acquire, or are not colonized by, detectable numbers of bacteria until after larval settlement and development of the juvenile polyp. This timing lays the groundwork for developing and testing new hypotheses regarding general regulatory mechanisms that control bacterial colonization and infection of corals, and how interactions among bacteria and juvenile polyps influence the structure of bacterial assemblages in corals.

  8. Tetracysteine-based fluorescent tags to study protein localization and trafficking in Plasmodium falciparum-infected erythrocytes.

    Directory of Open Access Journals (Sweden)

    Georgeta Crivat

    Full Text Available Plasmodium falciparum (Pf malaria parasites remodel host erythrocytes by placing membranous structures in the host cell cytoplasm and inserting proteins into the surrounding erythrocyte membranes. Dynamic imaging techniques with high spatial and temporal resolutions are required to study the trafficking pathways of proteins and the time courses of their delivery to the host erythrocyte membrane.Using a tetracysteine (TC motif tag and TC-binding biarsenical fluorophores (BAFs including fluorescein arsenical hairpin (FlAsH and resorufin arsenical hairpin (ReAsH, we detected knob-associated histidine-rich protein (KAHRP constructs in Pf-parasitized erythrocytes and compared their fluorescence signals to those of GFP (green fluorescent protein-tagged KAHRP. Rigorous treatment with BAL (2, 3 dimercaptopropanol; British anti-Lewisite was required to reduce high background due to nonspecific BAF interactions with endogenous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. The fluorescence from FlAsH and ReAsH-labeled protein bleached at faster rates than the fluorescence from GFP-labeled protein.While TC/BAF labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods. Our observations are in agreement with the currently-accepted model of KAHRP movement through the cytoplasm, including transient association of KAHRP with Maurer's clefts before its incorporation into knobs in the host erythrocyte membrane.

  9. 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

  10. pH-dependent transient conformational states control optical properties in cyan fluorescent protein.

    Science.gov (United States)

    Laricheva, Elena N; Goh, Garrett B; Dickson, Alex; Brooks, Charles L

    2015-03-04

    A recently engineered mutant of cyan fluorescent protein (WasCFP) that exhibits pH-dependent absorption suggests that its tryptophan-based chromophore switches between neutral (protonated) and charged (deprotonated) states depending on external pH. At pH 8.1, the latter gives rise to green fluorescence as opposed to the cyan color of emission that is characteristic for the neutral form at low pH. Given the high energy cost of deprotonating the tryptophan at the indole nitrogen, this behavior is puzzling, even if the stabilizing effect of the V61K mutation in proximity to the protonation/deprotonation site is considered. Because of its potential to open new avenues for the development of optical sensors and photoconvertible fluorescent proteins, a mechanistic understanding of how the charged state in WasCFP can possibly be stabilized is thus important. Attributed to the dynamic nature of proteins, such understanding often requires knowledge of the various conformations adopted, including transiently populated conformational states. Transient conformational states triggered by pH are of emerging interest and have been shown to be important whenever ionizable groups interact with hydrophobic environments. Using a combination of the weighted-ensemble sampling method and explicit-solvent constant pH molecular dynamics (CPHMD(MSλD)) simulations, we have identified a solvated transient state, characterized by a partially open β-barrel where the chromophore pKa of 6.8 is shifted by over 20 units from that of the closed form (6.8 and 31.7, respectively). This state contributes a small population at low pH (12% at pH 6.1) but becomes dominant at mildly basic conditions, contributing as much as 53% at pH 8.1. This pH-dependent population shift between neutral (at pH 6.1) and charged (at pH 8.1) forms is thus responsible for the observed absorption behavior of WasCFP. Our findings demonstrate the conditions necessary to stabilize the charged state of the WasCFP chromophore

  11. A study of using luminophore-doped silica nanoparticles as fluorescent probe in protein microarray assay

    Institute of Scientific and Technical Information of China (English)

    LIU Haibo; ZHUANG Zhixia; YANG Huanghao; CHEN Chengxiang; TAN Fang; YAN Qingpi; WANG Xiaoru

    2007-01-01

    A convenient method for the synthesis of tris(2,2'-bipyridyl) dichlororuthenium(Ⅱ)hexahydrate-doped amino-modified double-layer silica nanoparticles is presented in this paper.The synthesized nanoparticles are uniform and photostable,and can be well dispersed in a water solution.Proteins could be directly immobilized onto these nanoparticles by a simple coupling process without losing their biological activities.These nanoparticles were further used as fluorescent probes in protein microarray assay for the quantitative detection of protein.The results obtained by these nanoparticles,with the detection limit of as low as 3.5μg/mL,were much better than those involving the use of conventional FITC probe.

