Science.gov

Sample records for fluorescent proteins engineered

  1. An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

    PubMed Central

    Yu, Xiaozhen; Strub, Marie-Paule; Barnard, Travis J.; Noinaj, Nicholas; Piszczek, Grzegorz; Buchanan, Susan K.; Taraska, Justin W.

    2014-01-01

    Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum. PMID:24752441

  2. An engineered monomeric Zoanthus sp. yellow fluorescent protein.

    PubMed

    Hoi, Hiofan; Howe, Elizabeth S; Ding, Yidan; Zhang, Wei; Baird, Michelle A; Sell, Brittney R; Allen, John R; Davidson, Michael W; Campbell, Robert E

    2013-10-24

    Protein engineering has created a palette of monomeric fluorescent proteins (FPs), but there remains an ~30 nm spectral gap between the most red-shifted useful Aequorea victoria green FP (GFP) variants and the most blue-shifted useful Discosoma sp. red FP (RFP) variants. To fill this gap, we have engineered a monomeric version of the yellow FP (YFP) from Zoanthus sp. coral. Our preferred variant, designated as mPapaya1, displays excellent fluorescent brightness, good photostability, and retains its monomeric character both in vitro and in living cells in the context of protein chimeras. We demonstrate that mPapaya1 can serve as a good Förster resonance energy transfer (FRET) acceptor when paired with an mTFP1 donor. mPapaya1 is a valuable addition to the palette of FP variants that are useful for multicolor imaging and FRET-based biosensing.

  3. Genetically encoded biosensors based on engineered fluorescent proteins.

    PubMed

    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.

  4. Engineering and Characterization of a Superfolder Green Fluorescent Protein

    SciTech Connect

    Pedelacq,J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.

    2006-01-01

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

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

    DOE PAGES

    Close, Devin W.; Paul, Craig Don; Langan, Patricia S.; ...

    2015-05-08

    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 ofmore » 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.« less

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

    SciTech Connect

    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.

    2015-05-08

    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.

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

    PubMed

    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.

  8. Newly engineered cyan fluorescent proteins with enhanced performances for live cell FRET imaging.

    PubMed

    Mérola, Fabienne; Fredj, Asma; Betolngar, Dahdjim-Benoît; Ziegler, Cornelia; Erard, Marie; Pasquier, Hélène

    2014-02-01

    Cyan fluorescent proteins (CFPs) derived from Aequorea victoria green fluorescent protein are the most widely used Förster resonant energy transfer (FRET) donors in genetically encoded biosensors for live-cell imaging and bioassays. However, the weak and complex fluorescence emission of cyan variants, such as enhanced cyan fluorescent protein (ECFP) or Cerulean, has long remained a major bottleneck in these FRET techniques. Recently, several CFPs with greatly improved performances, including mTurquoise, mTurquoise2, mCerulean3, and Aquamarine, have been engineered through a mixture of site-directed and large-scale random mutagenesis. This review summarizes the engineering and relative merits of these new cyan donors, which can readily replace popular CFPs in FRET imaging protocols, while reaching fluorescence quantum yields close to 90%, and unprecedented long, near-single fluorescence lifetimes of about 4 ns. These variants display an increased general photostability and much reduced environmental sensitivity, notably towards acid pH. These new, bright, and robust CFPs now open up exciting outlooks for fluorescence lifetime imaging microscopy and advanced quantitative FRET analyses in living cells. In addition, the stepwise engineering of Aquamarine shows that only two critical mutations in ECFP, and one in Cerulean, are required to achieve these performances, which brings new insights into the structural bases of their photophysical properties.

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  10. Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

    PubMed Central

    Ciruela, Francisco; Fernández-Dueñas, Víctor; Jacobson, Kenneth A.

    2015-01-01

    The use of G protein-coupled receptors fluorescent ligands is undergoing continuous expansion. In line with this, fluorescent agonists and antagonists of high affinity for G protein-coupled adenosine and P2Y receptors have been shown to be useful pharmacological probe compounds. Fluorescent ligands for A1R, A2AR, and A3R (adenosine receptors) and P2Y2R, P2Y4R, P2Y6R, and P2Y14R (nucleotide receptors) have been reported. Such ligands have been successfully applied to drug discovery and to GPCR characterization by flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer and scanning confocal microscopy. Here we summarize recently reported and readily available representative fluorescent ligands of purinergic receptors. In addition, we pay special attention on the use of this family of fluorescent ligands revealing two main aspects of purinergic receptor biology, namely ligand binding and receptor oligomerization. PMID:25890205

  11. Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic Tools Engineered from Phytochromes.

    PubMed

    Chernov, Konstantin G; Redchuk, Taras A; Omelina, Evgeniya S; Verkhusha, Vladislav V

    2017-04-12

    Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore. Because biliverdin possesses the largest electron-conjugated chromophore system among linear tetrapyrroles, BphPs exhibit the most NIR-shifted spectra that reside within the NIR tissue transparency window. Here we analyze phytochrome structure and photochemistry to describe the molecular mechanisms by which they function. We then present strategies to engineer BphP-based NIR fluorescent proteins and review their properties and applications in modern imaging technologies. We next summarize designs of reporters and biosensors and describe their use in the detection of protein-protein interactions, proteolytic activities, and posttranslational modifications. Finally, we provide an overview of optogenetic tools developed from phytochromes and describe their use in light-controlled cell signaling, gene expression, and protein localization. Our review provides guidelines for the selection of NIR probes and tools for noninvasive imaging, sensing, and light-manipulation applications, specifically focusing on probes developed for use in mammalian cells and in vivo.

  12. Computational prediction of absorbance maxima for a structurally diverse series of engineered green fluorescent protein chromophores.

    PubMed

    Timerghazin, Qadir K; Carlson, Haley J; Liang, Chen; Campbell, Robert E; Brown, Alex

    2008-02-28

    By virtue of its self-sufficiency to form a visible wavelength chromophore within the confines of its tertiary structure, the Aequorea victoria green fluorescent protein (GFP) is single-handedly responsible for the ever-growing popularity of fluorescence imaging of recombinant fusion proteins in biological research. Engineered variants of GFP with altered excitation or emission wavelength maxima have helped to expand the range of applications of GFP. The engineering of the GFP variants is usually done empirically by genetic modifications of the chromophore structure and/or its environment in order to find variants with new photophysical properties. The process of identifying improved variants could be greatly facilitated if augmented or guided by computational studies of the chromophore ground and excited-state properties and dynamics. In pursuit of this goal, we now report a thorough investigation of computational methods for prediction of the absorbance maxima for an experimentally validated series of engineered GFP chromophore analogues. The experimental dataset is composed of absorption maxima for 10 chemically distinct GFP chromophore analogues, including a previously unreported Y66D variant, measured under identical denaturing conditions. For each chromophore analogue, excitation energies and oscillator strengths were calculated using configuration interaction with single excitations (CIS), CIS with perturbative correction for double substitutions [CIS(D)], and time-dependent density functional theory (TD DFT) using several density functionals with solvent effects included using a polarizable continuum model. Comparison of the experimental and computational results show generally poor quantitative agreement with all methods attempted. However, good linear correlations between the calculated and experimental excitation energies (R2>0.9) could be obtained. Oscillator strengths obtained with TD DFT using pure density functionals also correlate well with the

  13. Engineering fluorescent proteins towards ultimate performances: lessons from the newly developed cyan variants

    NASA Astrophysics Data System (ADS)

    Mérola, Fabienne; Erard, Marie; Fredj, Asma; Pasquier, Hélène

    2016-03-01

    New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from the Aequorea victoria species. Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards the de novo design of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.

  14. A new bright green-emitting fluorescent protein: Engineered monomeric and dimeric forms

    PubMed Central

    Ilagan, Robielyn P.; Rhoades, Elizabeth; Gruber, David F.; Kao, Hung-Teh; Pieribone, Vincent A.; Regan, Lynne

    2010-01-01

    Summary Fluorescent proteins (FP) have become essential tools in molecular and biological applications. Here, we present a novel fluorescent protein isolated from warm water coral, Cyphastrea microphthalma. The protein, which we named VFP (vivid Verde FP), matures readily at 37 °C and emits bright green light. Further characterizations revealed that VFP has a tendency to form dimers. By creating a homology model of VFP, based on the structure of red fluorescent protein DsRed, we were able to make mutations that alter the protein’s oligomerization state. We present two proteins, mVFP and mVFP1, that are both exclusively monomeric, and one, dVFP, which is dimeric. We characterized the spectroscopic properties of VFP and its variants in comparison with enhance green fluorescent protein (EGFP), a widely use variant of GFP. All the VFP variants are at least twice as bright as EGFP. Finally, we demonstrate the effectiveness of the VFP variants both in vitro and in vivo detection applications. PMID:20345907

  15. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

    SciTech Connect

    Arpino, James A. J.; Rizkallah, Pierre J.; Jones, D. Dafydd

    2014-08-01

    The beneficial engineered single-amino-acid deletion variants EGFP{sup D190Δ} and EGFP{sup A227Δ} have been studied. Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP{sup D190Δ} containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP{sup A227Δ} revealed that a ‘flipping’ mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

  16. Imaging HIV-1 Tat Trafficking and Interactions by Engineered Green-Fluorescent-Protein Tagging

    NASA Astrophysics Data System (ADS)

    Beltram, Fabio

    2002-03-01

    The direct monitoring of protein function in live cells under physiologically relevant conditions is one of the most powerful and innovative methodologies for proteomics. Efficient florescent probes fully compatible with human-cell expression are the fundamental tools for these studies and their optimization opens the way to resolution at the single-protein level. Biological events involving protein pairs are also directly accessible thanks to tuning of protein-tag spectral properties and production of complementary pairs. Such pairs are characterized by overlapping absorption (for the acceptor tag) and emission (for the donor tag) spectra. By tagging the proteins of interest with acceptor and donor molecules, protein interaction can be directly visualized by FRET, fluorescent resonant energy transfer. In this talk we shall present the design by molecular dynamics calculations and the application of optimized green fluorescent proteins to the study of the human immunodeficiency virus HIV-1 proteomics. In particular trafficking and cellular interactions of HIV-1 transactivator protein Tat in live human cells will be presented. Tat localization and complex internalization pathways of exogenous molecules will be presented thanks to the peculiar optical properties of mutated GFPs. Cellular protein partners and subcellular interaction sites will be identified and directly visualized. The relevance of such results and of advanced spectroscopic and imaging techniques for a new level of understanding of biological processes and its significance for advancement in molecular biology will be underlined. A. Marcello et al., J. Biol. Chem. 276, 39220 (2001). R. Cinelli et al., Appl. Phys. Lett. 79, 3353 (2001).

  17. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome

    PubMed Central

    Piatkevich, Kiryl D.; Subach, Fedor V.; Verkhusha, Vladislav V.

    2013-01-01

    Ability to modulate fluorescence of optical probes can be used to enhance signal-to-noise ratio for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here we report two phytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize heme-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690 nm/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels. PMID:23842578

  18. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

    PubMed Central

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

    2016-01-01

    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. PMID:27857208

  19. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

    NASA Astrophysics Data System (ADS)

    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.

  20. Structure-guided Engineering Enhances a Phytochrome-based Infrared Fluorescent Protein*

    PubMed Central

    Auldridge, Michele E.; Satyshur, Kenneth A.; Anstrom, David M.; Forest, Katrina T.

    2012-01-01

    Phytochrome is a multidomain dimeric red light photoreceptor that utilizes a chromophore-binding domain (CBD), a PHY domain, and an output module to induce cellular changes in response to light. A promising biotechnology tool emerged when a structure-based substitution at Asp-207 was shown to be an infrared fluorophore that uses a biologically available tetrapyrrole chromophore. We report multiple crystal structures of this D207H variant of the Deinococcus radiodurans CBD, in which His-207 is observed to form a hydrogen bond with either the tetrapyrrole A-ring oxygen or the Tyr-263 hydroxyl. Based on the implications of this duality for fluorescence properties, Y263F was introduced and shown to have stronger fluorescence than the original D207H template. Our structures are consistent with the model that the Y263F change prevents a red light-induced far-red light absorbing phytochrome chromophore configuration. With the goal of decreasing size and thereby facilitating use as a fluorescent tag in vivo, we also engineered a monomeric form of the CBD. Unexpectedly, photoconversion was observed in the monomer despite the lack of a PHY domain. This observation underscores an interplay between dimerization and the photochemical properties of phytochrome and suggests that the monomeric CBD could be used for further studies of the photocycle. The D207H substitution on its own in the monomer did not result in fluorescence, whereas Y263F did. Combined, the D207H and Y263F substitutions in the monomeric CBD lead to the brightest of our variants, designated Wisconsin infrared phytofluor (Wi-Phy). PMID:22210774

  1. Highly thermostable fluorescent proteins

    DOEpatents

    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.

  2. Highly thermostable fluorescent proteins

    DOEpatents

    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.

  3. Highly thermostable fluorescent proteins

    DOEpatents

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

    2011-11-29

    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.

  4. Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

    PubMed Central

    2013-01-01

    Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co

  5. Fluorescent protein biosensors applied to microphysiological systems

    PubMed Central

    Senutovitch, Nina; Boltz, Robert; DeBiasio, Richard; Gough, Albert; Taylor, D Lansing

    2015-01-01

    This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with

  6. Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift

    PubMed Central

    Piatkevich, Kiryl D.; Malashkevich, Vladimir N.; Almo, Steven C.; Verkhusha, Vladislav V.

    2010-01-01

    LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs) which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 Å and 1.5 Å, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis analyses, as well as isotope and temperature dependences suggest that an excited state proton transfer (ESPT) is responsible for the LSSs observed in LSSmKates. Hydrogen bonding between the chromophore hydroxyl and Glu160 in LSSmKate1 and a proton relay involving the chromophore tyrosine hydroxyl, Ser158 and the Asp160 carboxylate in LSSmKate2 represent the putative ESPT pathways. Comparisons with mKeima LSS RFP suggest that similar proton relays could be engineered in other FPs. Accordingly, we mutated positions 158 and 160 in several conventional red-shifted FPs, including mNeptune, mCherry, mStrawberry, mOrange and mKO, and the resulting FP variants exhibited LSS fluorescence emission in a wide range of wavelengths from 560 to 640 nm. These data suggest that different chromophores formed by distinct tripeptides in different environments can be rationally modified to yield RFPs with novel photochemical properties. PMID:20681709

  7. Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift.

    PubMed

    Piatkevich, Kiryl D; Malashkevich, Vladimir N; Almo, Steven C; Verkhusha, Vladislav V

    2010-08-11

    LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs), which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 and 1.5 A, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis analyses, as well as isotope and temperature dependences, suggest that an excited-state proton transfer (ESPT) is responsible for the LSSs observed in LSSmKates. Hydrogen bonding between the chromophore hydroxyl and Glu160 in LSSmKate1 and a proton relay involving the chromophore tyrosine hydroxyl, Ser158, and the Asp160 carboxylate in LSSmKate2 represent the putative ESPT pathways. Comparisons with mKeima LSS RFP suggest that similar proton relays could be engineered in other FPs. Accordingly, we mutated positions 158 and 160 in several conventional red-shifted FPs, including mNeptune, mCherry, mStrawberry, mOrange, and mKO, and the resulting FP variants exhibited LSS fluorescence emission in a wide range of wavelengths from 560 to 640 nm. These data suggest that different chromophores formed by distinct tripeptides in different environments can be rationally modified to yield RFPs with novel photochemical properties.

  8. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression.

  9. Green fluorescent protein: A perspective

    PubMed Central

    Remington, S James

    2011-01-01

    A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994–2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationships in photoswitchable fluorescent proteins and nonfluorescent chromoproteins are also briefly covered. PMID:21714025

  10. Green fluorescent protein: a perspective.

    PubMed

    Remington, S James

    2011-09-01

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

  11. Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: A comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies

    SciTech Connect

    Khan, Faaizah; Gnudi, Luigi; Pickup, John C.

    2008-01-04

    Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor.

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

    DOEpatents

    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.

  13. Imaging individual green fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Pierce, Daniel W.; Hom-Booher, Nora; Vale, Ronald D.

    1997-07-01

    Recent advances in fluorescence microscopy techniques have allowed the video-time imaging of single molecules of fluorescent dyes covalently bound to proteins in aqueous environments. However, the techniques have not been exploited fully because proteins can be difficult to label, and dye modification may cause partial or complete loss of activity. These difficulties could be circumvented by fusing proteins to green fluorescent protein (GFP) of the jellyfish Aequorea victoria. Here we report that single S65T mutant GFP molecules can be imaged using total internal reflection microscopy, and that ATP-driven movement of an individual kinesin molecule (a microtubule motor protein) fused to GFP can be readily observed.

  14. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

    PubMed Central

    Shaner, Nathan C.; Lambert, Gerard G.; Chammas, Andrew; Ni, Yuhui; Cranfill, Paula J.; Baird, Michelle A.; Sell, Brittney R.; Allen, John R.; Day, Richard N.; Israelsson, Maria; Davidson, Michael W.; Wang, Jiwu

    2013-01-01

    Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins. PMID:23524392

  15. Quantitative Assessment of Fluorescent Proteins

    PubMed Central

    Cranfill, Paula J.; Sell, Brittney R.; Baird, Michelle A.; Allen, John R.; Lavagnino, Zeno; de Gruiter, H. Martijn; Kremers, Gert-Jan; Davidson, Michael W.; Ustione, Alessandro; Piston, David W.

    2016-01-01

    The advent of fluorescent proteins (FP) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning the blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has its own unique photophysical properties. Thus, there is no single “best” fluorescent protein for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for any given application, we have characterized quantitatively over 40 different FPs for their brightness, photostability, pH stability, and monomeric properties, which permits easy apples-to-apples comparisons between these FPs. We report the values for all of the FPs measured, but focus the discussion on the more popular and/or best performing FPs in each spectral region. PMID:27240257

  16. Lasing from fluorescent protein crystals.

    PubMed

    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.

  17. Going Viral with Fluorescent Proteins.

    PubMed

    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.

  18. Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

    PubMed

    Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

    2015-04-21

    Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

  19. Advances in fluorescent protein technology.

    PubMed

    Shaner, Nathan C; Patterson, George H; Davidson, Michael W

    2007-12-15

    Current fluorescent protein (FP) development strategies are focused on fine-tuning the photophysical properties of blue to yellow variants derived from the Aequorea victoria jellyfish green fluorescent protein (GFP) and on the development of monomeric FPs from other organisms that emit in the yellow-orange to far-red regions of the visible light spectrum. Progress toward these goals has been substantial, and near-infrared emitting FPs may loom over the horizon. The latest efforts in jellyfish variants have resulted in new and improved monomeric BFP, CFP, GFP and YFP variants, and the relentless search for a bright, monomeric and fast-maturing red FP has yielded a host of excellent candidates, although none is yet optimal for all applications. Meanwhile, photoactivatable FPs are emerging as a powerful class of probes for intracellular dynamics and, unexpectedly, as useful tools for the development of superresolution microscopy applications.

  20. A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

    PubMed

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

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

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

    ERIC Educational Resources Information Center

    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…

  2. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  3. Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto; Krol, Silke; Campanini, Barbara; Cannone, Fabio; Chirico, Giuseppe

    2006-10-01

    Discovery of Green Fluorescent Protein (GFP) constituted an important improvement for living cell studies on submicron resolution allowing in vivo fluorescence labeling. We studied the photo-physical properties of single GFP molecules incorporated in a charged polyelectrolyte environment by means of single molecule spectroscopy. The fluorescence characteristics change dramatically in terms of photo-stability,lifetime and blinking behavior so that the proteins scale up to quantum dots. The reported results highlight interesting applications in the design of fluorescent markers and in the development of optical data storage architectures.

  4. Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo: application of green and red fluorescent protein-expressing retroviral vector.

    PubMed

    Sales, Virna L; Mettler, Bret A; Lopez-Ilasaca, Marco; Johnson, John A; Mayer, John E

    2007-03-01

    An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus-based retroviral vector yields transduction efficiency of greater than 97% GFP(+) EPC- and RFP(+) mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a retroviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circulating endothelial and marrow-derived precursors, we simultaneously co-seeded 2 x 10(6) GFP(+) EPCs and 2 x 10(5) RFP(+) MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP(+) EPC- and RFP(+) MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE constructs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double-labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells.

  5. Protein engineering of subtilisin.

    PubMed

    Bryan, P N

    2000-12-29

    The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.

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

    PubMed

    Fu, Yi; Zhang, Jian; Lakowicz, Joseph R

    2008-11-28

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

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

    SciTech Connect

    Fu Yi; Zhang Jian; Lakowicz, Joseph R.

    2008-11-28

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

  8. Advanced Fluorescence Protein-Based Synapse-Detectors

    PubMed Central

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

    2016-01-01

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

  9. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  10. Photocontrollable Fluorescent Proteins for Superresolution Imaging

    PubMed Central

    Shcherbakova, Daria M.; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V.

    2014-01-01

    Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine. PMID:24895855

  11. Fluorescent proteins: shine on, you crazy diamond.

    PubMed

    Dedecker, Peter; De Schryver, Frans C; Hofkens, Johan

    2013-02-20

    In this Perspective we discuss recent trends in the development and applications of fluorescent proteins. We start by providing a historical and structural perspective of their spectroscopic and structural aspects and describe how these properties have made fluorescent proteins essential as 'smart labels' for biosensing and advanced fluorescence imaging. We show that the strong link between the spectroscopic properties and protein structure and properties is a necessary element in these developments and that this dependence makes the proteins excellent model systems for a variety of fields. We pay particular attention to emerging or future research opportunities and unsolved questions.

  12. Imaging proteins inside cells with fluorescent tags

    PubMed Central

    Crivat, Georgeta; Taraska, Justin W.

    2011-01-01

    Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use I) auto-fluorescent proteins, II) self-labeling enzymes, III) enzymes that catalyze the attachment of a probe to a target sequence, and IV) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells. PMID:21924508

  13. An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy.

    PubMed

    Murray, John M

    2017-03-22

    The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report, the construction, properties and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.

  14. Directed evolution of an extremely stable fluorescent protein.

    PubMed

    Kiss, Csaba; Temirov, Jamshid; Chasteen, Leslie; Waldo, Geoffrey S; Bradbury, Andrew R M

    2009-05-01

    In this paper we describe the evolution of eCGP123, an extremely stable green fluorescent protein based on a previously described fluorescent protein created by consensus engineering (CGP: consensus green protein). eCGP123 could not be denatured by a standard thermal melt, preserved almost full fluorescence after overnight incubation at 80 degrees C and possessed a free energy of denaturation of 12.4 kcal/mol. It was created from CGP by a recursive process involving the sequential introduction of three destabilizing heterologous inserts, evolution to overcome the destabilization and finally 'removal' of the destabilizing insert by gene synthesis. We believe that this approach may be generally applicable to the stabilization of other proteins.

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  16. Flavin-based fluorescent proteins: emerging paradigms in biological imaging.

    PubMed

    Mukherjee, Arnab; Schroeder, Charles M

    2015-02-01

    Flavin-based fluorescent proteins (FbFPs) are an emerging class of fluorescent reporters characterized by oxygen-independent fluorescence and a small size - key advantages compared to the green fluorescent protein (GFP). FbFPs are at a nascent stage of development. However, they have already been used as versatile reporters for studying anaerobic biosystems and viral assemblies. Recently, FbFPs with improved brightness and photostability have been engineered. In addition, several FbFPs show high degrees of thermal and pH stability. For these reasons, FbFPs hold strong promise to extend bioimaging to clinically and industrially significant systems that have been challenging to study using GFPs. In this review, we highlight recent developments in the FbFP toolbox and explore further improvements necessary to maximize the potential of FbFPs.

  17. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy.

    PubMed

    Stiel, Andre C; Andresen, Martin; Bock, Hannes; Hilbert, Michael; Schilde, Jessica; Schönle, Andreas; Eggeling, Christian; Egner, Alexander; Hell, Stefan W; Jakobs, Stefan

    2008-09-15

    Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.

  18. DNA Duplex Engineering for Enantioselective Fluorescent Sensor.

    PubMed

    Hu, Yuehua; Lin, Fan; Wu, Tao; Zhou, Yufeng; Li, Qiusha; Shao, Yong; Xu, Zhiai

    2017-02-21

    The rapid identification of biomacromolecule structure that has a specific association with chiral enantiomers especially from natural sources will be helpful in developing enantioselective sensor and in speeding up drug exploitation. Herein, owing to its existence also in living cells, apurinic/apyrimidinic site (AP site) was first engineered into ds-DNA duplex to explore its competence in enantiomer selectivity. An AP site-specific fluorophore was utilized as an enantioselective discrimination probe to develop a straightforward chiral sensor using natural tetrahydropalmatine (L- and D-THP) as enantiomer representatives. We found that only L-THP can efficiently replace the prebound fluorophore to cause a significant fluorescence increase due to its specific binding with the AP site (two orders magnitude higher in affinity than binding with D-THP). The AP site binding specificity of L-THP over D-THP was assessed via intrinsic fluorescence, isothermal titration calorimetry, and DNA stability. The enantioselective performance can be easily tuned by the sequences near the AP site and the number of AP sites. A single AP site provides a perfect binding pocket to differentiate the chiral atom-induced structure discrepancy. We expect that our work will inspire interest in engineering local structures into a ds-DNA duplex for developing novel enantioselective sensors.

  19. Fluorescent protein integrated white LEDs for displays

    NASA Astrophysics Data System (ADS)

    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.

  20. Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

    PubMed Central

    Smurthwaite, Cameron A.; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D.; Reed, Connor W.; Dharmawan, Andre; Wolkowicz, Roland

    2015-01-01

    Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded ´indefinitely´. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing. PMID:25938804

  1. Trace fluorescent labeling for protein crystallization

    PubMed Central

    Pusey, Marc; Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-01-01

    Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent. PMID:26144224

  2. Hydrogels Constructed from Engineered Proteins.

    PubMed

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed.

  3. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

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

  4. Trace fluorescent labeling for protein crystallization

    SciTech Connect

    Pusey, Marc Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-06-27

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

  5. Conjugation of fluorescent proteins with DNA oligonucleotides.

    PubMed

    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.

  6. Fluorescence spectroscopy of protein oligomerization in membranes.

    PubMed

    Gorbenko, Galyna P

    2011-05-01

    Fluorescence spectroscopy is one of the most powerful tools for characterization of a multitude of biological processes. Of these, the phenomenon of protein oligomerization attracts especial interest due to its crucial role in the formation of fibrillar protein aggregates (amyloid fibrils) involved in ethiology of so-called protein misfolding diseases. It is becoming increasingly substantiated that protein fibrillization in vivo can be initiated and modulated at membrane-water interface. All steps of membrane-assisted fibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition of polypeptide chain into a highly aggregation-prone partially folded conformation, assembly of oligomeric nucleus from membrane-bound monomeric species and fiber elongation can be monitored with a mighty family of fluorescence-based techniques. Furthermore, the mechanisms behind cytotoxicity of prefibrillar protein oligomers are highly amenable to fluorescence analysis. The applications of fluorescence spectroscopy to monitoring protein oligomerization in a membrane environment are exemplified and some problems encountered in such kinds of studies are highlighted.

  7. Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters.

    PubMed

    Mukherjee, Arnab; Walker, Joshua; Weyant, Kevin B; Schroeder, Charles M

    2013-01-01

    Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable

  8. Fluorescent proteins: maturation, photochemistry and photophysics.

    PubMed

    Remington, S James

    2006-12-01

    It has long been appreciated that green fluorescent protein (GFP) autocatalytically forms its chromophore in a host-independent process; several of the initial steps in the reaction have recently been elucidated. Nevertheless, the end points of the process are unexpectedly diverse, as six chemically distinct chromophores, including two with three rings, have been identified. All fluorescent proteins continuously produce a low level of reactive oxygen species under illumination, which, in some cases, can lead to host cell death. In one extreme but useful example, the red fluorescent protein KillerRed can be used to selectively destroy cells upon brief illumination. Finally, when photophysical processes such as excited-state proton transfer, reversible photobleaching and photoactivation are understood, useful research tools, for example, real-time biosensors and optical highlighters, can result; however, side effects of their use may lead to significant artifacts in time-dependent microscopy experiments.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2014-01-01

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

  11. Modern fluorescent proteins: from chromophore formation to novel intracellular applications

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Shcherbakova, Daria M.; Kuznetsova, Irina M.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

    2015-01-01

    The diverse biochemical and photophysical properties of fluorescent proteins (FPs) have enabled the generation of a growing palette of colors, providing unique opportunities for their use in a variety of modern biology applications. Modulation of these FP characteristics is achieved through diversity in both the structure of the chromophore as well as the contacts between the chromophore and the surrounding protein barrel. Here we review our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. Progress in understanding the interplay between FP structure and function has allowed the engineering of FPs with many desirable features, and enabled recent advances in microscopy techniques such as super-resolution imaging of single molecules, imaging of protein dynamics, photochromic FRET, deep-tissue imaging, and multicolor two-photon microscopy in live animals. PMID:22054544

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

    NASA Astrophysics Data System (ADS)

    Tolosa, Leah

    Proteins are the cell’s workhorses as they are involved in essentially all cellular activities. From the birth of a daughter cell until its death during apoptosis, proteins perform various functions as needed. Such versatility can be attributed to the seemingly infinite ways that the amino acids are sequenced in a polypeptide. The polypeptide backbone and each amino acid functional group contribute unique intermolecular interactions that determine the protein’s shape and size. However, more importantly, these interactions determine protein function, that is, its ability to interact with the external environment. For the same reasons, proteins make ideal biorecognition elements for sensors (see below and Fig. 1). Protein enzymes and receptors developed unmatched sensitivity and selectivity for their substrates through millions of years of natural selection. In contrast, chemically synthesized artificial receptors seldom approach the same level of sophistication in ligand selectivity, sensitivity range and ease of production. In addition, there is available to the sensor researcher an ever expanding library of proteins for various ligands important in all manner of application such as disease biomarkers, toxins, contaminants, drugs, etc. Using tools of genetic engineering, researchers can even go beyond known existing wild type proteins by introducing useful functional groups thereby tuning or even altering protein sensitivity and selectivity. This can be done by mutagenesis and directed evolution or by incorporation of unnatural amino acids during translation. Additionally, genes for the protein of interest can be inserted in vectors predesigned with handles for affinity purification or immobilization on a surface. Recombinant proteins can then be produced in large amounts in appropriate cellular hosts with very high reproducibility. In addition, chemical reactions involving the amino acids have become standard protocols. Thus, there are thousands of dyes

  13. Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions.

    PubMed

    Gijsbers, Abril; Nishigaki, Takuya; Sánchez-Puig, Nuria

    2016-10-21

    Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4',5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the

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

    PubMed

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

    2015-10-27

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

  15. A Guide to Fluorescent Protein FRET Pairs

    PubMed Central

    Bajar, Bryce T.; Wang, Emily S.; Zhang, Shu; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. PMID:27649177

  16. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

    PubMed

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-15

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

  17. Engineering of vault nanocapsules with enzymatic and fluorescent properties.

    PubMed

    Kickhoefer, Valerie A; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I; Dunn, Bruce; Stewart, Phoebe L; Rome, Leonard H

    2005-03-22

    One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

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

    PubMed Central

    Vasiljevic, Snezana; Ren, Junyuan; Yao, YongXiu; Dalton, Kevin; Adamson, Catherine S; Jones, Ian M

    2006-01-01

    Background The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein. Results Fusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence. Conclusion Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction. PMID:16939649

  19. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

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

    PubMed

    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.

  1. [The green fluorescent protein that glows in bioscience].

    PubMed

    Millán, María Inés; Becú-Villalobos, Damasia

    2009-01-01

    Green fluorescent protein (GFP) is a protein produced by the jellyfish Aequorea victoria, that emits bioluminescence in the green zone of the visible spectrum. The GFP gene has been cloned and is used in molecular biology as a marker. The three researchers that participated independently in elucidating the structure and function of this and its related proteins, Drs. Shimomura, Chalfie and Tsien were awarded the Nobel Prize in Chemistry 2008. Dr. Shimomura discovered and studied the properties of GFP. Using molecular biological techniques, Chalfie succeeded in introducing the GFP gene into the DNA of the small, almost transparent worm C. elegans, and initiated an era in which GFP would be used as a glowing marker for cellular biology. Finally, Dr.Tsien found precisely how GFP's structure produces the observed green fluorescence, and succeeded in modifying the structure to generate molecules that emit light at slightly different wavelengths, which gave tags of different colors. Fluorescent proteins are very versatile and are being used in many areas, such as microbiology, biotechnology, physiology, environmental engineering, development, etc. They can, for example, illuminate growing cancer tumours; show the development of Alzheimer's disease, or detect arsenic traces in water. Finding the key to how a marine organism produces light unexpectedly ended up providing researchers with a powerful array of tools with which to visualize cell biology in action.

  2. Generation of cloned transgenic cats expressing red fluorescence protein.

    PubMed

    Yin, Xi Jun; Lee, Hyo Sang; Yu, Xian Feng; Choi, Eugene; Koo, Bon Chul; Kwon, Mo Sun; Lee, Young S; Cho, Su Jin; Jin, Guang Zhen; Kim, Lyoung Hyo; Shin, Hyoung Doo; Kim, Teoan; Kim, Nam Hyung; Kong, Il Keun

    2008-03-01

    A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

  3. Protein design for pathway engineering

    SciTech Connect

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  4. Protein Design for Pathway Engineering

    PubMed Central

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  5. Protein design for pathway engineering.

    PubMed

    Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.

  6. Engineered Protein Polymers

    DTIC Science & Technology

    2010-05-31

    of each pure polymer, we plan to combine the various polymer solutions in different ratios to tune the composition and physico-chemical properties...protein materials as vehicles for storage and delivery of small molecules. Each protein polymer under concentrations for particle formation ( vida

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Fluorescence Studies of Protein Crystallization Interactions

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  9. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells

    PubMed Central

    Day, Richard N.; Davidson, Michael W.

    2012-01-01

    Summary The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts, and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. PMID:22396229

  10. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

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

    PubMed

    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.

  12. Versatile protein tagging in cells with split fluorescent protein

    PubMed Central

    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

  13. Generation of red fluorescent protein transgenic dogs.

    PubMed

    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.

  14. Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein.

    PubMed

    Fan, Ben; Chen, Xiao Hua; Budiharjo, Anto; Bleiss, Wilfrid; Vater, Joachim; Borriss, Rainer

    2011-02-20

    A single copy of the gfp gene linked with the P(spac) promoter and flanked by the terminal FZB42 amyE sequences was stably integrated into the chromosome of plant growth promoting bacterium Bacillus amyloliquefaciens FZB42 via homologous recombination. A spontaneous mutant, FB01mut, emitting bright fluorescence was detected among the transformants and found suitable for colonization experiments performed with Zea mays, Arabidopsis thaliana and Lemna minor. Real-time RT-PCR revealed that FB01mut expressed 2.5 times more of the gfp transcript than the original GFP-labeled strain. Confocal laser scanning microscopy of plant roots infected with gfp+ tagged FZB42 revealed that the bacterium behaves different in colonizing surfaces of plant roots of different species. In contrast to maize, FZB42 colonized preferentially root tips when colonizing Arabidopsis. FZB42 colonized heavily Lemna fronds and roots by forming biofilms consisting of extracellular matrix and cells with altered morphology. Surfactin, but no other lipopeptide or polyketide synthesized by FZB42 under laboratory conditions, was detected in extracts of Lemna plantlets colonized by FZB42. Due to its stable and long-lasting emission of bright fluorescence without antibiotic pressure FB01mut is an excellent tool for studying plant colonization under competitive, environmental conditions.

  15. Fluorescence Studies of Protein Crystal Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.

    1999-01-01

    Fluorescence can be used to study protein crystal nucleation through methods such as anisotropy, quenching, and resonance energy transfer (FRET), to follow pH and ionic strength changes, and follow events occurring at the growth interface. We have postulated, based upon a range of experimental evidence that the growth unit of tetragonal hen egg white lysozyme is an octamer. Several fluorescent derivatives of chicken egg white lysozyme have been prepared. The fluorescent probes lucifer yellow (LY), cascade blue, and 5-((2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS), have been covalently attached to ASP 101. All crystallize in the characteristic tetragonal form, indicating that the bound probes are likely laying within the active site cleft. Crystals of the LY and EDANS derivatives have been found to diffract to at least 1.7 A. A second group of derivatives is to the N-terminal amine group, and these do not crystallize as this site is part of the contact region between the adjacent 43 helix chains. However derivatives at these sites would not interfere with formation of the 43 helices in solution. Preliminary FRET studies have been carried out using N-terminal bound pyrene acetic acid (Ex 340 nm, Em 376 nm) lysozyme as a donor and LY (Ex -425 nm, Em 525 nm) labeled lysozyme as an acceptor. FRET data have been obtained at pH 4.6, 0.1 M NaAc buffer, at 5 and 7% NaCl, 4 C. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10(exp -5) M respectively). The data at both salt concentrations show a consistent trend of decreasing fluorescence intensity of the donor species (PAA) with increasing total protein concentration. This decrease is more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations reflected in the lower solubility. The calculated average distance between any two protein molecules at 5 x 10(exp -6) M is approximately 70nm, well beyond the

  16. Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

    NASA Astrophysics Data System (ADS)

    Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

    2014-06-01

    Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

  17. Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

    PubMed Central

    Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

    2014-01-01

    Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

  18. Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins.

    PubMed

    Joosen, L; Hink, M A; Gadella, T W J; Goedhart, J

    2014-12-01

    Fluorescence lifetime imaging microscopy can be used to study protein-protein interactions by Förster Resonance Energy Transfer or to perform lifetime-based multiplexing. Fixation of samples with cells producing fluorescent fusion proteins is commonly used for preservation of samples and for staining with membrane impermeable reagents such as antibodies. However, the effect of fixation methods and mounting media on fluorescence lifetime is poorly documented so far. Here, we demonstrate that fixation by formaldehyde or methanol itself does not affect the lifetime of fluorescent proteins produced in cells but that several widely used mounting media decrease the fluorescence lifetime by up to 20%. It is shown that fixed cells producing Aequorea victoria derived fluorescent proteins mounted in Tris buffer have fluorescence lifetimes indistinguishable from values measured in living cells. Tris buffer also allows accurate Förster Resonance Energy Transfer quantification in fixed cells, as shown with an mTurquoise2-SYFP2 fusion protein. Moreover, identical lifetime contrasts are measured in living and fixed cells mounted in Tris buffer after introducing a single plasmid expressing two lifetime variants of cyan fluorescent proteins, each targeted to different locations in the cell. Our findings will aid the preparation of fixed cells producing fluorescent proteins for reliable measurement of fluorescence lifetimes for Förster Resonance Energy Transfer determination, lifetime based multiplexing and for instrument calibration for standardization purposes.

  19. BIMOLECULAR FLUORESCENCE COMPLEMENTATION ANALYSIS OF INDUCIBLE PROTEIN INTERACTIONS: EFFECTS OF FACTORS AFFECTING PROTEIN FOLDING ON FLUORESCENT PROTEIN FRAGMENT ASSOCIATION

    PubMed Central

    Robida, Aaron M; Kerppola, Tom K

    2009-01-01

    Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. We investigated the temporal resolution and the quantitative accuracy of BiFC analysis using fragments of different fluorescent proteins. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of YFP fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 minutes after rapamycin addition and a ten-fold increase in the mean fluorescence intensity in 8 hours. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and YFP produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment of the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized prior to rapamycin addition formed BiFC complexes with the same efficiency as newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggest that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular

  20. Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine.

    PubMed

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2010-01-01

    Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by approximately 300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

  1. Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine

    NASA Astrophysics Data System (ADS)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2010-01-01

    Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by ~300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

  2. Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

    NASA Astrophysics Data System (ADS)

    Konold, Patrick Eugene

    Red-emitting homologues (RFPs) of the native Green Fluorescent Protein (GFP) with emission wavelengths beyond 650 nm are desirable probes for in vivo imaging experiments. They offer the potential for deeper tissue penetration and lower background scatter given a cleaner spectral window. However, bioimaging applications are hindered by poor photophysics ( e.g. low fluorescence quantum yield, high photobleaching), which limits experimental resolution and represents a significant obstacle towards utilization for low copy-number, long-duration imaging applications. In this thesis, a variety of femtosecond nonlinear electronic spectroscopies were employed jointly with site-directed mutagenesis to investigate the photophysical properties of RFPs. In one study, the molecular mechanism of red emission was pursued in two notable RFPs, mPlum and TagRFP675. Solvation dynamics observed with time-resolved transient grating spectroscopy were interpreted with the aid of molecular dynamics simulations to indicate that their red-emission is correlated with the ability of specific chromophore-sidechain hydrogen-bonding interactions to interconvert between direct and water-mediated states. In a second set of studies, two-dimensional double quantum coherence spectroscopy was used to probe the electronic transitions of mPlum. It was discovered that it displayed a response distinctly different from an organic dye in bulk solvent. Modeling indicate of these spectra indicate the spectral features may be attributed to the existence of multiple high-lying (n>1) excited states. The results provide new insight into the electronic structure of these widely used fluorescent probes.

  3. Developing new fluorescent proteins with stagger extension process

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Lu, Jinling; Luo, Haiming; Luo, Qingming; Zhang, Zhihong

    2009-02-01

    The Stagger Extension Process (StEP), a recombination of DNA technique, has been used as a rapid molecular mutagenesis strategy. In this study, for obtaining the fluorescence proteins with new properties, six fluorescence proteins (EYFP, EGFP, ECFP, mCitrine, mCerulean and Venus) were used as the templates to recombine the mutation library by the Stagger Extension Process (StEP) technique. Through screening this mutation library, we have obtained some useful new FPs which are different fluorescent properties with ancestor. These protein will extend fluorescent proteins application.

  4. Fluorescent proteins in microbial biotechnology--new proteins and new applications.

    PubMed

    Vizcaino-Caston, Isaac; Wyre, Chris; Overton, Tim W

    2012-02-01

    The recent advances over the past 5 years in the utilisation of fluorescent proteins in microbial biotechnology applications, including recombinant protein production, food processing, and environmental biotechnology, are reviewed. We highlight possible areas where fluorescent proteins currently used in other bioscience disciplines could be adapted for use in biotechnological applications and also outline novel uses for recently developed fluorescent proteins.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-19

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

  7. Quantitation of carcinogen bound protein adducts by fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Gan, Liang-Shang; Otteson, Michael S.; Doxtader, Mark M.; Skipper, Paul L.; Dasari, Ramachandra R.; Tannenbaum, Steven R.

    1989-01-01

    A highly significant correlation of aflatoxin B 1 serum albumin adduct level with daily aflatoxin B 1 intake was observed in a molecular epidemiological study of aflatoxin carcinogenesis which used conventional fluorescence spectroscopy methods for adduct quantitation. Synchronous fluorescence spectroscopy and laser induced fluorescence techniques have been employed to quantitate antibenzo[ a]pyrene diol epoxide derived globin peptide adducts. Fast and efficient methods to isolate the peptide adducts as well as eliminate protein fluorescence background are described. A detection limit of several femtomoles has been achieved. Experimental and technical considerations of low temperature synchronous fluorescence spectroscopy and fluorescence line narrowing to improve the detection sensitivities are also presented.

  8. Dual labeling of a binding protein allows for specific fluorescence detection of native protein.

    PubMed

    Karlström, A; Nygren, P A

    2001-08-01

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X, SE), respectively. Biosensor analysis of purified doubly labeled protein showed that high-affinity binding to the Fc region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of Fc3(1) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc3(1), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  9. Protein hydrogels with engineered biomolecular recognition

    NASA Astrophysics Data System (ADS)

    Mi, Lixin

    Extracellular matrices (ECMs) are the hydrated macromolecular gels in which cells migrate and proliferate and organize into tissues in vivo . The development of artificial ECM with the required mechanical, physico-chemical, and biological properties has long been a challenge in the biomaterial research field. In this dissertation, a novel set of bioactive protein hydrogels has been synthesized and characterized at both molecular and materials levels. The self-recognized and self-assembled protein copolymers have the ability to provide engineered biofunctionality through the controlled arrangement of bioactive domains on the nanoscale. Genetic engineering methods have been employed to synthesize these protein copolymers. Plasmid DNA carrying genes to express both di- and tri-block proteins have been constructed using molecular cloning techniques. These genes were expressed in bacterial E. coli to ensure homogeneous protein length and anticipated structure. Three diblock protein sequences having a leucine zipper construct on one end and polyelectrolyte (AGAGAGPEG)10 on the other, have been studied by circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and static light scattering to characterize their secondary structure, structural stability, and oligomeric state. The results show that ABC diblock mixtures form very stable heterotrimer aggregates via self-recognition and self-assembly of the coiled coil end domains. Tri-block proteins with two leucine zipper motif ends flanking the polyelectrolyte random coil in the middle have been investigated by circular dichroism and fluorescence spectroscopy, and the hydrogels formed by self-assembly of these tri-blocks have been studied using transmission electronic microscopy and diffusing wave spectroscopy. The reversible gelation behavior is the result of heterotrimeric aggregation of helices to form the physical crosslinks in the gel, with the polyelectrolyte region center block retaining

  10. Fluorescence Studies of Protein Crystal Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Sumida, John

    2000-01-01

    -association process is a function of the protein concentration relative to the saturation concentration, and observing it in dilute solution (conc. less than or equal to 10(exp -5)M) requires that the experiments be performed under low solubility conditions, i.e., low temperatures and high salt concentrations. Data from preliminary steady state FRET studies with N-terminal bound pyrene acetic acid (PAA-lys, donor, Ex 340 nm, Em 376 nm) and asp101 LY-lys as an acceptor showed a consistent trend of decreasing donor fluorescence intensity with increasing total protein concentration. The FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C. The corresponding C(sub sat) values are 0.471 and 0.362 mg/ml (approx. 3.3 and approx. 2.5 x 10(exp -5)M respectively). The donor fluorescence decrease is more pronounced at7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations as reflected in the lower solubility. Results from these and other ongoing studies will be discussed in conjunction with an emerging model for how tetragonal lysozyme crystals nucleate and the relevance of that model to other proteins.

  11. Detection of Aequorea victoria green fluorescent protein by capillary electrophoresis laser induced fluorescence detection.

    PubMed

    Craig, D B; Wong, J C; Dovichi, N J

    1997-01-01

    Aequorea victoria green fluorescent protein was assayed by capillary electrophoresis using post-capillary laser-induced fluorescence detection in a sheath flow cuvette. The limit of detection was 3.0 x 10(-12) M protein in an injection volume of 17 nL, corresponding to a mass of 3100 molecules.