  12. Visualization of protein RecR in Escherichia coli by fluorescent labelling

    Institute of Scientific and Technical Information of China (English)

    Jiefang Qiu; Xuefeng Pan

    2009-01-01

    RecR protein, a functional equivalent of Rad52 in eukaryotes, plays a critical role in the Reef pathway of homologous recombination in Escherichia coli. By constructing and expressing the recR-yfp hybrid gene, the distribution of the RecR-YFP fusion protein was visu-alized in E. coli by fluorescent microscopy. Our results showed that RecR proteins can be localized predominantly in the nucleoid region of E. coli. By measuring the UV resistance of a recR mutant carrying the recR-yfp gene in the plasmid, the expressed RecR-YFP was found to be functional in improving the UV resistance of the recR deficiency strain.

  13. 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...... 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....

  14. [Fluorescence reaction of the system of safranine T-sodium dodecylbenzene sulfonate-protein and its application].

    Science.gov (United States)

    Cheng, Ding-xi; Wang, Shao-hua

    2007-01-01

    There is a self-polymerization equilibrium system between the cation of the fluorescent dye-safranine T and its dimer. In the presence of appropriate amounts of anionic surfactant SDBS, the above mentioned equilibrium can be adjusted by a quantitative addition of protein, thus leading to a regular change in the fluorescence intensity of the system. Based on this phenomenon, the properties of the absorption spectrum and fluorescence spectrum of the system were studied, and a new fluorescence probe for the determination of protein was established. It was found that ST was apt to dimerize in the solution added with appropriate amounts of anionic surfactant. Because of the dimerization, its fluorescence intensity decreased, whereas the addition of protein caused the dimer to be depolymerized and the system fluorescence increased. A linear relationship between the fluorescence intensities and serum albumin concentration was found in the range of 0-40 microg x mL(-1) , and the detection limit found was 0.034 microg x mL(-1). The significant features of this method were its rapidity of reaction, and high sensitivity, accuracy and selectivity. The method was applied to the determination of total proteins in real human serum, and satisfactory results were obtained.

  15. Characterizing Magnetic Properties in Belize Corals

    Science.gov (United States)

    Urbalejo, A. A.; Bhattacharya, A.; Gee, J. S.; Mitra, R.; Carilli, J.; Hangsterfer, A.; Feinberg, J. M.

    2016-12-01

    Measurements of magnetic remanence and characterization of magnetic phases are widely applied to environmental and climate studies; however, magnetic tools have not been widely applied to coral studies. As such, there is a deficit in our understanding of magnetic materials and behavior in coral skeletons and consequently, of processes by which magnetic materials may get incorporated into coral skeletons. In this study, we present magnetic measurements conducted on freshly broken chips from coral cores; the cores were collected from Mesoamerican sites in Belize. Trace, minor, and major element concentration has been well studied in these two coral cores, using inductively coupled mass spectrometric techniques (ICP-MS). The goal of our current research is to determine are as follows: (a) Is there is a viable magnetic signal that can be obtained from measuring chips broken off of coral skeletons? (b) What are some of the dominant magnetic behaviors? (c) What are the carrier phases of magnetic material? (c) can we determine possible variations in the type and quantity of magnetic materials over time and ultimately, (e) can magnetic fingerprinting of corals can be used as tracers of environmental, climate or biological processes? Here, we present preliminary magnetic remanence measurements (IRM and double IRM) from the two coral cores collected from Belize and dating back to the mid 1800s. Early results using freshly broken chips from both coral cores indicate a magnetite-like soft magnetic component during IRM experiments. Double-IRM experiments on the same samples indicate uniaxial single domain behavior. Furthermore, SEM images suggest that the magnetic carrier phase could likely be magnetite. We will also present comparisons of our magnetic data with newly collected X-Ray Fluorescence (XRF) data on the same coral cores. The goal is to properly characterize the type of magnetic signals and determine possible environmental and/or biological impacts on magnetic carrier

  16. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    Science.gov (United States)

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.