  12. Integrin‐Targeting Fluorescent Proteins: Exploration of RGD Insertion Sites

    PubMed Central

    Sonntag, Michael H.; Schill, Jurgen

    2017-01-01

    Abstract The potential of the fluorescent protein scaffold to control peptide sequence functionality is illustrated by an exploration of fluorescent proteins as novel probes for targeting integrins. A library of fluorescent mCitrine proteins with RGD motifs incorporated at several positions in loops within the protein main chain was generated and characterized. Amino acid mutations to RGD as well as RGD insertions were evaluated: both led to constructs with typical mCitrine fluorescent properties. Screening experiments against four human integrin receptors revealed two strong‐binding constructs and two selective integrin binders. The effect of the site of RGD incorporation illustrates the importance of the protein scaffold on RGD sequence functionality, leading to fluorescent protein constructs with the potential for selective integrin targeting. PMID:28004511

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

    PubMed

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

    2010-04-22

    Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the xYG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals.

  14. Toward fluorescence detection of protein residues on surgical instruments

    NASA Astrophysics Data System (ADS)

    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.

  15. Engineering protein farnesyltransferase for enzymatic protein labeling applications.

    PubMed

    Dozier, Jonathan K; Khatwani, Santoshkumar L; Wollack, James W; Wang, Yen-Chih; Schmidt-Dannert, Claudia; Distefano, Mark D

    2014-07-16

    Creating covalent protein conjugates is an active area of research due to the wide range of uses for protein conjugates spanning everything from biological studies to protein therapeutics. Protein Farnesyltransferase (PFTase) has been used for the creation of site-specific protein conjugates, and a number of PFTase substrates have been developed to facilitate that work. PFTase is an effective catalyst for protein modification because it transfers Farnesyl diphosphate (FPP) analogues to protein substrates on a cysteine four residues from the C-terminus. While much work has been done to synthesize various FPP analogues, there are few reports investigating how mutations in PFTase alter the kinetics with these unnatural analogues. Herein we examined how different mutations within the PFTase active site alter the kinetics of the PFTase reaction with a series of large FPP analogues. We found that mutating either a single tryptophan or tyrosine residue to alanine results in greatly improved catalytic parameters, particularly in kcat. Mutation of tryptophan 102β to alanine caused a 4-fold increase in kcat and a 10-fold decrease in KM for a benzaldehyde-containing FPP analogue resulting in an overall 40-fold increase in catalytic efficiency. Similarly, mutation of tyrosine 205β to alanine caused a 25-fold increase in kcat and a 10-fold decrease in KM for a coumarin-containing analogue leading to a 300-fold increase in catalytic efficiency. Smaller but significant changes in catalytic parameters were also obtained for cyclo-octene- and NBD-containing FPP analogues. The latter compound was used to create a fluorescently labeled form of Ciliary Neurotrophic Factor (CNTF), a protein of therapeutic importance. Additionally, computational modeling was performed to study how the large non-natural isoprenoid analogues can fit into the active sites enlarged via mutagenesis. Overall, these results demonstrate that PFTase can be improved via mutagenesis in ways that will be useful

  16. Mesoscopic Fluorescence Molecular Tomography for Evaluating Engineered Tissues

    PubMed Central

    Ozturk, Mehmet S.; Chen, Chao-Wei; Ji, Robin; Zhao, Lingling; Nguyen, Bao-Ngoc B.; Fisher, John P.; Chen, Yu; Intes, Xavier

    2015-01-01

    Optimization of regenerative medicine strategies includes the design of biomaterials, development of cell-seeding methods, and control of cell-biomaterial interactions within the engineered tissues. Among these steps, one paramount challenge is to non-destructively image the engineered tissues in their entirety to assess structure, function, and molecular expression. It is especially important to be able to enable cell phenotyping and monitor the distribution and migration of cells throughout the bulk scaffold. Advanced fluorescence microscopic techniques are commonly employed to perform such tasks; however, they are limited to superficial examination of tissue constructs. Therefore, the field of tissue engineering and regenerative medicine would greatly benefit from the development of molecular imaging techniques which are capable of non-destructive imaging of three-dimensional cellular distribution and maturation within a tissue-engineered scaffold beyond the limited depth of current microscopic techniques. In this review, we focus on an emerging depth-resolved optical mesoscopic imaging technique, termed Laminar Optical Tomography (LOT) or Mesoscopic Fluorescence Molecular Tomography (MFMT), which enables longitudinal imaging of cellular distribution in thick tissue engineering constructs at depths of a few millimeters and with relatively high resolution. The physical principle, image formation, and instrumentation of LOT/MFMT systems are introduced. Representative applications in tissue engineering include imaging the distribution of human mesenchymal stem cells (hMSCs) embedded in hydrogels, imaging of bio-printed tissues, and in vivo applications. PMID:26645079

  17. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    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.

  18. CHARACTERIZATION OF A CRYPTIC PLASMID FROM ESCHERICHIA COLI O157:H7 AND EVALUATION OF THE EXPRESSION OF GREEN-FLUORESCENT PROTEIN FROM GENETICALLY ENGINEERED DERIVATIVES OF THIS PLASMID

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7 strain 86-24 harbors a 3.3 kb cryptic plasmid (pSP). Our objectives were to clone a DNA cassette expressing a green-fluorescent protein (GFP) from a lac promoter and an ampicillin resistance (Amp**r) gene on pSP, and to monitor both the expression of GFP and the stability o...

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

    PubMed Central

    George Abraham, Bobin; Sarkisyan, Karen S.; Mishin, Alexander S.; Santala, Ville; Tkachenko, Nikolai V.; Karp, Matti

    2015-01-01

    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). PMID:26237400

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

    SciTech Connect

    Nam, Ki-Hyun; Kwon, Oh Yeun; Sugiyama, Kanako; Lee, Won-Ho; Kim, Young Kwan; Song, Hyun Kyu; Kim, Eunice Eunkyung; Park, Sam-Yong; Jeon, Hyesung . E-mail: hjeon@kist.re.kr; Hwang, Kwang Yeon . E-mail: chahong@korea.ac.kr

    2007-03-23

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

  1. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

    PubMed Central

    Gruber, David F.; Gaffney, Jean P.; Mehr, Shaadi; DeSalle, Rob; Sparks, John S.; Platisa, Jelena; Pieribone, Vincent A.

    2015-01-01

    We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. PMID:26561348

  2. Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime.

    PubMed

    Sarkisyan, Karen S; Goryashchenko, Alexander S; Lidsky, Peter V; Gorbachev, Dmitry A; Bozhanova, Nina G; Gorokhovatsky, Andrey Yu; Pereverzeva, Alina R; Ryumina, Alina P; Zherdeva, Victoria V; Savitsky, Alexander P; Solntsev, Kyril M; Bommarius, Andreas S; Sharonov, George V; Lindquist, Jake R; Drobizhev, Mikhail; Hughes, Thomas E; Rebane, Aleksander; Lukyanov, Konstantin A; Mishin, Alexander S

    2015-07-21

    Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

  3. Two-photon absorption properties of fluorescent proteins

    PubMed Central

    Drobizhev, Mikhail; Makarov, Nikolay S.; Tillo, Shane E.; Hughes, Thomas E.; Rebane, Aleksander

    2016-01-01

    Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest, and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right FP and excitation wavelength for two-photon applications. PMID:21527931

  4. Protein engineering approaches to chemical biotechnology.

    PubMed

    Chen, Zhen; Zeng, An-Ping

    2016-12-01

    Protein engineering for the improvement of properties of biocatalysts and for the generation of novel metabolic pathways plays more and more important roles in chemical biotechnology aiming at the production of chemicals from biomass. Although widely used in single-enzyme catalysis process, protein engineering is only being increasingly explored in recent years to achieve more complex in vitro and in vivo biocatalytic processes. This review focuses on major contributions of protein engineering to chemical biotechnology in the field of multi-enzymatic cascade catalysis and metabolic engineering. Especially, we discuss and highlight recent strategies for combining pathway design and protein engineering for the production of novel products.

  5. Fluorescence of Alexa fluor dye tracks protein folding.

    PubMed

    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.

  6. Synthesis of fluorescent dipeptidomimetics and their ribosomal incorporation into green fluorescent protein.

    PubMed

    Chowdhury, Sandipan Roy; Maini, Rumit; Dedkova, Larisa M; Hecht, Sidney M

    2015-11-01

    The synthesis and incorporation into position 66 of green fluorescent protein (GFP) by in vitro protein translation of novel oxazole and thiazole based dipeptidomimetics are described. The compounds may be regarded as GFP chromophore analogues, and are strongly fluorescent. An α-amido-β-ketoester intermediate was obtained via bisacylation of a protected glycine. The intermediate underwent dehydrative cyclization to afford the 1,3-oxazole and was treated with Lawesson's reagent to furnish the 1,3-thiazole. When these fluorophores were introduced into position 66 of GFP in place of Tyr66, the resulting GFP analogues exhibited fluorescence emission several-fold greater than wild-type GFP; the emission was also shifted to shorter wavelength. It may be noted that compared to the typical fluorophores formed in the natural and modified fluorescent proteins, the oxazole and thiazole fluorophores are completely stable and do not require activation by posttranslational modification to exhibit fluorescence.

  7. Fluorescence response profiling for small molecule sensors utilizing the green fluorescent protein chromophore and its derivatives.

    PubMed

    Lee, Jun-Seok; Baldridge, Anthony; Feng, Suihan; SiQiang, Yang; Kim, Yun Kyung; Tolbert, Laren M; Chang, Young-Tae

    2011-01-10

    Using a fluorescence response profile, a systematic examination was performed for synthetic chromophores of the green fluorescent protein (GFP) to discover new small molecule sensors. A group of 41 benzylideneimidazolinone compounds (BDI) was prepared and screened toward 94 biologically relevant analytes to generate fluorescence response profiles. From the response pattern, compounds containing aminobenzyl and heteroaromatic cyclic substructures revealed a pH dependent emission decrease effect, and unlike other fluorescence scaffolds, most BDIs showed fluorescence quenching when mixed with proteins. On the basis of the primary response profile, we obtained three selective fluorescence turn-on sensors for pH, human serum albumin (HSA), and total ribonucleic acid (RNA). Following analysis, a fluorescence response profile testing four nucleic acids revealed the alkyloxy (Ph-OR) functional group in the para position of benzyl analogues contributes to RNA selectivity. Among the primary hit compounds, BDI 2 showed outstanding selectivity toward total RNA with 5-fold emission enhancement. Finally, BDI 24 showed selective fluorescence increase to HSA (K(d) = 3.57 μM) with a blue-shifted emission max wavelength (Δλ(em) = 15 nm). These examples of fluorescence sensor discovery by large-scale fluorescence response profiling demonstrate the general applicability of this approach and the usefulness of the response profiles.

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

    ERIC Educational Resources Information Center

    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…

  9. Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements.

    PubMed

    Nakabayashi, Takakazu; Wang, Hui-Ping; Kinjo, Masataka; Ohta, Nobuhiro

    2008-06-01

    We have shown that the intracellular pH of a single HeLa cell expressing the enhanced green fluorescent protein (EGFP) can be imaged using the fluorescence lifetime of EGFP, which can be interpreted in terms of the pH-dependent ionic equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP.

  10. Strategies for In Vivo Imaging Using Fluorescent Proteins.

    PubMed

    Hoffman, Robert M

    2016-08-26

    Fluorescent proteins have enabled the color-coding of cells growing in vivo. Noninvasive imaging of cells expressing fluorescent proteins has allowed the real-time determination of the behavior on cancer cells, the progression of infection, the differentiation of stem cells, and interaction of stromal and cancer cells. Cancer cells in the nucleus and cytoplasm can visualize in vivo nuclear-cytoplasmic dynamics in vivo including: mitosis, apoptosis, cell-cycle phase, and differential behavior of nucleus and cytoplasm that occurs during cancer-cell deformation. Linking spectrally-distinct fluorescent proteins with cell-cycle-specific proteins results in color-coding the phases of the cell cycle. With the use of fluorescent proteins, literally any cellular or molecular function can be imaged in vivo. This article is protected by copyright. All rights reserved.

  11. mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities

    PubMed Central

    McEvoy, Ann L.; Hoi, Hiofan; Bates, Mark; Platonova, Evgenia; Cranfill, Paula J.; Baird, Michelle A.; Davidson, Michael W.; Ewers, Helge; Liphardt, Jan; Campbell, Robert E.

    2012-01-01

    Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct. PMID:23240015

  12. Description of Hydration Water in Protein (Green Fluorescent Protein) Solution

    SciTech Connect

    Perticaroli, Stefania; Ehlers, Georg; Stanley, Christopher B.; Mamontov, Eugene; O’Neill, Hugh; Zhang, Qiu; Cheng, Xiaolin; Myles, Dean A. A.; Katsaras, John; Nickels, Jonathan D.

    2016-10-26

    The structurally and dynamically perturbed hydration shells that surround proteins and biomolecules have a substantial influence upon their function and stability. This makes the extent and degree of water perturbation of practical interest for general biological study and industrial formulation. Here, we present an experimental description of the dynamical perturbation of hydration water around green fluorescent protein in solution. Less than two shells (~5.5 Å) were perturbed, with dynamics a factor of 2–10 times slower than bulk water, depending on their distance from the protein surface and the probe length of the measurement. Furthermore, this dependence on probe length demonstrates that hydration water undergoes subdiffusive motions (τ ∝ q–2.5 for the first hydration shell, τ ∝ q–2.3 for perturbed water in the second shell), an important difference with neat water, which demonstrates diffusive behavior (τ ∝ q–2). Our results help clarify the seemingly conflicting range of values reported for hydration water retardation as a logical consequence of the different length scales probed by the analytical techniques used.

  13. Description of Hydration Water in Protein (Green Fluorescent Protein) Solution

    DOE PAGES

    Perticaroli, Stefania; Ehlers, Georg; Stanley, Christopher B.; ...

    2016-10-26

    The structurally and dynamically perturbed hydration shells that surround proteins and biomolecules have a substantial influence upon their function and stability. This makes the extent and degree of water perturbation of practical interest for general biological study and industrial formulation. Here, we present an experimental description of the dynamical perturbation of hydration water around green fluorescent protein in solution. Less than two shells (~5.5 Å) were perturbed, with dynamics a factor of 2–10 times slower than bulk water, depending on their distance from the protein surface and the probe length of the measurement. Furthermore, this dependence on probe length demonstratesmore » that hydration water undergoes subdiffusive motions (τ ∝ q–2.5 for the first hydration shell, τ ∝ q–2.3 for perturbed water in the second shell), an important difference with neat water, which demonstrates diffusive behavior (τ ∝ q–2). Our results help clarify the seemingly conflicting range of values reported for hydration water retardation as a logical consequence of the different length scales probed by the analytical techniques used.« less

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

    ERIC Educational Resources Information Center

    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,…

  15. LucY: A versatile new fluorescent reporter protein

    DOE PAGES

    Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; ...

    2015-04-23

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

  16. LucY: A versatile new fluorescent reporter protein

    SciTech Connect

    Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, Jr., George N.; Mead, David; Steinmetz, Eric J.; Michnick, Stephen W.

    2015-04-23

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

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

    PubMed Central

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

    2016-01-01

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

  18. From green to blue: site-directed mutagenesis of the green fluorescent protein to teach protein structure-function relationships.

    PubMed

    Girón, María D; Salto, Rafael

    2011-07-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the site-directed mutagenesis of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The GFP is ideal because the students are able to correlate the changes introduced into the structure of the protein with the observable modification of its fluorescence properties. By using noncommercial kits, we set up a non PCR-thermocycling reaction using mutagenic primers, followed by removal of the original plasmid template by DpnI digestion. By introducing only one (Y66H) or two mutations (Y66H/Y145F) in the "cycle 3" variant of GFP (F99S, M153T, and V163A) or GFPuv, students are able to analyze the changes from green to blue in the fluorescence emission of the mutated proteins and to correlate these differences in fluorescence with the structural changes using three-dimensional structure visualization software. This inexpensive laboratory course familiarizes the students with the design of mutagenic oligonucleotides, site-directed mutagenesis, bacterial transformation, restriction analysis of the mutated plasmids, and protein characterization by SDS-PAGE and fluorescence spectroscopy.

  19. Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy.

    PubMed

    Andresen, Martin; Stiel, Andre C; Fölling, Jonas; Wenzel, Dirk; Schönle, Andreas; Egner, Alexander; Eggeling, Christian; Hell, Stefan W; Jakobs, Stefan

    2008-09-01

    Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.

  20. X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals.

    PubMed

    Perozzo, M A; Ward, K B; Thompson, R B; Ward, W W

    1988-06-05

    The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.

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

    PubMed

    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. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants.

    PubMed

    Davis, S J; Vierstra, R D

    1998-03-01

    Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is 'brighter' because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.

  3. Can the fluorescence of green fluorescent protein chromophore be related directly to the nativity of protein structure?

    PubMed

    Melnik, B S; Povarnitsyna, T V; Melnik, T N

    2009-12-25

    In studies of green fluorescence protein (GFP) or other proteins with the use of GFP as a marker, the fluorescence of GFP is for the most part related directly to the nativity of its structure. Naturally, such a relation does exist since the chromophore of this protein is formed autocatalytically only just after GFP acquires its native structure. However, the fluorescence method may not yield reliable information on protein structure when studying renaturation and denaturation of this protein (with the formed chromophore). Using proteolysis, denaturant gradient gel electrophoresis and circular dichroism, we demonstrate herein that at major disturbances of the native structure of protein GFP-cycle3 the intensity of fluorescence of its chromophore can change insignificantly. In other words, the chromophore fluorescence does not reliably mirror alterations in protein structure. Since the main conclusions of this study are especially qualitative, it can be suggested that during renaturation/denaturation of wild-type GFP and its "multicolored" mutants their fluorescence is also not always associated with the changes in the structure of these proteins.

  4. Fluorescence properties of o-aminobenzoyl-labeled proteins.

    PubMed

    Churchich, J E

    1993-09-01

    Isatoic anhydride reacts with nucleophile groups of proteins to yield o-aminobenzoyl protein conjugates. The fluorescence emitted by the chromophore decays in a multiexponential manner with average fluorescence lifetimes ranging from 9.9 to 10.7 ns. The steady emission anisotropy, measured upon excitation at 330 nm, is influenced by the molecular mass of the protein to which o-aminobenzoyl is attached. The fluorescence properties of o-aminobenzoyl are suitable for rotational correlation time measurements of proteins smaller than 65 kDa. The technique of emission anisotropy can be used to detect interactions between proteins in solution, provided one of the proteins is labeled with o-aminobenzoyl.

  5. Latest methods of fluorescence-based protein crystal identification

    SciTech Connect

    Meyer, Arne; Betzel, Christian

    2015-01-28

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

  6. Mechanisms of protein evolution and their application to protein engineering.

    PubMed

    Glasner, Margaret E; Gerlt, John A; Babbitt, Patricia C

    2007-01-01

    Protein engineering holds great promise for the development of new biosensors, diagnostics, therapeutics, and agents for bioremediation. Despite some remarkable successes in experimental and computational protein design, engineered proteins rarely achieve the efficiency or specificity of natural enzymes. Current protein design methods utilize evolutionary concepts, including mutation, recombination, and selection, but the inability to fully recapitulate the success of natural evolution suggests that some evolutionary principles have not been fully exploited. One aspect of protein engineering that has received little attention is how to select the most promising proteins to serve as templates, or scaffolds, for engineering. Two evolutionary concepts that could provide a rational basis for template selection are the conservation of catalytic mechanisms and functional promiscuity. Knowledge of the catalytic motifs responsible for conserved aspects of catalysis in mechanistically diverse superfamilies could be used to identify promising templates for protein engineering. Second, protein evolution often proceeds through promiscuous intermediates, suggesting that templates which are naturally promiscuous for a target reaction could enhance protein engineering strategies. This review explores these ideas and alternative hypotheses concerning protein evolution and engineering. Future research will determine if application of these principles will lead to a protein engineering methodology governed by predictable rules for designing efficient, novel catalysts.

  7. Two-photon excited UV fluorescence for protein crystal detection

    SciTech Connect

    Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

    2011-10-01

    Complementary measurements using SONICC and TPE-UVF allow the sensitive and selective detection of protein crystals. Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

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

    PubMed

    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.

  9. Photoacoustic analysis of proteins: volumetric signals and fluorescence quantum yields.

    PubMed Central

    Kurian, E; Prendergast, F G; Small, J R

    1997-01-01

    A series of proteins has been examined using time-resolved, pulsed-laser volumetric photoacoustic spectroscopy. Photoacoustic waveforms were collected to measure heat release for calculation of fluorescence quantum yields, and to explore the possibility of photoinduced nonthermal volume changes occurring in these protein samples. The proteins studied were the green fluorescent protein (GFP); intestinal fatty acid binding protein (IFABP), and adipocyte lipid-binding protein (ALBP), each labeled noncovalently with 1-anilinonaphthalene-8-sulfonate (1,8-ANS) and covalently with 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan); and acrylodan-labeled IFABP and ALBP with added oleic acid. Of this group of proteins, only the ALBP labeled with 1,8-ANS showed significant nonthermal volume changes at the beta = 0 temperature (approximately 3.8 degrees C) for the buffer used (10 mM Tris-HCI, pH 7.5) (beta is the thermal cubic volumetric expansion coefficient). For all of the proteins except for acrylodan-labeled IFABP, the fluorescence quantum yields calculated assuming simple energy conservation were anomalously high, i.e., the apparent heat signals were lower than those predicted from independent fluorescence measurements. The consistent anomalies suggest that the low photoacoustic signals may be characteristic of fluorophores buried in proteins, and that photoacoustic signals derive in part from the microenvironment of the absorbing chromophore. Images FIGURE 1 PMID:9199809

  10. Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Protein specific fluorescent microspheres for labelling a protein

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor)

    1982-01-01

    Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.

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

    PubMed Central

    Deng, Qiliang; Wu, Jianhua; Zhai, Xiaorui; Fang, Guozhen; Wang, Shuo

    2013-01-01

    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. PMID:24077318

  14. Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein

    NASA Astrophysics Data System (ADS)

    Heim, Roger; Prasher, Douglas C.; Tsien, Roger Y.

    1994-12-01

    The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (≈4 hr at 22^circC and atmospheric pO_2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.

  15. Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening.

    PubMed

    Ma, Fuqiang; Fischer, Michael; Han, Yunbin; Withers, Stephen G; Feng, Yan; Yang, Guang-Yu

    2016-09-06

    In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins.

  16. Exploration of sequence space for protein engineering.

    PubMed

    Gustafsson, C; Govindarajan, S; Emig, R

    2001-01-01

    The process of protein engineering is currently evolving towards a heuristic understanding of the sequence-function relationship. Improved DNA sequencing capacity, efficient protein function characterization and improved quality of data points in conjunction with well-established statistical tools from other industries are changing the protein engineering field. Algorithms capturing the heuristic sequence-function relationships will have a drastic impact on the field of protein engineering. In this review, several alternative approaches to quantitatively assess sequence space are discussed and the relatively few examples of wet-lab validation of statistical sequence-function characterization/correlation are described.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  20. Computer Modeling of the Structure and Spectra of Fluorescent Proteins

    PubMed Central

    Grigorenko, B.L.; Savitsky, A.P.

    2009-01-01

    Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers. PMID:22649601

  1. Improving membrane voltage measurements using FRET with new fluorescent proteins.

    PubMed

    Tsutsui, Hidekazu; Karasawa, Satoshi; Okamura, Yasushi; Miyawaki, Atsushi

    2008-08-01

    We used two new coral fluorescent proteins as fluorescence resonance energy transfer (FRET) donor and acceptor to develop a voltage sensor, named Mermaid, that displays approximately 40% changes in emission ratio per 100 mV, allowing for direct visualization of electrical activities in cultured excitable cells. Notably, Mermaid has fast on-off kinetics at warm (approximately 33 degrees C) temperatures and can report voltage spikes comparable to action potentials.

  2. Fluorescence resonance energy transfer between green fluorescent protein and doxorubicin enabled by DNA nanotechnology.