  17. Algal symbiont type affects gene expression in juveniles of the coral Acropora tenuis exposed to thermal stress.

    Science.gov (United States)

    Yuyama, Ikuko; Harii, Saki; Hidaka, Michio

    2012-05-01

    Reef-building corals harbor symbiotic dinoflagellates, Symbiodinium spp., which are currently divided into several clades. The responses of corals associated with different Symbiodinium clades to thermal stress are not well understood, especially at a gene expression level. Juveniles of the coral Acropora tenuis inoculated with different algal types (clade A or D) were exposed to thermal stress and the expression levels of four putative stress-responsive genes, including genes coding green and red fluorescent proteins, an oxidative stress-responsive protein, and an ascorbic acid transporter, were analyzed by quantitative real-time PCR. The expression levels of the four genes decreased at high temperatures if juveniles were associated with clade A symbionts but increased if the symbionts were in clade D. The intensity of green fluorescence increased with temperature in clade D symbionts harboring juveniles, but not in juveniles associated with clade A symbionts. The present results suggest that genotypes of endosymbiotic algae affect the thermal stress responses of the coral juveniles.

  18. A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

    Directory of Open Access Journals (Sweden)

    Jakobsdottir Klara B

    2002-06-01

    Full Text Available Abstract Background The jellyfish green fluorescent protein (GFP can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. Results We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. Conclusion This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

  19. 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.

  20. Optical absorption of the blue fluorescent protein: a first-principles study.

    Science.gov (United States)

    Lopez, Xabier; Marques, Miguel A L; Castro, Alberto; Rubio, Angel

    2005-09-07

    An extensive study of the optical absorption spectra of the blue fluorescent protein (BFP) is presented. We investigate different protonation states of the chromophore (neutral, anionic, and cationic) and analyze the role of the protein environment and of thermal fluctuations. The role of the environment is 2-fold: (i) it induces structural modifications of the gas-phase chromophore, the most important being the torsion of the imida rings; and (ii) it makes a local-field modification of the external electromagnetic field. It turns out that the torsion of the imida rings shifts significantly the gas-phase spectra to lower energies, whereas the consistent inclusion of the closest residues field produces only minor modifications on the spectra. From all of the configurations studied, the neutral cis-HSD and the anionic HSA seem to be the most likely candidates to explain the experimental spectrum. Furthermore, the present results clearly rule out the presence of the cationic protonation state (HSP) of the chromophore. However, a better description of the measured experimental absorption data may be obtained when the temperature fluctuations of the floppy torsional motion of the two imida rings are included. Our results, together with previous work on the green fluorescent protein, demonstrate the power of combining time-dependent density functional calculations and optical absorption measurements to discern the relevant chemical information on the nature and state of chromopeptides.

  1. Development of an X-ray fluorescence holographic measurement system for protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Sato-Tomita, Ayana, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Shibayama, Naoya, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Okabe, Takahiro [Division of Biophysics, Department of Physiology, Jichi Medical University, Yakushiji, Shimotsuke 329-0498 (Japan); Happo, Naohisa [Department of Computer and Network Engineering, Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-Ku, Hiroshima 731-3194 (Japan); Kimura, Koji [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Matsushita, Tomohiro [Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Sayo, Hyogo 679-5198 (Japan); Park, Sam-Yong [Drug Design Laboratory, Department of Medical Life Science, Yokohama City University, Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Sasaki, Yuji C. [Department of Advanced Material Science, Graduate School of Frontier Science, The University of Tokyo, Kashiwanoha, Kashiwa 277-8561 (Japan); Hayashi, Kouichi, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Frontier Research Institute for Materials Science, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan)

    2016-06-15

    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α{sub 2}β{sub 2} 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 mm{sup 3}) 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.

  2. [Reabsorption of yellow fluorescent protein in the Rana temporaria kidney by receptor-mediated endocytosis].

    Science.gov (United States)

    Seliverstova, E V; Prutskova, N P

    2014-01-01

    The absorption of yellow fluorescent protein (YFP) and the expression of the endocytic receptors, megalin and cubilin, were investigated in the renal proximal tubules (PT) in frogs Rana temporaria after parenteral YFP injections. The methods of confocal microscopy and immunohistochemistry were used. The dynamics of YFP absorption was analyzed 2 h after injection. The logarithmic time dependence of the accumulation of YFP-containing endocytic vesicles in PT cells and the completion of absorption process 90-120 min after injection were shown. Unlike substantial megalin and cubilin expression 15-30 min after YFP introduction, immunolabeled endocytic receptors were not detected in PT cells after 2 h. The re-injection of YFP led to the appearance of apical endocytic vesicles containing megalin or cubilin colocalized with YFP. At the same time, the decrease of YFP uptake associated with reduction in the number of receptor-containing vesicles was demonstrated, suggesting a failure of megalin and cubilin expression. The decrease of absorption capacity of PT cells after YFP re-injection was similar to that found previously under conditions of the competitive absorption of green fluorescent protein (GFP) and YFP injected in different sequences. The data are the further demonstration of the proposed mechanism limiting the tubular protein absorption in the frog kidney and suggest the involvement of megalin and cubilin in uptake and vesicular transport of YFP.

  3. Correlative imaging of fluorescent proteins in resin-embedded plant material.

    Science.gov (United States)

    Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

    2013-04-01

    Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology.

  4. Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

    Directory of Open Access Journals (Sweden)

    Thijs Roebroek

    2017-09-01

    Full Text Available Reversibly switchable fluorescent proteins (RSFPs enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties.

  5. 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.

  6. Design and synthesis of ICT-based fluorescent probe for high-sensitivity protein detection and application to rapid protein staining for SDS-PAGE.

    Science.gov (United States)

    Suzuki, Yoshio; Yokoyama, Kenji

    2008-07-01

    A novel fluorescent molecular probe possessing styryl, sulfonyl, and cyanopyranyl moieties that was termed compound 1 was designed and synthesized to detect proteins through noncovalent bonding. Compound 1 did not produce fluorescence emission in the absence of proteins. However, its fluorescence spectrum showed a dramatic increase in the fluorescence intensity and strong orange emission after the addition of BSA. These changes were caused by intramolecular charge transfer (ICT). The fluorescence intensities of compound 1 were plotted as a function of the protein concentrations. A good linear relationship was observed up to a protein concentration of 325 mug/mL, and the detection limit was 70 ng/mL under the given assay conditions; this detection limit was higher than that of previously reported compounds. To demonstrate the application of compound 1, proteins in an SDS-PAGE gel were stained with compound 1 and were successfully imaged with a higher sensitivity and shorter staining operation time as compared to those of the silver staining method and SYPRO Ruby staining method. Thus, easy and high-sensitivity protein detection can be performed with the fluorescent probe, and this probe is ideally suited to proteomic applications.

  7. 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.

  8. 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.

  9. Decoupling Electronic versus Nuclear Photoresponse of Isolated Green Fluorescent Protein Chromophores Using Short Laser Pulses

    Science.gov (United States)

    Kiefer, Hjalte V.; Pedersen, Henrik B.; Bochenkova, Anastasia V.; Andersen, Lars H.

    2016-12-01

    The photophysics of a deprotonated model chromophore for the green fluorescent protein is studied by femtosecond laser pulses in an electrostatic ion-storage ring. The laser-pulse duration is much shorter than the time for internal conversion, and, hence, contributions from sequential multiphoton absorption, typically encountered with ns-laser pulses, are avoided. Following single-photon excitation, the action-absorption maximum is shown to be shifted within the S0 to S1 band from its origin at about 490 to 450 nm, which is explained by the different photophysics involved in the detected action.