    PubMed

    Heger, Zbynek; Kominkova, Marketa; Cernei, Natalia; Krejcova, Ludmila; Kopel, Pavel; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2014-12-01

    DNA nanotechnology is a rapidly growing research area, where DNA may be used for wide range of applications such as construction of nanodevices serving for large scale of diverse purposes. Likewise a panel of various purified fluorescent proteins is investigated for their ability to emit their typical fluorescence spectra under influence of particular excitation. Hence these proteins may form ideal donor molecules for assembly of fluorescence resonance emission transfer (FRET) constructions. To extend the application possibilities of fluorescent proteins, while using DNA nanotechnology, we developed nanoconstruction comprising green fluorescent protein (GFP) bound onto surface of surface active nanomaghemite and functionalized with gold nanoparticles. We took advantage of natural affinity between gold and thiol moieties, which were modified to bind DNA fragment. Finally we enclosed doxorubicin into fullerene cages. Doxorubicin intercalated in DNA fragment bound on the particles and thus we were able to connect these parts together. Because GFP behaved as a donor and doxorubicin as an acceptor using excitation wavelength for GFP (395 nm) in emission wavelength of doxorubicin (590 nm) FRET was observed. This nanoconstruction may serve as a double-labeled transporter of doxorubicin guided by force of external magnetic force owing to the presence of nanomaghemite. Further nanomaghemite offers the possibility of using this technology for thermotherapy.

  3. Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy

    PubMed Central

    2016-01-01

    Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ2 orientation factor arising from differences in donor–acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor–acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET. PMID:28217242

  4. Construction of genetically engineered bacteria that degrades organophosphorus pesticide residues and can be easily detected by the fluorescence.

    PubMed

    Li, Qin; Wang, Pan; Chen, Rui; Li, Wei; Wu, Yi-Jun

    2014-01-01

    Organophosphorus compounds (OPs) are widely used in agriculture and industry and there is increased concern about their toxicological effects in the environment. Bioremediation can offer an efficient and cost-effective option for the removal of OPs. Herein, we describe the construction of a genetically engineered microorganism (GEM) that can degrade OPs and be directly detected and monitored in the environment using an enhanced green fluorescent protein (EGFP) fusion strategy. The coding regions of EGFP, a reporter protein that can fluoresce by itself, and organophosphorus hydrolase (OPH), which has a broad substrate specificity and is able to hydrolyse a number of organophosphorus pesticides, were cloned into the expression vector pET-28b. The fusion protein of EGFP-OPH was expressed in E. coli BL21 (DE3) and the protein expression reached the highest level at 11 h after isopropyl beta-D-thiogalactopyranoside induction. The fluorescence of the GEM was detected by fluorescence spectrophotometry and microscopy, and its ability to degrade OPs was determined by OPH activity assay. Those GEM that express the fusion protein (EGFP and OPH) exhibited strong fluorescence intensity and also potent hydrolase activity, which could be used to degrade organophosphorus pesticide residues in the environment and can also be directly monitored by fluorescence.

  5. Deletion mapping of the Aequorea victoria green fluorescent protein.

    PubMed

    Dopf, J; Horiagon, T M

    1996-01-01

    Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2-232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300-500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.

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

    PubMed

    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.

  7. Structural Determinants of Improved Fluorescence in a Family of Bacteriophytochrome-Based Infrared Fluorescent Proteins: Insights from Continuum Electrostatic Calculations and Molecular Dynamics Simulations.

    PubMed

    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.

  8. A palette of fluorescent proteins optimized for diverse cellular environments

    PubMed Central

    Costantini, Lindsey M.; Baloban, Mikhail; Markwardt, Michele L.; Rizzo, Mark; Guo, Feng; Verkhusha, Vladislav V.; Snapp, Erik L.

    2015-01-01

    To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Yet, within the biochemically distinct environments of cellular organelles, popular fluorescent proteins (FPs), including EGFP, can be unreliable for quantitative imaging, resulting in underestimation of protein levels and incorrect localization. Specifically, within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. Furthermore, transmembrane FP fusion constructs can disrupt organelle architecture due to oligomerizing tendencies of numerous common FPs. Here, we describe a powerful set of bright and inert FPs optimized for use in multiple cellular compartments, especially oxidizing environments and biological membranes. Also, we provide new insights into use of red FPs in the secretory pathway. Our monomeric "oxFPs" finally resolve long standing, underappreciated, and important problems of cell biology and should be useful for a number of applications. PMID:26158227

  9. Development of a photoactivatable fluorescent protein from Aequorea victoria GFP

    NASA Astrophysics Data System (ADS)

    Patterson, George H.; Lippincott-Schwartz, Jennifer

    2004-06-01

    Photoactivation, the rapid conversion of photoactivatable molecules to a fluorescent state by intense irradiation, can be used to mark and monitor selected molecules within cells1. We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) based on a mutation at position 203 that upon intense irradiation with 413 nm light exhibits a stable 60-100 fluorescence increase under 488 nm excitation. The photoactivated form of this mutant named photoactivatable GFP (PA-GFP), is stable under a number of conditions. PA-GFP can be used to analyze protein dynamics in living cells, offering enormous potential for addressing outstanding questions in protein trafficking and turnover, organelle dynamics, and cell lineage patterns.

  10. Distance-dependent intrinsic fluorescence of proteins on aluminum nanostructures

    NASA Astrophysics Data System (ADS)

    Akbay, Nuriye; Lakowicz, Joseph R.; Ray, Krishanu

    2012-03-01

    In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using ensemble spectroscopic studies. In the represented study, we explored the distance effect on intrinsic fluorescence of proteins adsorbed on our layer-by-layer assembled metallic nanostructures. The study is expected to provide more information on the importance of positioning the proteins at a particular distance for enhanced fluorescence from metallic structures. For the present study, we considered using easy and inexpensive LbL technique to provide welldefined distance from metallic surface. The explored proteins were adsorbed on different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). SA and BSA were electrostatically attached to the positively charged PAH layer. We obtained a maximum of ~11-fold and 9-fold increase in fluorescence intensity from SA and BSA, respectively. And also we observed ~3-fold decrease in BSA lifetime on metallic nanostructures than those on bare control quartz slides. This study reveals the distance dependence of protein fluorescence.

  11. Monomeric red fluorescent proteins with a large Stokes shift.

    PubMed

    Piatkevich, Kiryl D; Hulit, James; Subach, Oksana M; Wu, Bin; Abdulla, Arian; Segall, Jeffrey E; Verkhusha, Vladislav V

    2010-03-23

    Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 mum the breast cancer cells show significant polarization towards vessels in living mice.

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

    PubMed

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

    2016-11-05

    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.

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

    PubMed

    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.

  14. Imaging cellular dynamics in vivo with multicolor fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2005-04-01

    The new field of in vivo cell biology is being developed with multi-colored fluorescent proteins. With the use of fluorescent proteins, the behavior of individual cells can be visualized in the living animal. 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 the tumor-stroma cell-cell interaction 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 and their interaction can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo with fluorescent proteins. Mice, in which the regulatory elements of the stem-cell marker nestin drive GFP expression, can be used to visualize hair follicle stem cells including their ability to form hair follicles as well as blood vessels. 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. Dual-color cells also enable the in vivo imaging of cell and nuclear deformation as well as trafficking in capillaries in living animals. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including cell-cell interaction, gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still studied in vitro.

  15. Fluorescence imaging preparation methods for tissue scaffolds implanted into a green fluorescent protein porcine model.

    PubMed

    Smith, Sarah E; White, Richard A; Grant, David A; Grant, Sheila A

    2015-10-01

    Green fluorescent protein (GFP) animal models have become increasingly popular due to their potential to enhance in vivo imaging and their application to many fields of study. We have developed a technique to observe host tissue integration into scaffolds using GFP expressing swine and fluorescence imaging. Current fluorescence imaging preparation methods cannot be translated to a full GFP animal model due to several challenges and limitations that are investigated here. We have implanted tissue scaffolds into GFP expressing swine and have prepared explanted scaffolds for fluorescence imaging using four different methods including formalin fixation and paraffin embedding, vapor fixation, freshly prepared paraformaldehyde fixation, and fresh frozen tissue. Explanted scaffolds and tissue were imaged using confocal microscopy with spectral separation to evaluate the GFP animal model for visualization of host tissue integration into explanted scaffolds. All methods except fresh frozen tissue induced autofluorescence of the scaffold, preventing visualization of detail between host tissue and scaffold fibers. Fresh frozen tissue preparation allowed for the most reliable visualization of fluorescent host tissue integration into non-fluorescent scaffolds. It was concluded that fresh frozen tissue preparation is the best method for fluorescence imaging preparation when using scaffolds implanted into GFP whole animal models.

  16. Design of a confocal fluorescence spectrometer for transdermal observation of green fluorescent protein expression in cartilage

    NASA Astrophysics Data System (ADS)

    Gareau, Daniel S.; Ma, Hong; Horton, William A.; Jacques, Steven L.

    2001-04-01

    We have developed a noninvasive confocal fluorescence spectrometer to measure in vivo fluorescence spectra in mice. Our motivation is the study of the healing process of wounded cartilage in the murine model. The spectrometer measures the expression of green fluorescent protein (GFP) which is linked to the promoter of type II collagen as a marker for the expression of the chondrocyte phenotype. The confocal system uses an argon ion laser (488 nm) to excite fluorescence in a confocal volume about 200 um below the skin surface in the cartilage of the xyphoid process. In vitro studies showed how the refractive mismatch at the surface boundary and the light scattering of the tissue affect the depth and size of the confocal volume. A thin fluorescent layer was detected through a 130 um tissue phantom (murine skin) by moving the confocal volume axially through the phantom. The presence of skin increased the axial full width at half maximum of the confocal response from 7 um to 72 um. In conclusion, we achieve a low-spatial-resolution confocal spectrometer, which yields a fluorescence spectrum that maximizes the GFP fluorescence of cartilage and minimizes the skin autofluorescence.

  17. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    NASA Astrophysics Data System (ADS)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  1. The photochemistry of fluorescent proteins: implications for their biological applications.

    PubMed

    Seward, Harriet E; Bagshaw, Clive R

    2009-10-01

    Green fluorescent protein from Aequorea victoria, its relatives and derivatives are ubiquitous in their use as biological probes. In this tutorial review, we discuss the photochemistry of this fascinating class of proteins and illustrate some of their advantages and drawbacks in a range of applications. In particular, we focus on the ionisation states of the chromophore and how they are affected by internal and external proton transfer. Light-induced reversible and irreversible events are discussed in terms of the underlying chromophore structure. These phenomena have an influence on the interpretation of FRET (Förster resonance energy transfer), FRAP (fluorescence recovery after photobleaching), as well as single molecule studies.

  2. Engineering RNA-binding proteins for biology.

    PubMed

    Chen, Yu; Varani, Gabriele

    2013-08-01

    RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequence specificity will provide valuable tools for biochemical research as well as potential therapeutic applications. In this review, we discuss the suitability of various RNA-binding domains for engineering RNA-binding specificity, based on the structural basis for their recognition. We also compare various protein engineering and design methods applied to RNA-binding proteins, and discuss future applications of these proteins.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Fluorescent protein marker lines in maize: generation and applications.

    PubMed

    Wu, Qingyu; Luo, Anding; Zadrozny, Tara; Sylvester, Anne; Jackson, Dave

    2013-01-01

    Fluorescent proteins (FP) have significantly impacted the way that we study plants in the past two decades. In the post-genomics era, these FP tools are in higher demand by plant scientists for studying the dynamics of protein localization, function, and interactions, and to translate sequence information to biological knowledge that can benefit humans. Although FP tools have been widely used in the model plant Arabidopsis, few FP resources have been developed for maize, one of the most important food crops worldwide, and an ideal species for genetic and developmental biology research. In an effort to provide the maize and cereals research communities with a comprehensive set of FP resources for different purposes of study, we generated more than 100 stable transformed maize FP marker lines, which mark most compartments in maize cells with different FPs. Additionally, we are generating driver and reporter lines, based on the principle of the pOp-LhG4 transactivation system, allowing specific expression or mis-expression of any gene of interest to precisely study protein functions. These marker lines can be used not only for static protein localization studies, but will be useful for studying protein dynamics and interactions using kinetic microscopy methods, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET).

  5. Protein amyloids develop an intrinsic fluorescence signature during aggregation†

    PubMed Central

    Chan, Fiona T. S.; Kaminski Schierle, Gabriele S.; Kumita, Janet R.; Bertoncini, Carlos W.; Dobson, Christopher M.; Kaminski, Clemens F.

    2017-01-01

    We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1–40) and (1–42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner. PMID:23420088

  6. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence.

    PubMed

    Li, X; Zhang, G; Ngo, N; Zhao, X; Kain, S R; Huang, C C

    1997-11-07

    The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small alpha helix at the N terminus of GFP and ends immediately following the last beta sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. Change of the Glu residues to other amino acids results in reduction of GFP fluorescence. Position 229 at the C terminus of GFP, however, is nonspecific: the Ile can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino acids can be deleted from GFP without disrupting fluorescence, consistent with findings of a previous study of GFP crystal structure (Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. (1996) Science 273, 1392-1395 and Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246-1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence.

  7. Generic comparison of protein inference engines.

    PubMed

    Claassen, Manfred; Reiter, Lukas; Hengartner, Michael O; Buhmann, Joachim M; Aebersold, Ruedi

    2012-04-01

    Protein identifications, instead of peptide-spectrum matches, constitute the biologically relevant result of shotgun proteomics studies. How to appropriately infer and report protein identifications has triggered a still ongoing debate. This debate has so far suffered from the lack of appropriate performance measures that allow us to objectively assess protein inference approaches. This study describes an intuitive, generic and yet formal performance measure and demonstrates how it enables experimentalists to select an optimal protein inference strategy for a given collection of fragment ion spectra. We applied the performance measure to systematically explore the benefit of excluding possibly unreliable protein identifications, such as single-hit wonders. Therefore, we defined a family of protein inference engines by extending a simple inference engine by thousands of pruning variants, each excluding a different specified set of possibly unreliable identifications. We benchmarked these protein inference engines on several data sets representing different proteomes and mass spectrometry platforms. Optimally performing inference engines retained all high confidence spectral evidence, without posterior exclusion of any type of protein identifications. Despite the diversity of studied data sets consistently supporting this rule, other data sets might behave differently. In order to ensure maximal reliable proteome coverage for data sets arising in other studies we advocate abstaining from rigid protein inference rules, such as exclusion of single-hit wonders, and instead consider several protein inference approaches and assess these with respect to the presented performance measure in the specific application context.

  8. Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.

    PubMed

    Matsuda, Tomoki; Nagai, Takeharu

    2014-12-01

    Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques.

  9. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing

    PubMed Central

    Shi, Siyuan; Thomas, Abu; Corzo, Neil V.; Kumar, Prem; Huang, Yuping; Lee, Kim Fook

    2016-01-01

    Recent studies in quantum biology suggest that quantum mechanics help us to explore quantum processes in biological system. Here, we demonstrate generation of photon pairs through spontaneous four-wave mixing process in naturally occurring fluorescent proteins. We develop a general empirical method for analyzing the relative strength of nonlinear optical interaction processes in five different organic fluorophores. Our results indicate that the generation of photon pairs in green fluorescent proteins is subject to less background noises than in other fluorophores, leading to a coincidence-to-accidental ratio ~145. As such proteins can be genetically engineered and fused to many biological cells, our experiment enables a new platform for quantum information processing in a biological environment such as biomimetic quantum networks and quantum sensors. PMID:27076032

  10. Broadband photon pair generation in green fluorescent proteins through spontaneous four-wave mixing

    NASA Astrophysics Data System (ADS)

    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.

  11. Two-photon directed evolution of green fluorescent proteins

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  12. Production of green fluorescent protein in transgenic rice seeds.

    PubMed

    Li, Ding; Gao, Jing; Shen, Chunxiu; Fang, Zhen; Xia, Yumei; Yuan, Longping; Cao, Mengliang

    2013-03-01

    Immature embryos from immature seeds of rice (Oryza sativa L.) were transformed by biolistic bombardment with the plasmid carrying the coding region of the hygromycin phosphotransferase gene under the control of the 5' region of the cauliflower mosaic virus 35S promoter and the synthetic green fluorescence protein gene (sgfp) under the control of the maize ubiquitine promoter. Southern blot analysis confirmed the stable integration of hpt and sgfp genes in transformants. Subsequently leaves from regenerated plants were resistant to hygromycin, and microscopic observation of the green fluorescence and immunoblotting analysis revealed that green fluorescence protein was not only detected in the leaf and pollen of primary transformants but also in mature seeds. The results bear out the importance of the suitability of GFP as an in vivo marker to follow the processes of selection of somatic hybrid embryos and plants.

  13. Characterization of a Fluorescent Protein Reporter System

    DTIC Science & Technology

    2008-03-01

    pathways are initiated with the binding of a small molecule to a catalytic ribonucleic acid molecule (RNA), called a ribozyme (Thodima et al., 2006). The... ribozyme is part of a larger RNA construct, called a riboswitch, which initiates translation of a specific genetic sequence on a plasmid (circular...protein gene. Yen et al. (2004) reported insertion of a self-cleaving ribozyme upstream of the reporter gene. In the absence of a regulator (“off

  14. Fluorescence studies of beer protein uptake by silica

    NASA Astrophysics Data System (ADS)

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

    2001-05-01

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

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

    PubMed

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

    2016-02-18

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

  16. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  17. Thrombin detection in murine plasma using engineered fluorescence resonance energy transfer aptadimers

    NASA Astrophysics Data System (ADS)

    Trapaidze, Ana; Brut, Marie; Mazères, Serge; Estève, Daniel; Gué, Anne-Marie; Bancaud, Aurélien

    2015-12-01

    Biodetection strategies, in which two sides of one target protein are targeted simultaneously, have been shown to increase specificity, selectivity, and affinity, and it has been suggested that they constitute excellent candidates for protein sensing in complex media. In this study we propose a method to engineer the sequence of a DNA construct dedicated to reversible thrombin detection. This construct, called Fluorescence Resonance Energy Transfer (FRET) aptadimer, is assembled with two aptamers, which target different epitopes of thrombin, interconnected with a DNA linker that contains a FRET couple and a reversible double helix stem. In the absence of target, the stem is stable maintaining a FRET couple in close proximity, and fluorescence is unquenched upon thrombin addition due to the dehybridization of the stem. We define design rules for the conception of FRET aptadimers, and develop a software to optimize their functionality. One engineered FRET aptadimer sequence is subsequently characterized experimentally by temperature scanning fluorimetry, demonstrating the relevance of our technology for thrombin sensing in bulk and diluted murine plasma.

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

    ERIC Educational Resources Information Center

    Larkin, Patrick D.; Hartberg, Yasha

    2005-01-01

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

  19. Novel steroid-sensing model and characterization of protein interactions based on fluorescence anisotropy decay.

    PubMed

    Chang, Ko-Shing; Luo, Liyang; Chang, Chih-Wei; Huang, Yen-Chieh; Cheng, Chih-Yu; Hung, Chen-Shiung; Diau, Eric Wei-Guang; Li, Yaw-Kuen

    2010-04-01

    Intramolecular binding of a ligand with an alkyl link, (-CH(2))(3), covalently bound to a residue near the active site of the protein forms a novel steroid-sensing model. A genetically engineered big up tri, open(5)-3-ketosteroid isomerase (KSI) was designed to conjugate uniquely with this ligand at its Cys-86 through the formation of a disulfide bond. The steady-state protein-ligand binding, mediated by hydrophobic interactions, was confirmed with fluorescence spectra, and the fluorophore-labeled peptide sequence was identified with tandem mass spectra. A comparison of steady-state fluorescence spectra of various fluorophore-labeled KSI mutants reveals that the emission characteristics vary with environmental factors. An evaluation of the decay of the fluorescence anisotropy of the fluorophore indicates the existence of an intramolecular protein-ligand binding interaction. The measurement of time-resolved fluorescence anisotropy of various protein-ligand complexes yielded values of anisotropy decay representing the degrees of freedom of the fluorophore related to its location, inside or outside the steroid-binding domain. When 19-norandrostenedione (19-NA) was added to this protein-ligand system, competitive binding between the ligand and the steroid was observed; this finding confirms the feasibility of the design of steroid detection with engineered KSI. On integration of this protein-ligand system with a silicon-based nanodevice (a p-type field-effect transistor with an ultrathin body), a noncharged steroid, 19-NA, became detectable at a micromolar level ( Biosens. Bioelectron. 2008 , 23 , 1883 ).

  20. Fluorescent rare earth solutions as intrinsic wavelength standards for protein fluorescence spectroscopy.

    PubMed

    Anderle, Heinz; Weber, Alfred

    2017-02-01

    Trivalent Gd, Tm, and Dy solutions can be used as intrinsic excitation and emission standards to validate the UV and violet-blue wavelength accuracy of a spectrofluorimeter. Europium extends the range into the red. To attain sufficient sensitivity, these luminescent rare earth ions require deuterated reagents or carbonate complexation, which allow the use of ordinary water and thus preparation in virtually any laboratory. Such solutions are particularly valuable as system suitability standards (SST) for protein fluorescence spectroscopy to detect red shifts of the intrinsic fluorescence maximum in stability and storage studies.

  1. Natural and Genetically Engineered Proteins for Tissue Engineering

    PubMed Central

    Gomes, Sílvia; Leonor, Isabel B.; Mano, João F.; Reis, Rui L.

    2011-01-01

    To overcome the limitations of traditionally used autografts, allografts and, to a lesser extent, synthetic materials, there is the need to develop a new generation of scaffolds with adequate mechanical and structural support, control of cell attachment, migration, proliferation and differentiation and with bio-resorbable features. This suite of properties would allow the body to heal itself at the same rate as implant degradation. Genetic engineering offers a route to this level of control of biomaterial systems. The possibility of expressing biological components in nature and to modify or bioengineer them further, offers a path towards multifunctional biomaterial systems. This includes opportunities to generate new protein sequences, new self-assembling peptides or fusions of different bioactive domains or protein motifs. New protein sequences with tunable properties can be generated that can be used as new biomaterials. In this review we address some of the most frequently used proteins for tissue engineering and biomedical applications and describe the techniques most commonly used to functionalize protein-based biomaterials by combining them with bioactive molecules to enhance biological performance. We also highlight the use of genetic engineering, for protein heterologous expression and the synthesis of new protein-based biopolymers, focusing the advantages of these functionalized biopolymers when compared with their counterparts extracted directly from nature and modified by techniques such as physical adsorption or chemical modification. PMID:22058578

  2. Engineered Proteins: Redox Properties and Their Applications

    PubMed Central

    Prabhulkar, Shradha; Tian, Hui; Wang, Xiaotang; Zhu, Jun-Jie

    2012-01-01

    Abstract Oxidoreductases and metalloproteins, representing more than one third of all known proteins, serve as significant catalysts for numerous biological processes that involve electron transfers such as photosynthesis, respiration, metabolism, and molecular signaling. The functional properties of the oxidoreductases/metalloproteins are determined by the nature of their redox centers. Protein engineering is a powerful approach that is used to incorporate biological and abiological redox cofactors as well as novel enzymes and redox proteins with predictable structures and desirable functions for important biological and chemical applications. The methods of protein engineering, mainly rational design, directed evolution, protein surface modifications, and domain shuffling, have allowed the creation and study of a number of redox proteins. This review presents a selection of engineered redox proteins achieved through these methods, resulting in a manipulation in redox potentials, an increase in electron-transfer efficiency, and an expansion of native proteins by de novo design. Such engineered/modified redox proteins with desired properties have led to a broad spectrum of practical applications, ranging from biosensors, biofuel cells, to pharmaceuticals and hybrid catalysis. Glucose biosensors are one of the most successful products in enzyme electrochemistry, with reconstituted glucose oxidase achieving effective electrical communication with the sensor electrode; direct electron-transfer-type biofuel cells are developed to avoid thermodynamic loss and mediator leakage; and fusion proteins of P450s and redox partners make the biocatalytic generation of drug metabolites possible. In summary, this review includes the properties and applications of the engineered redox proteins as well as their significance and great potential in the exploration of bioelectrochemical sensing devices. Antioxid. Redox Signal. 17, 1796–1822. PMID:22435347

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

    PubMed

    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.