  10. Post-mortem re-cloning of a transgenic red fluorescent protein dog.

    Science.gov (United States)

    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

    2011-12-01

    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. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

  11. Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

    Science.gov (United States)

    Nakanishi, J; Nakajima, T; Sato, M; Ozawa, T; Tohda, K; Umezawa, Y

    2001-07-01

    We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

  12. A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

    Directory of Open Access Journals (Sweden)

    Khorchid Ahmad

    2003-07-01

    Full Text Available Abstract Background The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. Results Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. Conclusions Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

  13. 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.

  14. 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.

  15. Fluorescent nanodiamond as a probe for the intercellular transport of proteins in vivo.

    Science.gov (United States)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Chang, Huan-Cheng

    2013-11-01

    This study investigates the intercellular transport of yolk lipoproteins in Caenorhabditis elegans by using fluorescence lifetime imaging microscopy (FLIM) and fluorescent nanodiamonds (FNDs) as photostable labels and tracers. The yolk lipoproteins in the nematode are similar to human serum low-density lipoproteins (LDLs), serving as an intercellular transporter of fat molecules and cholesterol. To study this fundamentally important process, FNDs were first coated with yolk lipoprotein complexes (YLCs) and then microinjected into the intestinal cells of the living organism. Real-time imaging over a time period of more than 50 min with FLIM revealed the process of YLC-FND secretion from the intestine to the pseudocoelomic space, followed by transporting into oocytes and subsequent accumulation in the multi-cellular embryos derived from the oocytes. Colocalization studies of the rme-2 adult hermaphrodites expressing green fluorescent protein (GFP)-tagged YLCs confirmed that the injected YLC-FNDs were taken up by oocytes through endocytosis mediated by the LDL receptor, RME-2, functioning as an YLC receptor. Our results demonstrate that FND is useful as a biomolecular nanocarrier without significantly altering the functionality of the cargos for intercellular transport, cell-specific targeting, and long-term imaging applications in vivo.

  16. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  17. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Sriram Sokalingam

    Full Text Available Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

  18. Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

    Science.gov (United States)

    Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

    2016-10-01

    Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

  19. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein: Protein Redesign to Lower Protein-lignin Binding

    Energy Technology Data Exchange (ETDEWEB)

    Haarmeyer, Carolyn N. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Smith, Matthew D. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Chundawat, Shishir P. S. [Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, East Lansing Michigan; Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway New Jersey; Sammond, Deanne [Biosciences Center, National Renewable Energy Laboratory, Golden Colorado; Whitehead, Timothy A. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing Michigan 48824

    2016-11-07

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active

  20. 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.

  1. Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

    Science.gov (United States)

    Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

    2016-01-01

    Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

  2. Coral contact dermatitis

    OpenAIRE

    Jefferson, Julie; Thompson, Curtis; Hinshaw, Molly; Rich, Phoebe

    2015-01-01

    Corals can elicit both toxic and allergic reactions upon contact with the skin. Clinical presentations vary depending on whether the reaction is acute, delayed, or chronic. Literature concerning cutaneous reactions to corals and other Cnidarians is scarce. Herein we report a case of delayed contact hypersensitivity reaction to coral and review the clinical and histopathological features of coral contact dermatitis.

  3. Coral contact dermatitis.

    Science.gov (United States)

    Jefferson, Julie; Thompson, Curtis; Hinshaw, Molly; Rich, Phoebe

    2015-04-16

    Corals can elicit both toxic and allergic reactions upon contact with the skin. Clinical presentations vary depending on whether the reaction is acute, delayed, or chronic. Literature concerning cutaneous reactions to corals and other Cnidarians is scarce. Herein we report a case of delayed contact hypersensitivity reaction to coral and review the clinical and histopathological features of coral contact dermatitis.

  4. 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.

  5. An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein

    Science.gov (United States)

    Baker, Stokes S.; Vidican, Cleo B.; Cameron, David S.; Greib, Haittam G.; Jarocki, Christine C.; Setaputri, Andres W.; Spicuzza, Christopher H.; Burr, Aaron A.; Waqas, Meriam A.; Tolbert, Danzell A.

    2012-01-01

    Background and aims Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants. Methodology Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically. Principal findings Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29). Conclusions Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with

  6. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    Science.gov (United States)

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras. Copyright 2004 Wiley-Liss, Inc.

  7. 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.

  8. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

    Directory of Open Access Journals (Sweden)

    Intekhab Islam

    2016-01-01

    Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

  9. 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

  10. Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution.

    Science.gov (United States)

    Clavel, Damien; Gotthard, Guillaume; von Stetten, David; De Sanctis, Daniele; Pasquier, Hélène; Lambert, Gerard G; Shaner, Nathan C; Royant, Antoine

    2016-12-01

    Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

  11. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

    Science.gov (United States)

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

    2016-11-01

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

  12. Corals form characteristic associations with symbiotic nitrogen-fixing bacteria.

    Science.gov (United States)

    Lema, Kimberley A; Willis, Bette L; Bourne, David G

    2012-05-01

    The complex symbiotic relationship between corals and their dinoflagellate partner Symbiodinium is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (Acropora millepora, Acropora muricata, and Pocillopora damicormis) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the nifH gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse nifH phylotypes that differ between tissue and mucus microhabitats. Coral mucus nifH sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to Symbiodinium.

  13. 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.

  14. Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

    Science.gov (United States)

    Ghisaidoobe, Amar B. T.; Chung, Sang J.

    2014-01-01

    Förster 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 (λEX ∼ 280 nm, λEM ∼ 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 protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster 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. PMID:25490136

  15. 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

  16. Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

    Directory of Open Access Journals (Sweden)

    Jeong Y

    2015-11-01

    Full Text Available Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface

  17. 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.

  18. Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume.

    Science.gov (United States)

    Pedersen, Meike; Carmosino, Monica; Forbush, Biff

    2008-02-01

    To examine the structure and function of the Na-K-Cl cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl(-) provided a ratiometric indicator of intracellular [Cl(-)]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed contemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

  19. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    Directory of Open Access Journals (Sweden)

    Alexander Asanov

    2012-02-01

    Full Text Available We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax.

  20. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    Science.gov (United States)

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

  1. pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins.

    Science.gov (United States)

    Betolngar, Dahdjim-Benoît; Erard, Marie; Pasquier, Hélène; Bousmah, Yasmina; Diop-Sy, Awa; Guiot, Elvire; Vincent, Pierre; Mérola, Fabienne

    2015-05-01

    It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.

  2. 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.

  3. POLA EKSPRESI GEN ENHANCED GREEN FLUORESCENT PROTEIN PADA EMBRIO DAN LARVA IKAN PATIN SIAM (Pangasianodon hypophthalmus

    Directory of Open Access Journals (Sweden)

    Raden Roro Sri Pudji Sinarni Dewi

    2016-04-01

    menggunakan gen reporter berguna untuk mendesain konstruksi gen yang akan digunakan pada penelitian transgenesis. Gen reporter yang umum digunakan dalam penelitian ekspresi sementara transgen adalah gen GFP (green fluorescent protein. Pengamatan gen EGFP (enhanced green fluorescent protein pada embrio dan larva ikan patin siam (Pangasianodon hypophthalmus ditujukan untuk mendapatkan informasi mengenai kemampuan promoter -aktin ikan mas dalam mengendalikan ekspresi gen EGFP. Gen EGFP diintroduksikan ke dalam sperma ikan patin siam menggunakan metode elektroporasi. Sperma yang telah dielektroporasi digunakan untuk membuahi sel telur ikan patin siam. Pengamatan ekspresi gen EGFP dilakukan setiap enam jam dimulai dari embrio fase 2 sel sampai larva. Berdasarkan hasil penelitian, gen EGFP terekspresi pada fase embrio dan larva ikan patin siam. Puncak ekspresi gen EGFP terjadi pada fase neurula dan menurun pada fase larva. Berdasarkan penelitian ini maka ikan patin siam transgenik telah berhasil dibentuk dan promoter -aktin ikan mas terbukti aktif dalam mengarahkan ekspresi gen asing (GFP di dalam tubuh ikan patin siam.

  4. Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

    Science.gov (United States)

    Sang, Chen-Hsiang; Chou, Shu-Jen; Pan, F M; Sheu, Jeng-Tzong

    2016-01-15

    In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection.