  4. FMDV replicons encoding green fluorescent protein are replication competent.

    PubMed

    Tulloch, Fiona; Pathania, Uday; Luke, Garry A; Nicholson, John; Stonehouse, Nicola J; Rowlands, David J; Jackson, Terry; Tuthill, Toby; Haas, Juergen; Lamond, Angus I; Ryan, Martin D

    2014-12-01

    The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.

  5. Natural Photoreceptors as a Source of Fluorescent Proteins, Biosensors, and Optogenetic Tools

    PubMed Central

    Shcherbakova, Daria M.; Shemetov, Anton A.; Kaberniuk, Andrii A.; Verkhusha, Vladislav V.

    2015-01-01

    Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein–like probes, including near-infrared fluorescence, independence of oxygen, small size, and photo-sensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells. PMID:25706899

  6. In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS).

    PubMed

    Krupka, Simone S; Wiltschi, Birgit; Reuning, Ute; Hölscher, Kerstin; Hara, Masahiko; Sinner, Eva-Kathrin

    2006-08-15

    Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

  7. Engineered coiled-coil protein microfibers.

    PubMed

    Hume, Jasmin; Sun, Jennifer; Jacquet, Rudy; Renfrew, P Douglas; Martin, Jesse A; Bonneau, Richard; Gilchrist, M Lane; Montclare, Jin Kim

    2014-10-13

    The fabrication of de novo proteins able to self-assemble on the nano- to meso-length scales is critical in the development of protein-based biomaterials in nanotechnology and medicine. Here we report the design and characterization of a protein engineered coiled-coil that not only assembles into microfibers, but also can bind hydrophobic small molecules. Under ambient conditions, the protein forms fibers with nanoscale structure possessing large aspect ratios formed by bundles of α-helical homopentameric assemblies, which further assemble into mesoscale fibers in the presence of curcumin through aggregation. Surprisingly, these biosynthesized fibers are able to form in conditions of remarkably low concentrations. Unlike previously designed coiled-coil fibers, these engineered protein microfibers can bind the small molecule curcumin throughout the assembly, serving as a depot for encapsulation and delivery of other chemical agents within protein-based 3D microenvironments.

  8. Exciton dynamics in solid-state green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Dietrich, Christof P.; Siegert, Marie; Betzold, Simon; Ohmer, Jürgen; Fischer, Utz; Höfling, Sven

    2017-01-01

    We study the decay characteristics of Frenkel excitons in solid-state enhanced green fluorescent protein (eGFP) dried from solution. We further monitor the changes of the radiative exciton decay over time by crossing the phase transition from the solved to the solid state. Complex interactions between protonated and deprotonated states in solid-state eGFP can be identified from temperature-dependent and time-resolved fluorescence experiments that further allow the determination of activation energies for each identified process.

  9. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  10. A model for multiexponential tryptophan fluorescence intensity decay in proteins.

    PubMed Central

    Bajzer, Z; Prendergast, F G

    1993-01-01

    Tryptophan fluorescence intensity decay in proteins is modeled by multiexponential functions characterized by lifetimes and preexponential factors. Commonly, multiple conformations of the protein are invoked to explain the recovery of two or more lifetimes from the experimental data. However, in many proteins the structure seems to preclude the possibility of multiple conformers sufficiently different from one another to justify such an inference. We present here another plausible multiexponential model based on the assumption that an energetically excited donor surrounded by N acceptor molecules decays by specific radiative and radiationless relaxation processes, and by transferring its energy to acceptors present in or close to the protein matrix. If interactions between the acceptors themselves and back energy transfer are neglected, we show that the intensity decay function contain 2N exponential components characterized by the unperturbed donor lifetime, by energy transfer rates and a probability of occurrence for the corresponding process. We applied this model to the fluorescence decay of holo- and apoazurin, ribonuclease T1, and the reduced single tryptophan mutant (W28F) of thioredoxin. Use of a multiexponential model for the analysis of the fluorescence intensity decay can therefore be justified, without invoking multiple protein conformations. Images FIGURE 1 PMID:8312471

  11. Crystal structure of the Aequorea victoria green fluorescent protein.

    PubMed

    Ormö, M; Cubitt, A B; Kallio, K; Gross, L A; Tsien, R Y; Remington, S J

    1996-09-06

    The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

  12. Crystal Structure of the Aequorea victoria Green Fluorescent Protein

    NASA Astrophysics Data System (ADS)

    Ormo, Mats; Cubitt, Andrew B.; Kallio, Karen; Gross, Larry A.; Tsien, Roger Y.; Remington, S. James

    1996-09-01

    The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded β barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

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

    PubMed

    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.

  14. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  15. Protein engineering methods applied to membrane protein targets.

    PubMed

    Lluis, M W; Godfroy, J I; Yin, H

    2013-02-01

    Genes encoding membrane proteins have been estimated to comprise as much as 30% of the human genome. Among these membrane, proteins are a large number of signaling receptors, transporters, ion channels and enzymes that are vital to cellular regulation, metabolism and homeostasis. While many membrane proteins are considered high-priority targets for drug design, there is a dearth of structural and biochemical information on them. This lack of information stems from the inherent insolubility and instability of transmembrane domains, which prevents easy obtainment of high-resolution crystals to specifically study structure-function relationships. In part, this lack of structures has greatly impeded our understanding in the field of membrane proteins. One method that can be used to enhance our understanding is directed evolution, a molecular biology method that mimics natural selection to engineer proteins that have specific phenotypes. It is a powerful technique that has considerable success with globular proteins, notably the engineering of protein therapeutics. With respect to transmembrane protein targets, this tool may be underutilized. Another powerful tool to investigate membrane protein structure-function relationships is computational modeling. This review will discuss these protein engineering methods and their tremendous potential in the study of membrane proteins.

  16. Local fitness landscape of the green fluorescent protein.

    PubMed

    Sarkisyan, Karen S; Bolotin, Dmitry A; Meer, Margarita V; Usmanova, Dinara R; Mishin, Alexander S; Sharonov, George V; Ivankov, Dmitry N; Bozhanova, Nina G; Baranov, Mikhail S; Soylemez, Onuralp; Bogatyreva, Natalya S; Vlasov, Peter K; Egorov, Evgeny S; Logacheva, Maria D; Kondrashov, Alexey S; Chudakov, Dmitry M; Putintseva, Ekaterina V; Mamedov, Ilgar Z; Tawfik, Dan S; Lukyanov, Konstantin A; Kondrashov, Fyodor A

    2016-05-19

    Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

  17. Local fitness landscape of the green fluorescent protein

    PubMed Central

    Sarkisyan, Karen S.; Bolotin, Dmitry A.; Meer, Margarita V.; Usmanova, Dinara R.; Mishin, Alexander S.; Sharonov, George V.; Ivankov, Dmitry N.; Bozhanova, Nina G.; Baranov, Mikhail S.; Soylemez, Onuralp; Bogatyreva, Natalya S.; Vlasov, Peter K.; Egorov, Evgeny S.; Logacheva, Maria D.; Kondrashov, Alexey S.; Chudakov, Dmitry M.; Putintseva, Ekaterina V.; Mamedov, Ilgar Z.; Tawfik, Dan S.; Lukyanov, Konstantin A.; Kondrashov, Fyodor A.

    2016-01-01

    Fitness landscapes1,2, depictions of how genotypes manifest at the phenotypic level, form the basis for our understanding of many areas of biology2–7 yet their properties remain elusive. Studies addressing this issue often consider specific genes and their function as proxy for fitness2,4, experimentally assessing the impact on function of single mutations and their combinations in a specific sequence2,5,8–15 or in different sequences2,3,5,16–18. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here, we chart an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function, fluorescence, of tens of thousands of derivative genotypes of avGFP. We find that its fitness landscape is narrow, with half of genotypes with two mutations showing reduced fluorescence and half of genotypes with five mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations arising mostly through the cumulative impact of slightly deleterious mutations causing a threshold-like decrease of protein stability and concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for a number of fields including molecular evolution, population genetics and protein design. PMID:27193686

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

    PubMed Central

    Volkmer, A; Subramaniam, V; Birch, D J; Jovin, T M

    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 anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore. PMID:10692343

  19. Fluorescent protein senses and reports mechanical damage in glass-fiber-reinforced polymer composites.

    PubMed

    Makyła, Katarzyna; Müller, Christoph; Lörcher, Samuel; Winkler, Thomas; Nussbaumer, Martin G; Eder, Michaela; Bruns, Nico

    2013-05-21

    Yellow fluorescent protein (YFP) is used as a mechanoresponsive layer at the fiber/resin interface in glass-fiber-reinforced composites. The protein loses its fluorescence when subjected to mechanical stress. Within the material, it reports interfacial shear debonding and barely visible impact damage by a transition from a fluorescent to a non-fluorescent state.

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

    SciTech Connect

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

    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.

  1. Engineering nanoparticle-protein associations for protein crystal nucleation and nanoparticle arrangement

    NASA Astrophysics Data System (ADS)

    Benoit, Denise N.

    Engineering the nanoparticle - protein association offers a new way to form protein crystals as well as new approaches for arrangement of nanoparticles. Central to this control is the nanoparticle surface. By conjugating polymers on the surface with controlled molecular weights many properties of the nanoparticle can be changed including its size, stability in buffers and the association of proteins with its surface. Large molecular weight poly(ethylene glycol) (PEG) coatings allow for weak associations between proteins and nanoparticles. These interactions can lead to changes in how proteins crystallize. In particular, they decrease the time to nucleation and expand the range of conditions over which protein crystals form. Interestingly, when PEG chain lengths are too short then protein association is minimized and these effects are not observed. One important feature of protein crystals nucleated with nanoparticles is that the nanoparticles are incorporated into the crystals. What results are nanoparticles placed at well-defined distances in composite protein-nanoparticle crystals. Crystals on the size scale of 10 - 100 micrometers exhibit optical absorbance, fluorescence and super paramagnetic behavior derivative from the incorporated nanomaterials. The arrangement of nanoparticles into three dimensional arrays also gives rise to new and interesting physical and chemical properties, such as fluorescence enhancement and varied magnetic response. In addition, anisotropic nanomaterials aligned throughout the composite crystal have polarization dependent optical properties.

  2. Fluorescence quenching in proteins: some applications to protein-DNA and protein-lipid interactions

    NASA Astrophysics Data System (ADS)

    Knutson, Jay R.; Chen, Raymond F.; Porter, D. K.; Hensley, Preston; Han, Myun K.; Kim, S. J.; Wilson, Samuel H.; Clague, M.; Williamson, Cynthia K.

    1992-04-01

    The authors outline some examples of the advantages found in subdividing overall quenching into heterogeneous contributions. Subdivision is accomplished by overdetermination (global) and association (DAS, decay associated spectral) methods. In some cases, the subdivision of fluorescence leads to the unique identification of different fluorophores in different sites. Alternatively, the recovered components may reflect conformational heterogeneity at each site. For intrinsic protein fluorescence, it is often noted in the literature that single Trp proteins may be multiexponential. Genetic substitution in multi-Trp proteins, however, often leads to very strong (if not complete) lifetime-to-Trp assignment. Even if a single Trp experiences two or more microenvironments, it can be a useful reporter. The linkage of multiple lifetimes and amplitudes to changes in global conformation often reveals a more `sensitive' subpopulation or lifetime component that becomes a better indicator for important conformational states than aggregate intensity can provide. This has proven useful in studying pH transitions of proteins both in solution and embedded in membranes. Energy transfer is particularly useful in differentiating sites at different distances. Further, the disclosure of heterogeneity in distance is clearly superior to the reporting of a mean distance. This report surveys several systems that have been examined via emission DAS techniques, showing how each protein is better understood when viewed in terms of discrete spectral contributions. We conclude with an overview and some details about our construction of an EDAS (excitation-DAS) instrument; i.e., how excitation scans can be incorporated into a time-resolved instrument.

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

    PubMed

    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. Dual-color fluorescence imaging of tumor/host interaction with green and red fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Amoh, Yasuyuki; Li, Lingna; Baranov, Eugene; Wang, Jin Wei; Jiang, Ping; Moossa, A. R.; Hoffman, Robert M.

    2004-06-01

    Dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP) expressing transgenic mice has been shown to be a powerful technology to study tumor-host interaction. Host animals include mice which express the GFP transgene in essentially all cells as well as animals in which the regulatory elements of the stem cell marker nestin drive GFP. The general GFP-transgenic mouse is available in both the normal and athymic nude (nu/nu) background. These models show with great clarity the details of the tumor-stroma interaction especially tumor induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. GFP-expressing tumor vasculature could be visualized interacting with the RFP-expressing tumor cells transplanted to the nestin-driven GFP transgenic mice which expressed nestin-GFP in nascent blood vessels was shown as a marker of nascent tumor angiogenesis. Dual-color fluorescence imaging, which visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations (1).

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Structural basis of fluorescence fluctuation dynamics of green fluorescent proteins in acidic environments.

    PubMed

    Liu, Yuexin; Kim, Hye-Ryong; Heikal, Ahmed A

    2006-11-30

    Green fluorescent proteins (GFPs) have become powerful markers for numerous biological studies due to their robust fluorescence properties, site-specific labeling, pH sensitivity, and mutations for multiple-site labeling. Fluorescence correlation spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic GFP mutants takes place on a time scale of 45-300 ms, depending on pH, and have been attributed to external proton transfer. Here we present experimental evidence indicating that conformational change in the protein &beta-barrel is a determining step for the external protonation of GFP-S65T (at low pH) using time-resolved fluorescence and polarization anisotropy measurements. While the average anionic fluorescence lifetime of GFP-S65T is reduced by approximately 18% over a pH range of 3.6-10.0, the fluorescence polarization anisotropy decays mostly as a single exponential with a rotational time of phi = 17 +/- 1 ns, which indicates an intact beta-barrel with a hydrodynamic volume of 78 +/- 5 nm3. In contrast, the total fluorescence (525 +/- 50 nm) of the excited neutral state of S65T reveals a strong correlation between the fluorescence lifetime, structural conformation, and pH. The average fluorescence lifetime of the excited neutral state of S65T as a function of pH yields pKa approximately 5.9 in agreement with literature values using steady-state techniques. In contrast to the intact beta-barrel at high pH, the anisotropy of neutral S65T (at pH protein. The segmental motion of the S65T chromophore becomes faster with an enhanced amplitude ratio as pH is reduced. For comparative purposes, we also provide complementary FCS results on fluorescence blinking of the excited neutral state of an EGFP mutant (F64L/S65T) on a much

  7. Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Habuchi, Satoshi; Whitier, Jennifer E.; Werner, James H.; De Schryver, Frans C.; Hofkens, Johan; Goodwin, Peter M.

    2006-02-01

    We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all

  8. Multi-color imaging of the bacterial nucleoid and division proteins with blue, orange, and near-infrared fluorescent proteins

    PubMed Central

    Wu, Fabai; Van Rijn, Erwin; Van Schie, Bas G. C.; Dekker, Cees

    2015-01-01

    Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange (TagRFP-T, mKO2), and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified in vivo for their introduction into bacteria; (2) spectral crosstalk and inter-variant interactions between FPs should be carefully examined for multi-color imaging; and (3) successful genomic fusion to the 5′-end of a gene strongly depends on the translational read-through of the inserted coding sequence. PMID:26136737

  9. [Isolation and purification of enhanced green fluorescent protein using chromatography].

    PubMed

    Hou, Qinghua; Song, Shuliang; Liang, Hao; Wang, Weili; Ji, Aiguo

    2013-02-01

    Enhanced green fluorescent protein (EGFP) is a common biological marker. In this research, on the foundation of successful clone and expression of EGFP, a two-step chromatographic method was established to separate and purify EGFP, which includes the use of HisTrap HP immobilized metal affinity chromatography (IMAC) and Sephadex G-10 HR size exclusion chromatography in sequence. Sephacryl S-300 HR size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to check out the purity of EGFP. At last, it was found that EGFP still had fluorescent activity using fluorescence spectrophotometric detection and Native-PAGE detection. This method can effectively separate the active EGFP. The purity of the obtained EGFP was more than 98%.

  10. Colloidal quantum dots for fluorescent labels of proteins

    NASA Astrophysics Data System (ADS)

    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.

  11. Enhanced protein production by engineered zinc finger proteins.

    PubMed

    Reik, Andreas; Zhou, Yuanyue; Collingwood, Trevor N; Warfe, Lyndon; Bartsevich, Victor; Kong, Yanhong; Henning, Karla A; Fallentine, Barrett K; Zhang, Lei; Zhong, Xiaohong; Jouvenot, Yann; Jamieson, Andrew C; Rebar, Edward J; Case, Casey C; Korman, Alan; Li, Xiao-Yong; Black, Amelia; King, David J; Gregory, Philip D

    2007-08-01

    Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings. Expression vectors engineered to carry up to 10 ZFP binding sites further enhanced ZFP-mediated increases in protein production up to approximately 500%. The multimerized ZFP binding sites function independently of the promoter, and therefore across vector platforms. CHO cell lines stably expressing ZFP TFs demonstrated growth characteristics similar to parental cell lines. ZFP TF expression and gains in protein production were stable over >30 generations in the absence of antibiotic selection. Our results demonstrate that ZFP TFs can rapidly and stably increase protein production in mammalian cells.

  12. Biosensing and protein fluorescence enhancement by functionalized porous silicon devices.

    PubMed

    Palestino, Gabriela; Agarwal, Vivechana; Aulombard, Roger; Pérez, Elías; Gergely, Csilla

    2008-12-02

    Porous silicon (PSi) is a promising biomaterial presenting the advantage of being biocompatible and bioresorbable. Due to the large specific surface area and unique optical features, these microporous structures are excellent candidates for biosensing applications. Investigating device functionality and developing simple Si-based transducers need to be addressed in novel biological detection. Our work demonstrates that, among the various PSi configurations for molecular detection, PSi microcavity structure demonstrates the best biosensing performance, reflected through the enhanced luminescence response and the changes in the refractive index. For successful immobilization, molecular infiltration and confinement are the two key factors that are controlled by the pore size distribution of the PSi microcavities and by the surface modification obtained by silane-glutaraldehyde chemistry. Enhancement of the fluorescence emission of confined fluorescent biomolecules in the active layer of PSi microcavities was observed for a nonlabeled protein with a natural green fluorescence, the glucose oxidase enzyme (GOX). An increase in the fluorescence emission was also observed when functionalized PSi material was used to detect specific binding between biotin and a low concentration of labeled streptavidin. Evidence for the enzymatic activity of GOX in its adsorbed form is also presented. Use of smart silicon devices, enabling enhancement of fluorescence emission of biomolecules, offers easy-to-use biosensing, based on the luminescence response of the molecules to be detected.

  13. A knot in the protein structure - probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome.

    PubMed

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

    2014-05-01

    The possibility of engineering near-infrared fluorescent proteins and biosensors from bacterial phytochrome photoreceptors (BphPs) has led to substantial interest in this family of proteins. The near-infrared fluorescent proteins have allowed non-invasive bio-imaging of deep tissues and whole organs in living animals. BphPs and derived near-infrared fluorescent proteins contain a structural element, called a knot, in their polypeptide chains. The formation of knot structures in proteins was refuted for a long time. Here, we studied the denaturation and renaturation processes of the near-infrared fluorescent probe iRFP, engineered from RpBphP2, which utilizes a heme-derived tetrapyrrole compound biliverdin as a chromophore. iRFP contains a unique figure-of-eight knot. The denaturation and renaturation curves of the iRFP apoform coincided well, suggesting efficient refolding. However, the iRFP holoform exhibited irreversible unfolding and aggregation associated with the bound chromophore. The knot structure in the apoform did not prevent subsequent binding of biliverdin, resulting in the functional iRFP holoform. We suggest that the irreversibility of protein unfolding is caused by post-translational protein modifications, such as chromophore binding, rather than the presence of the knot. These results are essential for future design of BphP-based near-infrared probes, and add important features to our knowledge of protein folding.

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

    PubMed

    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.

  15. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

    PubMed

    Kahana, J A; Silver, P A

    2001-05-01

    Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

  16. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

    PubMed

    Kahana, J A; Silver, P A

    2001-05-01

    Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has heretofore been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

  17. Synthesis of a fluorescent 7-methylguanosine analog and a fluorescence spectroscopic study of its reaction with wheatgerm cap binding proteins.

    PubMed Central

    Ren, J; Goss, D J

    1996-01-01

    In the initiation of protein synthesis, the mRNA 5'-terminal 7-methylguanosine cap structure and several recognition proteins play a pivotal role. For the study of this cap binding reaction, one approach is to use fluorescence spectroscopy. A ribose diol-modified fluorescent cap analog, anthraniloyl-m7GTP (Ant-m7GTP), was designed and synthesized for this purpose. This fluorescent cap analog was found to have a high quantum yield, resistance to photobleaching and avoided overlap of excitation and emission wavelengths with those of proteins. The binding of Ant-m7GTP with wheatgerm initiation factors elF-4F and elF-(iso)4F was determined. The fluorescent cap analog and m7GTP had similar interactions with both cap binding proteins. Fluorescence quenching experiments showed that the microenvironment of Ant-m7GTP when bound to protein was hydrophobic. PMID:8836193

  18. Fluorescence-based characterization of non-fluorescent transient states of tryptophan – prospects for protein conformation and interaction studies

    NASA Astrophysics Data System (ADS)

    Hevekerl, Heike; Tornmalm, Johan; Widengren, Jerker

    2016-10-01

    Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies.

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

    PubMed Central

    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

  20. Optical studies of dynamical processes in fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Liebig, Carl; Dennis, William; Kirkpatrick, Sean; Naik, Rajesh; Stone, Morley

    2002-03-01

    Green fluorescent protein (GFP) extracted from the bioluminescent jellyfish Aequorea Victoria[1] and its mutants are novel nanoscale systems, which have been shown to exhibit desirable linear and nonlinear optical properties[2]. In this paper, a combination of both linear and nonlinear optical spectroscopic techniques was used to investigate dynamical processes in fluorescent proteins in both aqueous solution and an organic polymer matrix. Experimental results were analyzed in terms of a Brownian oscillator model[3] and by comparison to computer simulations. [1] M. Chalfie, G. Euskirchen, W. W. Ward and D. C. Prasher, Science 263 (1994) 802. [2] Sean M. Kirkpatrick, Rajesh R. Naik, Morley O. Stone, J. Phys. Chem. B 105 (2001) 2867. [3] S. Mukamel, "Nonlinear Optical Spectroscopy", (Oxford University Press, New York, 1995) pp. 227.

  1. Primary structure of the Aequorea victoria green-fluorescent protein.

    PubMed

    Prasher, D C; Eckenrode, V K; Ward, W W; Prendergast, F G; Cormier, M J

    1992-02-15

    Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

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

    SciTech Connect

    Watkins, Jennifer L.; Kim, Hanseong; Markwardt, Michele L.; Chen, Liqing; Fromme, Raimund; Rizzo, Mark A.; Wachter, Rebekka M.

    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.

  3. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

    PubMed Central

    Shcherbakova, Daria M.; Baloban, Mikhail; Emelyanov, Alexander V.; Brenowitz, Michael; Guo, Peng; Verkhusha, Vladislav V.

    2016-01-01

    Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2–5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein–protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes. PMID:27539380

  4. Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-alpha induced apoptosis.

    PubMed

    Ito, Toshiyuki; Oshita, Shugo; Nakabayashi, Takakazu; Sun, Fan; Kinjo, Masataka; Ohta, Nobuhiro

    2009-06-01

    Fluorescence lifetime images of HeLa cells expressing enhanced green fluorescent protein (EGFP) have been measured as apoptosis is induced by tumor necrosis factor-alpha (TNF-alpha) in combination with cycloheximide. The fluorescence lifetime of EGFP is found to decrease after the induction of apoptosis, indicating that the change in environment occurs around the chromophore of EGFP with the apoptosis process. The fluorescence lifetime imaging technique can be used to perform in vivo observation of cell death processes. Fluorescence lifetime measurements are useful to examine the induction of the apoptosis process, even when a morphological change of each cell cannot be observed because of a low spatial resolution.

  5. Bifunctional chimeric fusion proteins engineered for DNA delivery: Optimization of the protein to DNA ratio

    PubMed Central

    Gao, Shan; Simon, Melissa J.; Morrison, Barclay; Banta, Scott

    2009-01-01

    Background Cell penetrating peptides (CPPs) have been used to deliver nucleotide-based therapeutics to cells, but this approach has produced mixed results. Ionic interactions and covalent bonds between the CPPs and the cargos may inhibit the effectiveness of the CPPs or interfere with the bioactivity of the cargos. Methods We have created a bifunctional chimeric protein that binds DNA using the p50 domain of the NF-κB transcription factor and is functionalized for delivery with the TAT CPP. The green fluorescent protein (GFP) has been incorporated for tracking delivery. The new chimeric protein, p50-GFP-TAT, was compared to p50-GFP, GFP-TAT and GFP as controls for the ability to transduce PC12 cells with and without oligonucleotide cargos. Results The p50-GFP-TAT construct can deliver 30bp and 293bp oligonucleotides to PC12 cells with an optimal ratio of 1.89 protein molecules per base pair of DNA length. This correlation was validated through the delivery of a fluorescent protein transgene encoded in a plasmid to PC12 cells. Conclusion Self-assembling CPP-based bifunctional fusion proteins can be engineered for the non-viral delivery of nucleotide-based cargos to mammalian cells. General significance This work represents an important step forward in the rational design of protein-based systems for the delivery of macromolecular cargos. PMID:19402206

  6. Complementation and reconstitution of fluorescence from circularly permuted and truncated green fluorescent protein.

    PubMed

    Huang, Yao-ming; Bystroff, Christopher

    2009-02-10

    Green fluorescent protein (GFP) has been used as a proof of concept for a novel "leave-one-out" biosensor design in which a protein that has a segment omitted from the middle of the sequence by circular permutation and truncation binds the missing peptide and reconstitutes its function. Three variants of GFP have been synthesized that are each missing one of the 11 beta-strands from its beta-barrel structure, and in two of the variants, adding the omitted peptide sequence in trans reconstitutes fluorescence. Detailed biochemical analysis indicates that GFP with beta-strand 7 "left out" (t7SPm) exists in a partially unfolded state. The apo form t7SPm binds the free beta-strand 7 peptide with a dissociation constant of approximately 0.5 microM and folds into the native state of GFP, resulting in fluorescence recovery. Folding of t7SPm, both with and without the peptide ligand, is at least a three-state process and has a rate comparable to that of the full-length and unpermuted GFP. The conserved kinetic properties strongly suggest that the rate-limiting steps in the folding pathway have not been altered by circular permutation and truncation in t7SPm. This study shows that structural and functional reconstitution of GFP can occur with a segment omitted from the middle of the chain, and that the unbound form is in a partially unfolded state.

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

    PubMed

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

    2011-03-01

    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.

  8. Thermal effect on Aequorea green fluorescent protein anionic and neutral chromophore forms fluorescence.

    PubMed

    dos Santos, Andrea Martins

    2012-01-01

    The emission behaviour of Aequorea green fluorescent protein (A-GFP) chromophore, in both neutral (N) and anionic (A) form, was studied in the temperature range from 20 °C to 75 °C and at pH = 7. Excitation wavelengths of 399 nm and 476 nm were applied to probe the N and A forms environment, respectively. Both forms exhibit distinct fluorescence patterns at high temperature values. The emission quenching rate, following a temperature increase, is higher for the chromophore N form as a result of the hydrogen bond network weakening. The chromophore anionic form emission maximum is red shifted, upon temperature increase, due to a charge transfer process occurring after A form excitation.

  9. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    SciTech Connect

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  10. Fluorescence lifetime imaging of symbionts and fluorescent proteins in reef corals

    NASA Astrophysics Data System (ADS)

    Cox, Guy; Salih, Anya

    2005-03-01

    Reef-building corals are dependent on dinoflagellate algal symbionts (zooxanthellae). Within the range of habitats of any one coral species there can be huge variations in light intensities, so there is a risk of photoinhibition from excess light. In extremes of light and heat, senescent algae are expelled en masse, a phenomenon known as coral bleaching. In freshly isolated tissue the chlorophyll fluorescence has a lifetime of ~1.1ns. 6 hours and 15 hours after isolation the zooxanthellae looked visually healthy, but the lifetimes had increased to 2ns after 6 hours and 2.2ns after 15 hours. Zooxanthellae which were visibly damaged or necrotic had a mean lifetime of 3ns. Lifetime of chlorophyll fluorescence is thus a sensitive indicator, revealing effects in cell metabolism before any structural changes are evident. The occurrence of FRET between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504 and amilFP593 (names indicate emission peaks) were 2.8ns, 2.9ns and 2.9ns respectively. In the coral sample, imaging the entire emission spectrum from 420nm, the mean lifetime was reduced to 1.5ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3ns, supporting this interpretation.

  11. Fluorescence imaging of angiogenesis in green fluorescent protein-expressing tumors

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Baranov, Eugene; Jiang, Ping; Li, Xiao-Ming; Wang, Jin W.; Li, Lingna; Yagi, Shigeo; Moossa, A. R.; Hoffman, Robert M.

    2002-05-01

    The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

  12. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein.

    PubMed

    Brejc, K; Sixma, T K; Kitts, P A; Kain, S R; Tsien, R Y; Ormö, M; Remington, S J

    1997-03-18

    The 2.1-A resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.

  13. Structural Basis for Dual Excitation and Photoisomerization of the Aequorea victoria Green Fluorescent Protein

    NASA Astrophysics Data System (ADS)

    Brejc, Katjusa; Sixma, Titia K.; Kitts, Paul A.; Kain, Steven R.; Tsien, Roger Y.; Ormo, Mats; Remington, S. James

    1997-03-01

    The 2.1- angstrom resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.

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

    ERIC Educational Resources Information Center

    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…

  15. [Preparation and penetrating effect of the polyarginine-enhanced green fluorescence protein fusion protein].

    PubMed

    Zhang, Nan; Bai, Yin; Zhao, Jingzhuang; Ye, Xianlong; Wang, Wenfei; Ren, Guiping; Li, Deshan; Jing, Yan

    2013-11-01

    The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.

  16. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

    PubMed Central

    Tillberg, Paul W.; Chen, Fei; Piatkevich, Kiryl D.; Zhao, Yongxin; Yu, Chih-Chieh (Jay); English, Brian P.; Gao, Linyi; Martorell, Anthony; Suk, Ho-Jun; Yoshida, Fumiaki; DeGennaro, Ellen M.; Roossien, Douglas H.; Gong, Guanyu; Seneviratne, Uthpala; Tannenbaum, Steven R.; Desimone, Robert; Cai, Dawen; Boyden, Edward S.

    2016-01-01

    Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first expansion microscopy method was unable to retain native proteins in the gel and used custom made reagents not widely available. Here, we describe protein retention ExM (proExM), a variant of ExM that anchors proteins to the swellable gel allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validate and demonstrate utility of proExM for multi-color super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes. PMID:27376584

  17. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

    PubMed

    Kahana, J A; Silver, P A

    2001-05-01

    Fluorescent molecules serve as valuable tools for the detection of numerous biochemical phenomena and have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting and determination of chemical concentrations. However, the use of such techniques generally requires significant nonphysiological perturbations to the biological system being studied; therefore, they are not always appropriate for the observation of dynamic phenomena. Green fluorescent protein (GFP), cloned from jellyfish, has been used to overcome many of these problems. It is a small, extremely stable fluorescent protein that has been successfully expressed and detected in a wide variety of organisms, both in intact form and fused to other proteins. This overview unit describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena.

  18. Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    PubMed Central

    Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

    2011-01-01

    In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed. PMID:20885379

  19. Recent advances in engineering proteins for biocatalysis.

    PubMed

    Li, Ye; Cirino, Patrick C

    2014-07-01

    Protein engineers are increasingly able to rely on structure-function insights, computational methods, and deeper understanding of natural biosynthesis processes, to streamline the design and applications of enzymes. This review highlights recent successes in applying new or improved protein engineering strategies toward the design of improved enzymes and enzymes with new activities. We focus on three approaches: structure-guided protein design, computational design, and the use of novel scaffolding and compartmentalization techniques to improve performance of multienzyme systems. Examples described address problems relating to enzyme specificity, stability, and/or activity, or aim to balance sequential reactions and route intermediates by co-localizing multiple enzymes. Specific applications include improving production of biofuels using enzymes with altered cofactor specificity, production of high-value chiral compounds by enzymes with tailored substrate specificities, and accelerated cellulose degradation via multi-enzyme scaffold assemblies. Collectively, these studies demonstrate a growing variety of computational and molecular biology tools. Continued advances on these fronts coupled with better mindfulness of how to apply proteins in unique ways offer exciting prospects for future protein engineering and biocatalysis research.

  20. Engineering of filamentous bacteriophage for protein sensing

    NASA Astrophysics Data System (ADS)

    Brasino, Michael

    Methods of high throughput, sensitive and cost effective quantification of proteins enables personalized medicine by allowing healthcare professionals to better monitor patient condition and response to treatment. My doctoral research has attempted to advance these methods through the use of filamentous bacteriophage (phage). These bacterial viruses are particularly amenable to both genetic and chemical engineering and can be produced efficiently in large amounts. Here, I discuss several strategies for modifying phage for use in protein sensing assays. These include the expression of bio-orthogonal conjugation handles on the phage coat, the incorporation of specific recognition sequences within the phage genome, and the creation of antibody-phage conjugates via a photo-crosslinking non-canonical amino acid. The physical and chemical characterization of these engineered phage and the results of their use in modified protein sensing assays will be presented.

  1. Dentin Matrix Proteins in Bone Tissue Engineering.

    PubMed

    Ravindran, Sriram; George, Anne

    2015-01-01

    Dentin and bone are mineralized tissue matrices comprised of collagen fibrils and reinforced with oriented crystalline hydroxyapatite. Although both tissues perform different functionalities, they are assembled and orchestrated by mesenchymal cells that synthesize both collagenous and noncollagenous proteins albeit in different proportions. The dentin matrix proteins (DMPs) have been studied in great detail in recent years due to its inherent calcium binding properties in the extracellular matrix resulting in tissue calcification. Recent studies have shown that these proteins can serve both as intracellular signaling proteins leading to induction of stem cell differentiation and also function as nucleating proteins in the extracellular matrix. These properties make the DMPs attractive candidates for bone and dentin tissue regeneration. This chapter will provide an overview of the DMPs, their functionality and their proven and possible applications with respect to bone tissue engineering.

  2. A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

    PubMed Central

    Valuchova, Sona; Fulnecek, Jaroslav; Petrov, Alexander P.; Tripsianes, Konstantinos; Riha, Karel

    2016-01-01

    Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions. PMID:28008962

  3. External optical imaging of freely moving mice with green fluorescent protein-expressing metastatic tumors

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Baranov, Eugene; Shimada, Hiroshi; Moossa, A. R.; Hoffman, Robert M.

    2000-04-01

    We report here a new approach to genetically engineering tumors to become fluorescence such that they can be imaged externally in freely-moving animals. We describe here external high-resolution real-time fluorescent optical imaging of metastatic tumors in live mice. Stable high-level green flourescent protein (GFP)-expressing human and rodent cell lines enable tumors and metastasis is formed from them to be externally imaged from freely-moving mice. Real-time tumor and metastatic growth were quantitated from whole-body real-time imaging in GFP-expressing melanoma and colon carcinoma models. This GFP optical imaging system is highly appropriate for high throughput in vivo drug screening.

  4. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  5. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

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

    DOEpatents

    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. Fluorescent Probe Encapsulated in Avidin Protein to Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity in Blood Serum.

    PubMed

    Wu, Ting-Wei; Lee, Fang-Hong; Gao, Ruo-Cing; Chew, Chee Ying; Tan, Kui-Thong

    2016-08-16

    Quantitative detection of trace amounts of a biomarker in protein rich human blood plasma using fluorescent probes is a great challenge as the real signal is usually obscured by nonspecific fluorescence. This problem occurs because most of the fluorescent dyes bind very tightly with blood proteins to produce a large fluorescence increase, resulting in overestimation of the biomarker concentrations and false positive diagnosis. In this paper, we report that biotinylated fluorescent probes encapsulated in avidin protein can generate very specific fluorescence in blood serum by blocking out nonspecific dye-protein interactions. We applied our novel probe design to detect two different types of biomolecules, hydrogen sulfide and nitroreductase. Our Avidin conjugated probes achieved quantitative analyte detection in blood serum; whereas concentrations were overestimated up to 320-fold when bare fluorescent probes were employed. As compared to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple approach successfully overcomes 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.

  8. Protein Engineering and Its Applications in Food Industry.

    PubMed

    Kapoor, Swati; Rafiq, Aasima; Sharma, Savita

    2015-06-11

    Protein engineering is a young discipline that has been branched out from the field of genetic engineering. Protein engineering is based on the available knowledge about the proteins structure/function(s), tools/instruments, software, bioinformatics database, available cloned gene, knowledge about available protein, vectors, recombinant strains and other materials that could lead to change in the protein backbone. Protein produced properly from genetic engineering process means a protein that is able to fold correctly and to do particular function(s) efficiently even after being subjected to engineering practices. Protein is modified through its gene or chemically. However, modification of protein through gene is easier. There is no specific limitation of Protein Engineering tools; any technique that can lead to change the protein constituent of amino acid and result in the modification of protein structure/function is in the frame of Protein Engineering. Meanwhile, there are some common tools used to reach a specific target. More active industrial and pharmaceutical based proteins have been invented by the field of Protein Engineering to introduce new function as well as to change its interaction with surrounding environment. A variety of protein engineering applications have been reported in the literature. These applications range from biocatalysis for food and industry to environmental, medical and nanobiotechnology applications. Successful combinations of various protein engineering methods had led to successful results in food industries and have created a scope to maintain the quality of finished product after processing.

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

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2014-04-01

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

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

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2015-07-14

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

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

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-07

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

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

    PubMed

    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.

  13. Green Fluorescent Protein as a Visual Marker in Somatic Hybridization

    PubMed Central

    OLIVARES‐FUSTER, O.; PEÑA, L.; DURAN‐VILA, N.; NAVARRO, L.

    2002-01-01

    Using a transgenic citrus plant expressing Green Fluorescent Protein (GFP) as a parent in somatic fusion experiments, we investigated the suitability of GFP as an in vivo marker to follow the processes of protoplast fusion, regeneration and selection of hybrid plants. A high level of GFP expression was detected in transgenic citrus protoplasts, hybrid callus, embryos and plants. It is demonstrated that GFP can be used for the continuous monitoring of the fusion process, localization of hybrid colonies and callus, and selection of somatic hybrid embryos and plants. PMID:12096810

  14. Protein Turnover during in vitro Tissue Engineering

    PubMed Central

    Li, Qiyao; Chang, Zhen; Oliveira, Gisele; Xiong, Maiyer; Smith, Lloyd M.; Frey, Brian L.; Welham, Nathan V.

    2015-01-01

    Repopulating acellular biological scaffolds with phenotypically appropriate cells is a promising approach for regenerating functional tissues and organs. Under this tissue engineering paradigm, reseeded cells are expected to remodel the scaffold by active protein synthesis and degradation; however, the rate and extent of this remodeling remain largely unknown. Here, we present a technique to measure dynamic proteome changes during in vitro remodeling of decellularized tissue by reseeded cells, using vocal fold mucosa as the model system. Decellularization and recellularization were optimized, and a stable isotope labeling strategy was developed to differentiate remnant proteins constituting the original scaffold from proteins newly synthesized by reseeded cells. Turnover of matrix and cellular proteins and the effects of cell-scaffold interaction were elucidated. This technique sheds new light on in vitro tissue remodeling and the process of tissue regeneration, and is readily applicable to other tissue and organ systems. PMID:26724458

  15. Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae

    PubMed Central

    Baranowski, Kaitlyn; Lee, Wei-Lih

    2016-01-01

    The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast due to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Due to problems associated with fusing genes encoding fluorescent proteins (FPs) to the native a-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome integrated FP-tubulin fusions negatively affect microtubule functions. Thus, a simple, economical, and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP-Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of thirty FP-Tub1 plasmids that employ various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function. PMID:25711127

  16. Ground-State Proton Transfer Kinetics in Green Fluorescent Protein

    PubMed Central

    2015-01-01

    Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional 13C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a 13C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site, likely the terminal proton acceptor of ESPT, Glu222. Additionally, experiments on a selection of GFP circular permutants suggest an important role played by the structural dynamics of the seventh β-strand in gating proton transfer from bulk solution to the buried chromophore. PMID:25184668

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.

    PubMed Central

    Young, R M; Arnette, J K; Roess, D A; Barisas, B G

    1994-01-01

    We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved. PMID:7948701

  19. Aequorea green fluorescent protein analysis by flow cytometry.

    PubMed

    Ropp, J D; Donahue, C J; Wolfgang-Kimball, D; Hooley, J J; Chin, J Y; Hoffman, R A; Cuthbertson, R A; Bauer, K D

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.

  20. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

    Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.; Wolfgang-Kimball, D.

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.

  1. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  2. Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

    PubMed Central

    Heppert, Jennifer K.; Dickinson, Daniel J.; Pani, Ariel M.; Higgins, Christopher D.; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R.; Goldstein, Bob

    2016-01-01

    Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. PMID:27385332

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

    PubMed

    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.

  4. Plasmon-enhanced emission from single fluorescent proteins

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Determination of protein concentration on substrates using fluorescence fluctuation microscopy

    NASA Astrophysics Data System (ADS)

    De Mets, Richard; Wang, Irène; Gallagher, Joseph; Destaing, Olivier; Balland, Martial; Delon, Antoine

    2014-03-01

    Micro-fabrication and surface functionalization imply to know the equilibrium surface concentration of various kinds of molecules. Paradoxically, this crucial parameter is often poorly controlled and even less quantified. We have used a technique belonging to the family of fluorescence fluctuation microscopy, namely Image Correlation Spectroscopy (ICS), to measure the absolute surface concentration of fibrinogen molecules adsorbed on glass substrates. As these molecules are immobile, the width of the autocorrelation of the confocal image obtained by scanning the sample only reflects that of the confocal Point Spread Function. Conversely, the amplitude of the autocorrelation is directly related to the average number of proteins simultaneously illuminated by the laser beam and therefore to their surface concentration. We have studied the surface concentration of fibrinogen proteins versus the initial concentration of these molecules, solubilized in the solution which has been deposited on the surface. The estimation of this relation can be biased for several reasons: the concentration of fibrinogen molecules in solution is difficult to control; the measurement of the surface concentration of adsorbed molecules can be strongly underestimated if the surface coverage or the molecular brightness is not uniform. We suggest methods to detect these artifacts and estimate the actual surface concentration, together with control parameters. Globally, fluorescence fluctuation microscopy is a powerful set of techniques when one wants to quantify the surface concentration of molecules at the micrometer scale.

  8. Engineering enhanced protein disaggregases for neurodegenerative disease

    PubMed Central

    Jackrel, Meredith E; Shorter, James

    2015-01-01

    Abstract Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by misfolded conformers. Moreover, a disruptive technology of this nature would eliminate self-templating conformers that spread pathology and catalyze formation of toxic, soluble oligomers. Here, we highlight our efforts to engineer Hsp104, a protein disaggregase from yeast, to more effectively disaggregate misfolded proteins connected with PD, ALS, and FTD. Remarkably subtle modifications of Hsp104 primary sequence yielded large gains in protective activity against deleterious α-synuclein, TDP-43, FUS, and TAF15 misfolding. Unusually, in many cases loss of amino acid identity at select positions in Hsp104 rather than specific mutation conferred a robust therapeutic gain-of-function. Nevertheless, the misfolding and toxicity of EWSR1, an RNA-binding protein with a prion-like domain linked to ALS and FTD, could not be buffered by potentiated Hsp104 variants, indicating that further amelioration of disaggregase activity or sharpening of substrate specificity is warranted. We suggest that neuroprotection is achievable for diverse neurodegenerative conditions via surprisingly subtle structural modifications of existing chaperones. PMID:25738979

  9. Development of an optical Zn2+ probe based on a single fluorescent protein

    DOE PAGES

    Qin, Yan; Sammond, Deanne W.; Braselmann, Esther; ...

    2016-07-28

    Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutationsmore » in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1 which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). In conclusion, this sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.« less

  10. Development of an optical Zn2+ probe based on a single fluorescent protein

    SciTech Connect

    Qin, Yan; Sammond, Deanne W.; Braselmann, Esther; Carpenter, Margaret C.; Palmer, Amy E.

    2016-07-28

    Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1 which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). In conclusion, this sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.

  11. Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

    SciTech Connect

    Rodríguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Akerboom, Jasper; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

    2008-07-01

    The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.

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

    PubMed

    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.

  13. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

    PubMed

    Teng, Chao-Yi; Wu, Tzong-Yuan

    2007-07-01

    The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

  14. Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Salomonnson, Emma; Mihalko, Laura Anne; Verkhusha, Vladislav V.; Luker, Kathryn E.; Luker, Gary D.

    2012-09-01

    Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760 nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy.

    PubMed

    Wang, Ke; Liu, Tzu-Ming; Wu, Juwell; Horton, Nicholas G; Lin, Charles P; Xu, Chris

    2012-09-01

    We demonstrate a fiber-based, three-color femtosecond source for simultaneous imaging of three fluorescent proteins (FPs) using two-photon fluorescence microscopy (2PM). The three excitation wavelengths at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation (SHG) of the 1550-nm pump laser and the 1728-nm and 1900-nm solitons generated through soliton self-frequency shift (SSFS) in a large-mode-area (LMA) fiber. These energetic pulses are well matched to the two-photon excitation peaks of red, cyan and yellow fluorescent proteins (TagRFPs, TagCFPs, and TagYFPs) for efficient excitation. We demonstrate simultaneous 2PM of human melanoma cells expressing a "rainbow" combination of these three fluorescent proteins.

  17. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.

    PubMed

    Filonov, Grigory S; Moon, Jared D; Svensen, Nina; Jaffrey, Samie R

    2014-11-19

    Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA-fluorophore complexes would extend the utility of this technology, the identification of novel RNA-fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA-fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.