  5. Recovery of red fluorescent protein chromophore maturation deficiency through rational design.

    Directory of Open Access Journals (Sweden)

    Matthew M Moore

    Full Text Available Red fluorescent proteins (RFPs derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore's covalent structure. We also created an optimized mPlum mutant (mPlum-E16P that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.

  6. 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.

  7. 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

    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...

  8. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    Science.gov (United States)

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  9. First molecular identification of the transgene red fluorescent protein (RFP) in transgenic ornamental zebrafish (Danio rerio) introduced in Peru

    OpenAIRE

    Carlos Scotto; Fernando Serna

    2013-01-01

    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...

  10. Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection.

    Science.gov (United States)

    Dumas, B; Centis, S; Sarrazin, N; Esquerré-Tugayé, M T

    1999-04-01

    The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.

  11. 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.

  12. Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.

    Science.gov (United States)

    Fortin, Dale A; Tillo, Shane E; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V; Guo, Caiying; Mao, Tianyi; Zhong, Haining

    2014-12-10

    Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. Copyright © 2014 the authors 0270-6474/14/3416698-15$15.00/0.

  13. 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

  14. 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.

  15. 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

  16. Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.

    Science.gov (United States)

    Sustarsic, Marko; Plochowietz, Anne; Aigrain, Louise; Yuzenkova, Yulia; Zenkin, Nikolay; Kapanidis, Achillefs

    2014-07-01

    Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.

  17. Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Shun-lin HU; Qin SUN; Qu-zhi WANG; Yul-iang LIU; Yan-tao WU; Xiu-fan LIU

    2007-01-01

    Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain,seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI,which contained the full-length cDNA of the NDV ZJI strain.The pNDV/ZJI,with three helper plasmids,pCIneoNP,pCIneoP and pCIneoL,were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase.After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock,an infectious NDV ZJI strain was successfully rescued.Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP.After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells,the recombinant NDV,NDV/ZJIGFP,was rescued.Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection,indicating that the GFP gene was expressed at a relatively high level.NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route.Four days post-infection,strong green fluorescence could be detected in the kidneys and tracheae,indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

  18. Use of fluorescent proteins and color-coded imaging to visualize cancer cells with different genetic properties.

    Science.gov (United States)

    Hoffman, Robert M

    2016-03-01

    Fluorescent proteins are very bright and available in spectrally-distinct colors, enable the imaging of color-coded cancer cells growing in vivo and therefore the distinction of cancer cells with different genetic properties. Non-invasive and intravital imaging of cancer cells with fluorescent proteins allows the visualization of distinct genetic variants of cancer cells down to the cellular level in vivo. Cancer cells with increased or decreased ability to metastasize can be distinguished in vivo. Gene exchange in vivo which enables low metastatic cancer cells to convert to high metastatic can be color-coded imaged in vivo. Cancer stem-like and non-stem cells can be distinguished in vivo by color-coded imaging. These properties also demonstrate the vast superiority of imaging cancer cells in vivo with fluorescent proteins over photon counting of luciferase-labeled cancer cells.

  19. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  20. Bio-optimized energy transfer in densely packed fluorescent protein enables near-maximal luminescence and solid-state lasers.

    Science.gov (United States)

    Gather, Malte C; Yun, Seok Hyun

    2014-12-08

    Bioluminescent organisms are likely to have an evolutionary drive towards high radiance. As such, bio-optimized materials derived from them hold great promise for photonic applications. Here, we show that biologically produced fluorescent proteins retain their high brightness even at the maximum density in solid state through a special molecular structure that provides optimal balance between high protein concentration and low resonance energy transfer self-quenching. Dried films of green fluorescent protein show low fluorescence quenching (-7 dB) and support strong optical amplification (gnet=22 cm(-1); 96 dB cm(-1)). Using these properties, we demonstrate vertical cavity surface emitting micro-lasers with low threshold (lasers) and self-assembled all-protein ring lasers. Moreover, solid-state blends of different proteins support efficient Förster resonance energy transfer, with sensitivity to intermolecular distance thus allowing all-optical sensing. The design of fluorescent proteins may be exploited for bio-inspired solid-state luminescent molecules or nanoparticles.