  18. Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Widengren, Jerker; Mets, Ülo; Rigler, Rudolf

    1999-12-01

    GFPs are upon excitation influenced by many different photophysical and photochemical processes effective over a very broad time scale. Much effort has been spent to investigate these processes. However, in the microsecond to millisecond time-range many processes still remain to be further characterized. This time-range can be conveniently covered by FCS, and is used here to study the photodynamical behaviour of wild-type (WT) and a F64L S65T mutant (BioST) of GFP. In addition to intersystem crossing to the triplet state, additional photophysical processes are seen, showing identical fluctuations in fluorescence to those found for a reversible photo-induced isomerization process, as well as fluctuations, not influenced by the electronic state of the chromophore unit. In the nanosecond time-range a contribution to the fluorescence correlation function is observed which can be attributed to rotational diffusion, suggesting a convenient way to measure rotational diffusion of proteins expressed with GFP on a microscopic scale.

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

    PubMed

    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.

  20. Rotation of plasma membrane proteins measured by polarized fluorescence depletion

    NASA Astrophysics Data System (ADS)

    Barisas, B. George; Rahman, Noorul A.; Yoshida, Thomas M.; Roess, Deborah A.

    1990-05-01

    We have implemented a new laser microscopic method, polarized fluorescence depletion (PFD), for measuring the rotational dynamics of functional membrane proteins on individual, microscopically selected cells under physiological conditions. This method combines the long lifetimes of triplet-state probes with the sensitivity of fluorescence detection to measure macromolecular rotational correlation times from 10 microsec to > 1 ms. As examples, the rotational correlation time of Fc receptors (FcR) on the surface of 2H3 rat basophilic leukemia cells is 79.9 4.4 microsec at 4°C when labeled with eosin conjugates of IgE. This value is consistent with the known 100 kDa receptor size. When labeled with intact F4 anti-FcR monoclonal antibody, the rotational correlation time for FcER is increased about 2-fold to 170.8 +/- 6.5 microsec, consistent with receptor dimer formation on the plasma membrane and with the ability of this antibody to form FcER dimers on 2H3 cell surfaces. We have also examined the rotational diffusion of the luteinizing hormone receptor on plasma membranes of small ovine luteal cells. Luteinizing hormone receptors (LHR), when occupied by ovine luteinizing hormone (oLH), have a rotational correlation time of 20.5 +/- 0.1 microsec at 4°C. When occupied by human chorionic gonadotropin (hCG), LHR have a rotational correlation time of 46.2 +/- 0.4 microsec suggesting that binding of hCG triggers additional LHR interactions with plasma membrane proteins. Together these studies suggest the utility of PFD measurements in assessing molecular size and molecular association of membrane proteins on individual cells. Relative advantages of time- and frequency-domain implementations of PFD are also discussed.

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

    DOEpatents

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-16

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

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

    PubMed

    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.

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

    PubMed Central

    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

  7. Fluorescence turn-on sensing of protein based on mannose functionalized perylene bisimides and its fluorescence imaging.

    PubMed

    Wang, Ke-Rang; An, Hong-Wei; Rong, Rui-Xue; Cao, Zhi-Ran; Li, Xiao-Liu

    2014-08-15

    A new water-soluble glycocluster based on perylene bisimides PBI-12-Man has been designed and synthesized, and its specific and selective binding property with Concanavalin A (Con A) has been investigated by fluorescence spectroscopy and circular dichroism (CD) spectroscopy, which showed strong binding affinity for Con A with the binding constant of 8.2×10(5)M(-1) for monomeric mannose unit, two orders of magnitude higher than the corresponding monosaccharide ligand. Most interestingly, a fluorescence enhancement of PBI-12-Man was observed upon binding with Con A because of deaggregation of the self-assembly of PBI-12-Man induced by carbohydrate-protein interaction, and the further study of the fluorescence enhancement with macrophage cells showed that PBI-12-Man as a biocompatible agent had fluorescence imaging of the surface mannose receptor of the cells. Such fluorescence turn-on sensing of protein based on carbohydrate-protein interactions would facilitate the development of new protein-specific fluorescent probe for diagnosis and molecular imaging under live cell conditions.

  8. Quenching of photoexcited states of the proteins chromophores and introduced into the protein macromolecules fluorescent probes by heavy metal ions

    NASA Astrophysics Data System (ADS)

    Melnikov, A. G.; Dyachuk, O. A.; Melnikov, G. V.

    2015-03-01

    We have studied the processes of quenching of photoexcited states of fluorescent probes and quenching of the fluorescence of the chromophores of human serum albumin (HSA) by heavy metal ions (HM): cations Tl+, Pb2+, Cu2+, Cd2+, and the anion of iodine (I-). We used the dye from xanthene series - eosin as a fluorescent probe. By quenching of the fluorescence of protein chromophores we found an influence of HM on the structure of proteins, resulting in a shift of the peak of the fluorescence of HSA tryptophanyl. This can be explained by proteins denaturation under the influence of heavy metals and penetration of water into the inner environment of HSA tryptophan. It was established that the constant of the quenching of the probe phosphorescence is much higher than the fluorescence, which is explained by significantly longer lifetime of the photoexcited states of fluorescent probes in the triplet state than in the singlet.

  9. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  10. Dual fluorescence detection of protein and RNA in Drosophila tissues

    PubMed Central

    Toledano, Hila; D’Alterio, Cecilia; Loza-Coll, Mariano; Jones, D Leanne

    2015-01-01

    Detection of RNAs by in situ hybridization (ISH) is a well-established technique that permits the study of specific RNA expression patterns in tissues; however, not all tissues are equally amenable to staining using the same procedure. Here we describe a protocol that combines whole-mount immunofluorescence (IF) and fluorescence in situ hybridization (FISH) for the simultaneous detection of specific RNA transcripts and proteins, greatly enhancing the spatial resolution of RNA expression in complex, intact fly tissues. To date, we have successfully used this protocol in adult testis, larval male gonads, adult intestine and Malpighian tubules. IF is conducted in RNase-free solutions, prior to the harsh conditions of FISH, in order to preserve protein antigenicity within dissected tissues. Separate protocols are described for mRNA and miRNA detection, which are based on robust digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, respectively. The combined IF-FISH procedure can be completed in 2 d for miRNA detection and 4 d for mRNA detection. Although optimized for Drosophila, this IF-FISH protocol should be adaptable to a wide variety of organisms, tissues, antibodies and probes, thus providing a reliable and simple means to compare RNA and protein abundance and localization. PMID:22976352

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2013-01-01

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

  13. Making genes green: creating green fluorescent protein (GFP) fusions with blunt-end PCR products.

    PubMed

    Lo, W; Rodgers, W; Hughes, T

    1998-07-01

    The jellyfish green fluorescent protein (GFP) has proven to be a useful tool in protein localization and trafficking studies. Fused to GFP, a protein of interest can be visualized and tracked in vivo through fluorescence microscopy. However, the process of making these fusion proteins is often tedious and painstaking. Here, we describe a simple and quick method for creating GFP fusion proteins using blunt-end PCR product ligation.

  14. Application of nanosecond pulsed electric fields into HeLa cells expressing enhanced green fluorescent protein and fluorescence lifetime microscopy.

    PubMed

    Awasthi, Kamlesh; Nakabayashi, Takakazu; Ohta, Nobuhiro

    2012-09-13

    An electrode microchamber has been constructed for applying nanosecond pulsed strong electric fields to living cells, and fluorescence lifetime microscopy (FLIM) has been used to investigate the effects of external electric fields on dynamics and function of HeLa cells expressing enhanced green fluorescent protein (EGFP). Both morphological change in cells and reduction of the fluorescence lifetime of EGFP have been observed after application of electric fields having a pulsed width of 50 ns and a strength of 4 MV m(-1), indicating that apoptosis, which is a programmed cell death, was induced by nanosecond pulsed electric fields and that fluorescence lifetime of EGFP decreased along with the induction of apoptosis. The reduction of the fluorescence lifetime occurred before the morphological change, indicating that FLIM provides a sensitive and noninvasive detection of the progress of apoptosis induced by application of nanosecond pulsed electric fields.

  15. Engineering cell-fluorescent ion track hybrid detectors

    PubMed Central

    2013-01-01

    Background The lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al2O3:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). Methods The biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u−1). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information. Results The FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information. Conclusions The Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy

  16. Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae.

    PubMed

    Lee, Sidae; Lim, Wendell A; Thorn, Kurt S

    2013-01-01

    Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.

  17. Fluorescence-based characterization of non-fluorescent transient states of tryptophan – prospects for protein conformation and interaction studies

    PubMed Central

    Hevekerl, Heike; Tornmalm, Johan; Widengren, Jerker

    2016-01-01

    Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies. PMID:27748381

  18. Pharmaceutical and industrial protein engineering: where we are?

    PubMed

    Amara, Amro Abd-Al-Fattah

    2013-01-01

    The huge amount of information, the big number of scientists and their efforts, labs, man/hrs, fund, companies all and others factors build the success of the amazing new branch of genetic engineering the 'protein engineering' (PE). It concerns with the modification of protein structure/function(s) or building protein from scratch. The engineered proteins usually have new criteria(s). Engineering proteins can be mediated on the level of genes or proteins. PE fined its way in different important sectors including industrial, pharmaceutical and medicinal ones. Aspects about PE and its applications will be discussed with this review. The concept, tools, and the industrial applications of the protein, engineered proteins and PE will be under focus. In order to get up to date knowledge about the applications of PE in basic protein and molecular biology, several examples are discussed. PE can play a significant role in different industrial and pharmaceutical sectors if used wisely and selectively.

  19. Protein engineering: a new frontier for biological therapeutics.

    PubMed

    Tobin, Peter H; Richards, David H; Callender, Randolph A; Wilson, Corey J

    2014-01-01

    Protein engineering holds the potential to transform the metabolic drug landscape through the development of smart, stimulusresponsive drug systems. Protein therapeutics are a rapidly expanding segment of Food and Drug Administration approved drugs that will improve clinical outcomes over the long run. Engineering of protein therapeutics is still in its infancy, but recent general advances in protein engineering capabilities are being leveraged to yield improved control over both pharmacokinetics and pharmacodynamics. Stimulus- responsive protein therapeutics are drugs which have been designed to be metabolized under targeted conditions. Protein engineering is being utilized to develop tailored smart therapeutics with biochemical logic. This review focuses on applications of targeted drug neutralization, stimulus-responsive engineered protein prodrugs, and emerging multicomponent smart drug systems (e.g., antibody-drug conjugates, responsive engineered zymogens, prospective biochemical logic smart drug systems, drug buffers, and network medicine applications).

  20. Protein Engineering: A New Frontier for Biological Therapeutics

    PubMed Central

    Tobin, Peter H.; Richards, David H.; Callender, Randolph A.

    2016-01-01

    Protein engineering holds the potential to transform the metabolic drug landscape through the development of smart, stimulus-responsive drug systems. Protein therapeutics are a rapidly expanding segment of Food and Drug Administration approved drugs that will improve clinical outcomes over the long run. Engineering of protein therapeutics is still in its infancy, but recent general advances in protein engineering capabilities are being leveraged to yield improved control over both pharmacokinetics and pharmacodynamics. Stimulus-responsive protein therapeutics are drugs which have been designed to be metabolized under targeted conditions. Protein engineering is being utilized to develop tailored smart therapeutics with biochemical logic. This review focuses on applications of targeted drug neutralization, stimulus-responsive engineered protein prodrugs, and emerging multicomponent smart drug systems (e.g., antibody-drug conjugates, responsive engineered zymogens, prospective biochemical logic smart drug systems, drug buffers, and network medicine applications). PMID:25495737

  1. Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

    PubMed Central

    Kalish, J E; Keller, G A; Morrell, J C; Mihalik, S J; Smith, B; Cregg, J M; Gould, S J

    1996-01-01

    Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity. Images PMID:8670828

  2. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity.

    PubMed

    Goryashchenko, Alexander S; Khrenova, Maria G; Bochkova, Anna A; Ivashina, Tatiana V; Vinokurov, Leonid M; Savitsky, Alexander P

    2015-07-22

    This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb(3+) and from sensitized Tb(3+) to acceptor--the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

  3. Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting.

    PubMed

    Zou, Jiawei; Huang, Xin; Wu, Lei; Chen, Gangyi; Dong, Juan; Cui, Xin; Tang, Zhuo

    2015-12-01

    Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence over Spinach only in vitro. However, aptamer ZT-324 is 36% brighter than Spinach when expressed in E. coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer.

  4. Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions

    PubMed Central

    Sung, Uhna; Sepehri-Rad, Masoud; Piao, Hong Hua; Jin, Lei; Hughes, Thomas; Cohen, Lawrence B.; Baker, Bradley J.

    2015-01-01

    FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different “Nabi1” constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz. PMID:26587834

  5. Photoswitchable red fluorescent protein with a large Stokes shift.

    PubMed

    Piatkevich, Kiryl D; English, Brian P; Malashkevich, Vladimir N; Xiao, Hui; Almo, Steven C; Singer, Robert H; Verkhusha, Vladislav V

    2014-10-23

    A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

  6. Fluorescence of a histidine-modified enhanced green fluorescent protein (EGFP) effectively quenched by copper(II) ions.

    PubMed

    Bálint, Emese-Éva; Petres, Judit; Szabó, Mária; Orbán, Csongor-Kálmán; Szilágyi, László; Ábrahám, Beáta

    2013-03-01

    Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45 °C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 °C) the mutant's Kd is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.

  7. Fluorescence imaging inside an internal combustion engine using tunable excimer lasers.

    PubMed

    Andresen, P; Meijer, G; Schlüter, H; Voges, H; Koch, A; Hentschel, W; Oppermann, W; Rothe, E

    1990-06-01

    Tunable excimer lasers are used to obtain 2-D images of molecular (and some state-specific) density distributions inside a cylinder of a modified four-cylinder in-line engine that has optical access. Natural fluorescence (i.e., without a laser) is used for some OH pictures, normal laser-induced fluorescence (LIF) for those of NO and of the isooctane fuel, and laser-induced predissociative fluorescence (LIPF) for other OH pictures and for those of O(2). Relevant spectroscopy is done to find the laser and fluorescence frequencies needed to measure isolated species. LIPF works well at high pressures, is state specific, and is ideally suited to follow turbulent processes. No similar measurements in engines have been previously reported. Pictures are taken in succeeding engine cycles. Their sequence is either at a particular point of the engine's cycle to show cyclic fluctuations, or at succeeding portions of the cycle to illustrate the progress of the gasdynamics or of the combustion.

  8. Engineering of chaperone systems and of the unfolded protein response

    PubMed Central

    Khan, Saeed U.

    2008-01-01

    Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR. PMID:19003179

  9. Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

    PubMed Central

    Beal, Jacob; Haddock-Angelli, Traci; Gershater, Markus; de Mora, Kim; Lizarazo, Meagan; Hollenhorst, Jim; Rettberg, Randy

    2016-01-01

    We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices. PMID:26937966

  10. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    PubMed Central

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes. PMID:26939886

  11. Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us...

  12. Mutual effects of disorder and order in fusion proteins between intrinsically disordered domains and fluorescent proteins.

    PubMed

    Lotti, Marina; Longhi, Sonia

    2012-01-01

    Intrinsically disordered proteins are being paid an increasing amount of interest due to the understanding of the crucial role that flexible regions play in molecular recognition and in signaling. Accordingly, reports focusing on the structural and functional characterization of intrinsically disordered proteins or regions are growing exponentially. Relatively few studies have however been reported on the mutual effects of ordered and disordered moieties in artificial fusion proteins. In this review, we focus on the few available experimental data based on the use of chimeras in which fluorescent proteins were fused to disordered domains of different lengths, compactness and propensity to form secondary structures. The impact of the artificial fusion on the conformational and functional properties of the resulting proteins is discussed.

  13. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

    2014-02-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

  14. The role of green fluorescent protein (GFP) in transgenic plants to reduce gene silencing phenomena.

    PubMed

    El-Shemy, Hany A; Khalafalla, Mutasim M; Ishimoto, Masao

    2009-01-01

    The green fluorescent protein (GFP) of jellyfish (Aequorea victoria) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. However, sGFP(S65T) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(S65T) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(S65T ) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.

  15. A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

    PubMed Central

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    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. PMID:22792305

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

    PubMed

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    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.

  17. A novel fluorescent protein from the deep-sea anemone Cribrinopsis japonica (Anthozoa: Actiniaria)

    PubMed Central

    Tsutsui, Kenta; Shimada, Eriko; Ogawa, Tomohisa; Tsuruwaka, Yusuke

    2016-01-01

    A fluorescent protein was identified and cloned from the deep-sea anemone Cribrinopsis japonica. Bioluminescence and fluorescence expression were examined by direct observations of live specimens and RNA-Seq analysis. Both approaches revealed a novel green fluorescent protein in the tentacles of the anemone, but bioluminescence was not observed. Behavioural observations revealed that a blue light excited the fluorescence in the tentacles, and initiated a behavioural response whereby the fluorescent tentacles became fully exposed to the blue light. The excitation and emission peaks of C. japonica’s fluorescent protein were at 500 and 510 nm, respectively, which were greener than those reported in homologs. Furthermore, this protein was highly tolerant of increased temperatures and repeated freeze–thaw treatments. The current study presents an example of fluorescence in a deep-sea cnidarian, demonstrating that fluorescent proteins could have important roles, regardless of the presence or absence of strong sunlight. It also demonstrates that this deep-sea fluorescent protein has unique characteristics, including high stability, perhaps as an adaptation to the extreme environment. PMID:27002644

  18. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%

    PubMed Central

    Goedhart, Joachim; von Stetten, David; Noirclerc-Savoye, Marjolaine; Lelimousin, Mickaël; Joosen, Linda; Hink, Mark A.; van Weeren, Laura; Gadella, Theodorus W.J.; Royant, Antoine

    2012-01-01

    Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein. PMID:22434194

  19. Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.

    PubMed

    Reichel, C; Mathur, J; Eckes, P; Langenkemper, K; Koncz, C; Schell, J; Reiss, B; Maas, C

    1996-06-11

    The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.

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

    PubMed Central

    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

  1. A laboratory exercise for visible gel filtration chromatography using fluorescent proteins.

    PubMed

    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, and used in a laboratory exercise to intuitively demonstrate GFC. Different bands, corresponding to RFP, RFP-CFP (RC), YFP-RFP-YFP (YRY), and pyruvate kinase II-GFP (PKG) were well separated on a Superdex 200 column from a 0.5-mL sample. Increasing the sample volume and changing the chromatographic resin to Sephadex G-100 resulted in lower resolution separation. Students enjoyed identifying combinations of colored proteins and found this exercise helpful for understanding the factors that affect GFC resolution.

  2. A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

    PubMed Central

    Abdelfattah, Ahmed S.; Farhi, Samouil L.; Zhao, Yongxin; Brinks, Daan; Zou, Peng; Ruangkittisakul, Araya; Platisa, Jelena; Pieribone, Vincent A.; Ballanyi, Klaus; Cohen, Adam E.

    2016-01-01

    Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue

  3. Redox heme-proteins mediated fluorescence of CdSe/ZnS quantum dots.

    PubMed

    Qin, Lixia; He, Luwei; Ji, Congcong; Li, Xiangqing; Kang, Shi-Zhao; Mu, Jin

    2014-04-05

    The redox properties of cytochrome c (Cyt c), hemoglobin (Hb) and myoglobin (Mb) were studied based on electrostatic interactions between Thioglycolic acid (TGA) capped CdSe/ZnS quantum dots (QDs) and proteins. Results indicated that only Cyt c quenched the fluorescence of the QDs at pH>8.0. Under the optimized conditions, a significant fluorescence recovery of the QDs' system was observed when the reduced form of Cyt c incubated with TGA capped QDs, however, the reduced state of Hb and Mb resulted in a more fluorescence quenching on the same size of QDs. Interestingly, the fluorescence changes of QDs-proteins could be switched by modulating the redox potentials of proteins-attached QDs. Moreover, only the oxidized Cyt c form was reduced by the generated O2(-) that significantly enhanced the fluorescence of the QDs' system, which was also demonstrated by fluorescence imaging in HeLa cells.

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

    PubMed Central

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

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction. Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors. PMID:23165043

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

    DOE PAGES

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  6. A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

    PubMed

    Cotlet, Mircea; Goodwin, Peter M; Waldo, Geoffrey S; Werner, James H

    2006-01-16

    We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.

  7. Modular protein domains: an engineering approach toward functional biomaterials.

    PubMed

    Lin, Charng-Yu; Liu, Julie C

    2016-08-01

    Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications.

  8. Protein engineering for metabolic engineering: current and next-generation tools

    PubMed Central

    Marcheschi, Ryan J.; Gronenberg, Luisa S.; Liao, James C.

    2014-01-01

    Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically-produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. This article reviews advances of selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use, produce non-natural amino acids, alcohols, and carboxylic acids, and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes. PMID:23589443

  9. Protein engineering for metabolic engineering: Current and next-generation tools

    SciTech Connect

    Marcheschi, RJ; Gronenberg, LS; Liao, JC

    2013-04-16

    Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. We review advances in selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use; produce non-natural amino acids, alcohols, and carboxylic acids; and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes.

  10. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

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

  11. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  12. The Genetic Engineering of Motor Proteins

    NASA Astrophysics Data System (ADS)

    Hartz, Rachael M.

    Molecular motors are a remarkable feature within living organisms that are responsible for directional mechanical motion, which is driven by adenosine triphosphate (ATP) hydrolysis. Actin-binding molecular motors are of specific interest in the field of nanotechnology as filamentous actin is capable of carrying cargo, such as quantum dots, while it is translocated along a motor coated surface. The binding regions of motor proteins, which are known to interact with actin, such as Myosin, have been thoroughly examined and identified. Rapid genetic engineering of the ATP-hydrolyzing enzyme, adenosine kinase, to incorporate these binding regions is possible through the use of site- directed mutagenesis. The sequences, which were mutated into the ADK wt gene, were incorporated in an unstructured loop region. During the phosphate transfer, the mutants switch between open and closed conformational states. The binding affinity of the sequences to the actin is altered during this conformational switch, thus causing the motor to move along actin filament. The ADK mutants and their interaction with filamentous actin was monitored by an in vitro motility assay. Two different mutants of ADK were found to have retained enzymatic functionality after the mutagenesis as well as function as actin-based motor proteins.

  13. Fluorescence characteristics of the fuel tracers triethylamine and trimethylamine for the investigation of fuel distribution in internal combustion engines.

    PubMed

    Lind, Susanne; Aßmann, Simon; Zigan, Lars; Will, Stefan

    2016-03-01

    Laser-induced fluorescence based on fuel tracers like amines is a suitable measurement technique for mixing studies in internal combustion (IC) engines. Triethylamine has often been used in gasoline IC engines; however, no detailed fluorescence characterization for excitation at 263 or 266 nm is available. Trimethylamine (TMA) exhibits high potential as a gaseous fuel tracer but little information about TMA fluorescence is currently available. A picosecond laser source combined with a streak camera equipped with a spectrograph was used to determine the spectral fluorescence emission and fluorescence decay time of both tracers. The tracers were investigated at various temperatures and pressures in a calibration cell with nitrogen as bath gas. The results provide an in-depth understanding of the fluorescence characteristics of both tracers and allow assessment of their application to the investigation of fuel distribution in IC engines.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    PubMed

    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.

  16. Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels.

    PubMed

    Kaushik, Shubhangi; Prokop, Zbynek; Damborsky, Jiri; Chaloupkova, Radka

    2017-01-01

    Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method's application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as α/β-hydrolases containing > 100 000 protein sequences based on the Pfam database.

  17. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

    PubMed Central

    Hostettler, Lola; Grundy, Laura; Käser-Pébernard, Stéphanie; Wicky, Chantal; Schafer, William R.; Glauser, Dominique A.

    2017-01-01

    The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression. PMID:28108553

  18. Rewiring Multidomain Protein Switches: Transforming a Fluorescent Zn(2+) Sensor into a Light-Responsive Zn(2+) Binding Protein.

    PubMed

    Aper, Stijn J A; Merkx, Maarten

    2016-07-15

    Protein-based sensors and switches provide attractive tools for the real-time monitoring and control of molecular processes in complex biological environments. Fluorescent sensor proteins have been developed for a wide variety of small molecules, but the construction of genetically encoded light-responsive ligand binding proteins remains mostly unexplored. Here we present a generic approach to reengineer a previously developed FRET-based Zn(2+) sensor into a light-activatable Zn(2+) binding protein using a design strategy based on mutually exclusive domain interactions. These so-called VividZn proteins consist of two light-responsive Vivid domains that homodimerize upon illumination with blue light, thus preventing the binding of Zn(2+) between two Zn(2+) binding domains, Atox1 and WD4. Following optimization of the linker between WD4 and the N-terminus of one of the Vivid domains, VividZn variants were obtained that show a 9- to 55-fold decrease in Zn(2+) affinity upon illumination, which is fully reversible following dark adaptation. The Zn(2+) affinities of the switch could be rationally tuned between 1 pM and 2 nM by systematic variation of linker length and mutation of one of the Zn(2+) binding residues. Similarly, introduction of mutations in the Vivid domains allowed tuning of the switching kinetics between 10 min and 7 h. Low expression levels in mammalian cells precluded the demonstration of light-induced perturbation of cytosolic Zn(2+) levels. Nonetheless, our results firmly establish the use of intramolecular Vivid dimerization as an attractive light-sensitive input module to rationally engineer light-responsive protein switches based on mutually exclusive domain interactions.

  19. 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.

    PubMed

    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

  20. Protein cyclization enhanced thermostability and exopeptidase-resistance of green fluorescent protein.

    PubMed

    Zhao, Zhonglin; Ma, Xin; Li, Liang; Zhang, Wei; Ping, Shuzhen; Xu, Ming-Qun; Lin, Min

    2010-03-01

    A mutant of green fluorescent protein (GFPmut3*) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of GFPmut3* was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear GFPmut3*. The circular GFPmut3* was 5 degrees C more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular GFPmut3* also displayed increased relative fluorescence intensity. In addition, chemical stability of GFPmut3* against GdnHCl revealed more stability of the circular form compared with the linear form.

  1. Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.

    PubMed

    Ahn, Junhyoung; Shin, Yong-Beom; Lee, JaeJong; Kim, Min-Gon

    2015-09-15

    The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).

  2. Coupling Infusion and Gyration for the Nanoscale Assembly of Functional Polymer Nanofibers Integrated with Genetically Engineered Proteins

    PubMed Central

    Zhang, Siqi; Karaca, Banu Taktak; VanOosten, Sarah Kay; Yuca, Esra; Mahalingam, Suntharavathanan

    2015-01-01

    Nanofibers featuring functional nanoassemblies show great promise as enabling constituents for a diverse range of applications in areas such as tissue engineering, sensing, optoelectronics, and nanophotonics due to their controlled organization and architecture. An infusion gyration method is reported that enables the production of nanofibers with inherent biological functions by simply adjusting the flow rate of a polymer solution. Sufficient polymer chain entanglement is obtained at Berry number > 1.6 to make bead‐free fibers integrated with gold nanoparticles and proteins, in the diameter range of 117–216 nm. Integration of gold nanoparticles into the nanofiber assembly is followed using a gold‐binding peptide tag genetically conjugated to red fluorescence protein (DsRed). Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the engineered red fluorescence protein with the nanofibers. The gold nanoparticle decorated nanofibers having red fluorescence protein as an integral part keep their biological functionality including copper‐induced fluorescence quenching of the DsRed protein due to its selective Cu+2 binding. Thus, coupling the infusion gyration method in this way offers a simple nanoscale assembly approach to integrate a diverse repertoire of protein functionalities into nanofibers to generate biohybrid materials for imaging, sensing, and biomaterial applications. PMID:26033345

  3. Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus

    PubMed Central

    Bomati, Erin K.; Haley, Joy E.; Noel, Joseph P.; Deheyn, Dimitri D.

    2014-01-01

    The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level. PMID:24968921

  4. Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus.

    PubMed

    Bomati, Erin K; Haley, Joy E; Noel, Joseph P; Deheyn, Dimitri D

    2014-06-27

    The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level.

  5. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

    PubMed Central

    Chu, Jun; Haynes, Russell D; Corbel, Stéphane Y; Li, Pengpeng; González-González, Emilio; Burg, John S; Ataie, Niloufar J; Lam, Amy J; Cranfill, Paula J; Baird, Michelle A; Davidson, Michael W; Ng, Ho-Leung; Garcia, K Christopher; Contag, Christopher H; Shen, Kang; Blau, Helen M; Lin, Michael Z

    2014-01-01

    A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail. PMID:24633408

  6. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system

    PubMed Central

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2012-01-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins. PMID:16603603

  7. In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy.

    PubMed

    Shang, Li; Nienhaus, G Ulrich

    2017-02-21

    Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the "protein corona", forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with

  8. A reliability measure of protein-protein interactions and a reliability measure-based search engine.

    PubMed

    Park, Byungkyu; Han, Kyungsook

    2010-02-01

    Many methods developed for estimating the reliability of protein-protein interactions are based on the topology of protein-protein interaction networks. This paper describes a new reliability measure for protein-protein interactions, which does not rely on the topology of protein interaction networks, but expresses biological information on functional roles, sub-cellular localisations and protein classes as a scoring schema. The new measure is useful for filtering many spurious interactions, as well as for estimating the reliability of protein interaction data. In particular, the reliability measure can be used to search protein-protein interactions with the desired reliability in databases. The reliability-based search engine is available at http://yeast.hpid.org. We believe this is the first search engine for interacting proteins, which is made available to public. The search engine and the reliability measure of protein interactions should provide useful information for determining proteins to focus on.

  9. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.

    PubMed

    Jin, Lei; Han, Zhou; Platisa, Jelena; Wooltorton, Julian R A; Cohen, Lawrence B; Pieribone, Vincent A

    2012-09-06

    Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100 mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites.

  10. DNA-directed assembly of supramolecular fluorescent protein energy transfer systems.

    PubMed

    Kukolka, Florian; Schoeps, Oliver; Woggon, Ulrike; Niemeyer, Christof M

    2007-01-01

    Fluorescent proteins with a wide variety of physicochemical properties have evolved in the past few years. The use of these proteins for applications in biomolecular nanosciences requires their precise positioning at the nanometer length scale. To address this challenge, we report here on the self-organization of DNA-tagged fluorescent probes to construct a set of photofunctional supramolecular complexes which include the enhanced yellow fluorescent protein (EYFP). The optical functionality is based on the strongly distance dependent fluorescence resonance energy transfer (FRET), occurring between the donor (EYFP) and an acceptor fluorophore, i.e., the fluorescent dye Atto647. The photophysical properties of four bimolecular FRET complexes, each possessing a well-defined donor-acceptor distance defined by the length of the interconnecting DNA backbone, are investigated by two-dimensional photoluminescence excitation spectroscopy (2D-PLE).

  11. Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans

    PubMed Central

    Hiatt, Susan M.; Shyu, Y. John; Duren, Holli M.; Hu, Chang-Deng

    2008-01-01

    Protein interactions are essential components of signal transduction in cells. With the progress in genome-wide yeast two hybrid screens and proteomics analyses, many protein interaction networks have been generated. These analyses have identified hundreds and thousands of interactions in cells and organisms, creating a challenge for further validation under physiological conditions. The bimolecular fluorescence complementation (BiFC) assay is such an assay that meets this need. The BiFC assay is based on the principle of protein fragment complementation, in which two non-fluorescent fragments derived from a fluorescent protein are fused to a pair of interacting partners. When the two partners interact, the two non-fluorescent fragments are brought into proximity and an intact fluorescent protein is reconstituted. Hence, the reconstituted fluorescent signals reflect the interaction of two proteins under study. Over the past six years, the BiFC assay has been used for visualization of protein interactions in living cells and organisms, including our application of the BiFC assay to the transparent nematode Caenorhabditis elegans. We have demonstrated that BiFC analysis in C. elegans provides a direct means to identify and validate protein interactions in living worms and allows visualization of temporal and spatial interactions. Here we provide a guideline for the implementation of BiFC analysis in living worms and discuss the factors that are critical for BiFC analysis. PMID:18586101

  12. Noninvasive imaging in vivo with fluorescent proteins from centimeters to micrometers

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Jiang, Ping; Al-Zaid, Manal; Hoffman, Robert M.

    2008-02-01

    Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Our laboratory pioneered in vivo imaging with fluorescent proteins (1) including noninvasive whole-body imaging (2). Whole-body imaging with fluorescent proteins depends in large part on the brightness of the protein. Brighter, red-shifted proteins can make whole-body imaging more sensitive due to reduced absorption by tissues and less scatter. Non-invasive imaging with fluorescent proteins has been shown to be able to quantitatively track tumor growth and metastasis, gene expression, angiogenesis, and bacterial infection (3) even at subcellular resolution depending on the position of the cells in the animal. Interference by skin autofluorescence is kept to a minimum with the use of proper filters. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow as well as drug response. This imageable technology should lead to many new insights of in vivo cancer cell biology.

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

    PubMed

    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.

  14. Simultaneous laser-induced fluorescence and Raman imaging inside a hydrogen engine.

    PubMed

    Engel, Sascha Ronald; Koch, Peter; Braeuer, Andreas; Leipertz, Alfred

    2009-12-10

    We report on the simultaneous and two-dimensional measurement of laser-induced fluorescence (LIF) and Raman scattering (Ramanography) applied inside a hydrogen internal combustion (IC) engine. Two different LIF tracer molecules, triethylamine (TEA) and trimethylamine (TMA), were used for the LIF experiments. The LIF and Raman results were found to be in very good agreement. The simultaneous application of Ramanography and LIF imaging indicated that TMA is the more suitable LIF tracer molecule, compared to TEA.

  15. Direct Observation of Oil Consumption Mechanisms in a Production Spark Ignition Engine Using Fluorescence Techniques

    DTIC Science & Technology

    1994-05-01

    investigated for different piston ring end-gap configurations. A radiotracer was used to perform direct measurement of the oil consumption while Laser- induced ...and Instrumentation . . . . 43 3.1 General .......... ................... .. 43 3.2 Engine Description ....... ............. .. 43 3.3 Laser Induced ...Duty Diesels h" Non-dimensionalized Ah. k Proportionality constant for surface tension 11 (NI [mK]). Kpa kilo-Pascal LIF Laser Induced Fluorescence

  16. Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation

    PubMed Central

    Grimley, Joshua S.; Li, Li; Wang, Weina; Wen, Lei; Beese, Lorena S.

    2013-01-01

    We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using a cell-free protein engineering automation methodology that bypasses gene cloning, we iteratively constructed, produced, and assayed hundreds of mutations in binding-site residues to identify improvements in Clomeleon, a first-generation, suboptimal sensor. Structural analysis revealed that these improvements involve halide contacts and distant side chain rearrangements. The development of optogenetic sensors that respond to neural activity enables cellular tracking of neural activity using optical, rather than electrophysiological, signals. Construction of such sensors using in vitro protein engineering establishes a powerful approach for developing new probes for brain imaging. PMID:24107961

  17. Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2013-01-01

    This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain. PMID:23351712

  18. Structural basis for the fast maturation of Arthropoda green fluorescent protein.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew E; Egorov, Nikolay S; Zaraisky, Andrey G; Yampolsky, Ilya V; Merzlyak, Ekaterina M; Shkoporov, Andrey N; Sander, Ian; Lukyanov, Konstantin A; Chudakov, Dmitriy M

    2006-10-01

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

  19. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors.

    PubMed

    Kilpatrick, Laura E; Hill, Stephen J

    2016-04-15

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand-receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

  20. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

    PubMed Central

    Kilpatrick, Laura E.; Hill, Stephen J.

    2016-01-01

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties. PMID:27068980

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

    PubMed

    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.

  2. [Fluorescent proteins: physical-chemical properties and application in cell biology].

    PubMed

    Stepanenko, O V; Verkhusha, V V; Kuznetsova, I M; Turoverov, K K

    2007-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. At present novel naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by stability, value of quantum yield, absorption and fluorescence spectra position and photochemical properties. GFP-like proteins are the fast growing family. This review is an attempt to characterize the main groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

  3. Molecular engineering of two-photon fluorescent probes for bioimaging applications.

    PubMed

    Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

    2017-03-22

    During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

  4. Molecular engineering of two-photon fluorescent probes for bioimaging applications

    NASA Astrophysics Data System (ADS)

    Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

    2017-03-01

    During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

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

    PubMed

    Lai, Hsien-Tsung; Chiang, Cheng-Ming

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

  6. Polyacrylamide hydrogel encapsulated E. coli expressing metal-sensing green fluorescent protein as a potential tool for copper ion determination

    PubMed Central

    Tantimongcolwat, Tanawut; Isarankura-Na-Ayudhya, Chartchalerm; Srisarin, Apapan; Galla, Hans-Joachim; Prachayasittikul, Virapong

    2014-01-01

    A simple, inexpensive and field applicable metal determination system would be a powerful tool for the efficient control of metal ion contamination in various sources e.g. drinking-water, water reservoir and waste discharges. In this study, we developed a cell-based metal sensor for specific and real-time detection of copper ions. E. coli expressing metal-sensing green fluorescent protein (designated as TG1/(CG)6GFP and TG1/H6CdBP4GFP) were constructed and served as a metal analytical system. Copper ions were found to exert a fluorescence quenching effect, while zinc and cadmium ions caused minor fluorescence enhancement in the engineered bacterial suspension. To construct a user-friendly and reagentless metal detection system, TG1/H6CdBP4GFP and TG1/(CG)6GFP were encapsulated in polyacrylamide hydrogels that were subsequently immobilized on an optical fiber equipped with a fluorescence detection module. The sensor could be applied to measure metal ions by simply dipping the encapsulated bacteria into a metal solution and monitoring fluorescence changes in real time as a function of the metal concentration in solution. The sensor system demonstrated high specificity toward copper ions. The fluorescence intensities of the encapsulated TG1/(CG)6GFP and TG1/H6CdBP4GFP were quenched by approximately 70 % and 80 % by a high-dose of copper ions (50 mM), respectively. The level of fluorescence quenching exhibited a direct correlation with the copper concentration, with a linear correlation coefficient (r) of 0.99. The cell-based metal sensor was able to efficiently monitor copper concentrations ranging between 5 M and 50 mM, encompassing the maximum allowed copper contamination in drinking water (31.15 M) established by the WHO. Furthermore, the cell-based metal sensor could undergo prolonged storage for at least 2 weeks without significantly influencing the copper sensitivity. PMID:26417267

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

    PubMed

    Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

    2013-01-01

    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).

  8. Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins.

    PubMed

    Canto, Tomas; Palukaitis, Peter

    2005-04-01

    The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubules.

  9. Metal-enhanced intrinsic fluorescence of proteins and label-free bioassays

    NASA Astrophysics Data System (ADS)

    Ray, Krishanu; Szmacinski, Henryk; Chowdhury, Mustafa H.; Lakowicz, Joseph R.

    2010-02-01

    Most of the applications of fluorescence require the use of labeled drugs and labeled biomolecules. Due to the need of labeling biomolecules with extrinsic fluorophores, there is a rapidly growing interest in methods which provide label-free detection (LFD). Proteins are highly fluorescent, which is due primarily to tryptophan residues. However, since most proteins contain tryptophan, this emission is not specific for proteins of interest in a biological sample. This is one of the reasons of not utilizing intrinsic tryptophan emission from proteins to detect specific proteins. Here, we present the intrinsic fluorescence for several proteins bound to the silver or aluminum metal nanostructured surfaces. We demonstrate the metal enhanced fluorescence (MEF) of proteins with different numbers of tryptophan residues. Large increases in fluorescence intensity and decreases in lifetime provide the means of direct detection of bound protein without separation from the unbound. We present specific detection of individual types of proteins and measure the binding kinetics of proteins such as IgG and streptavidin. Additionally, specific detection of IgG and streptavidin has been accomplished in the presence of large concentrations of other proteins in sample solutions. These results will allow design of surface-based assays with biorecognitive layer that specifically bind the protein of interest and thus enhance its intrinsic fluorescence. The present study demonstrates the occurrence of MEF in the UV region and thus opens new possibilities to study tryptophan-containing proteins without labeling with longer wavelength fluorophores and provides an approach to label-free detection of biomolecules.

  10. Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein.

    PubMed

    Huang, Yao-Ming; Nayak, Sasmita; Bystroff, Christopher

    2011-11-01

    Several versions of split green fluorescent protein (GFP) fold and reconstitute fluorescence, as do many circular permutants, but little is known about the dependence of reconstitution on circular permutation. Explored here is the capacity of GFP to fold and reconstitute fluorescence from various truncated circular permutants, herein called "leave-one-outs" using a quantitative in vivo solubility assay and in vivo reconstitution of fluorescence. Twelve leave-one-out permutants are discussed, one for each of the 12 secondary structure elements. The results expand the outlook for the use of permuted split GFPs as specific and self-reporting gene encoded affinity reagents.

  11. Flame front imaging in an internal-combustion engine simulator by laser-induced fluorescence of acetaldehyde.

    PubMed

    Arnold, A; Becker, H; Suntz, R; Monkhouse, P; Wolfrum, J; Maly, R; Pfister, W

    1990-08-01

    Acetaldehyde has been used as a fluorescent dopant for two-dimensional imaging of the flame front in an internalcombustion-engine simulator. The molecule was excited with a XeCl-laser-light sheet at 308 nm, and broadband fluorescence centered at 400 nm was detected. In this way, the flame front could be marked by mapping regions of unburned gas. Also, the intake process into the engine could be followed.

  12. Ultrafast fluorescence dynamics of FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) and its site-directed mutated proteins

    NASA Astrophysics Data System (ADS)

    Chosrowjan, Haik; Taniguchi, Seiji; Mataga, Noboru; Tanaka, Fumio; Todoroki, Daisuke; Kitamura, Masaya

    2008-09-01

    Ultrafast fluorescence dynamics of FMN in FMN-binding protein (FMN-bp), and its mutated proteins, W32Y and W32A, were investigated by the fluorescence up-conversion method. Fluorescence lifetimes were 167 fs (96%) and 1.5 ps (4%) in wild-type FMN-bp (WT), and 3.4 ps (23%), 18.2 ps (74%), and 96 ps (3%) at 530 nm in W32Y, and 30.1 ps in W32A. The fluorescence lifetime of W32A, in which Trp-32 was absent, was about 140 times longer than that of WT. Tyr-32 in W32Y was not so effective quencher as Trp-32 in WT. This was explained in terms of different ionization potentials of quenchers and average donor-acceptor distances in the protein.

  13. Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging.

    PubMed

    Ding, Changqin; Zhu, Anwei; Tian, Yang

    2014-01-21

    Nanoparticles are promising scaffolds for applications such as imaging, chemical sensors and biosensors, diagnostics, drug delivery, catalysis, energy, photonics, medicine, and more. Surface functionalization of nanoparticles introduces an additional dimension in controlling nanoparticle interfacial properties and provides an effective bridge to connect nanoparticles to biological systems. With fascinating photoluminescence properties, carbon dots (C-dots), carbon-containing nanoparticles that are attracting considerable attention as a new type of quantum dot, are becoming both an important class of imaging probes and a versatile platform for engineering multifunctional nanosensors. In order to transfer C-dots from proof-of-concept studies toward real world applications such as in vivo bioimaging and biosensing, careful design and engineering of C-dot probes is becoming increasingly important. A comprehensive knowledge of how C-dot surfaces with various properties behave is essential for engineering C-dots with useful imaging properties such as high quantum yield, stability, and low toxicity, and with desirable biosensing properties such as high selectivity, sensitivity, and accuracy. Several reviews in recent years have reported preparation methods and properties of C-dots and described their application in biosensors, catalysis, photovoltatic cells, and more. However, no one has yet systematically summarized the surface engineering of C-dots, nor the use of C-dots as fluorescent nanosensors or probes for in vivo imaging in cells, tissues, and living organisms. In this Account, we discuss the major design principles and criteria for engineering the surface functionality of C-dots for biological applications. These criteria include brightness, long-term stability, and good biocompatibility. We review recent developments in designing C-dot surfaces with various functionalities for use as nanosensors or as fluorescent probes with fascinating analytical performance

  14. Tolerance of a knotted near infrared fluorescent protein to random circular permutation

    PubMed Central

    Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

    2016-01-01

    Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

  15. A sensitive method based on fluorescence-detected circular dichroism for protein local structure analysis.

    PubMed

    Nehira, Tatsuo; Ishihara, Kaoru; Matsuo, Koichi; Izumi, Shunsuke; Yamazaki, Takeshi; Ishida, Atsuhiko

    2012-11-15

    We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca(2+) and/or interacting with binding peptides. Because FDCD appears to reflect the protein's local structure around the fluorophore, it may provide a useful means for "pinpoint analysis" of protein structures.

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

    PubMed

    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.

  17. The role of protein characteristics in the formation and fluorescence of Au nanoclusters

    NASA Astrophysics Data System (ADS)

    Xu, Yaolin; Sherwood, Jennifer; Qin, Ying; Crowley, Dorothy; Bonizzoni, Marco; Bao, Yuping

    2014-01-01

    Protein-encapsulated gold nanoclusters have shown many advantages over other gold nanocluster systems, including green synthesis, biocompatibility, high water solubility, and the ease of further conjugation. In this article, we systematically investigated the effects of the protein size and amino acid content on the formation and fluorescent properties of gold nanoclusters using four model proteins (bovine serum albumin, lysozyme, trypsin, and pepsin). We discovered that the balance of amine and tyrosine/tryptophan containing residues was critical for the nanocluster formation. Protein templates with low cysteine contents caused blue shifts in the fluorescent emissions and difference in fluorescent lifetimes of the gold nanoclusters. Furthermore, the protein size was found to be a critical factor for the photostability and long-term stability of gold nanoclusters. The size of the protein also affected the Au nanocluster behaviour after immobilization.Protein-encapsulated gold nanoclusters have shown many advantages over other gold nanocluster systems, including green synthesis, biocompatibility, high water solubility, and the ease of further conjugation. In this article, we systematically investigated the effects of the protein size and amino acid content on the formation and fluorescent properties of gold nanoclusters using four model proteins (bovine serum albumin, lysozyme, trypsin, and pepsin). We discovered that the balance of amine and tyrosine/tryptophan containing residues was critical for the nanocluster formation. Protein templates with low cysteine contents caused blue shifts in the fluorescent emissions and difference in fluorescent lifetimes of the gold nanoclusters. Furthermore, the protein size was found to be a critical factor for the photostability and long-term stability of gold nanoclusters. The size of the protein also affected the Au nanocluster behaviour after immobilization. Electronic supplementary information (ESI) available See DOI: 10

  18. Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

    PubMed

    Peter, Sébastien; Oven-Krockhaus, Sven Zur; Veerabagu, Manikandan; Rodado, Virtudes Mira; Berendzen, Kenneth W; Meixner, Alfred J; Harter, Klaus; Schleifenbaum, Frank E

    2017-03-10

    The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly

  19. In Vitro Dynamic Visualization Analysis of Fluorescently Labeled Minor Capsid Protein IX and Core Protein V by Simultaneous Detection

    PubMed Central

    Ugai, Hideyo; Wang, Minghui; Le, Long P.; Matthews, David A.; Yamamoto, Masato; Curiel, David T.

    2009-01-01

    Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation to human clinical trials requires careful evaluation of these viral characteristics. While the function of the adenovirus proteins have been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints which prevent adequate tracking of the adenovirus particles after infection. Fluorescent labeling of the adenoviral particles is one new strategy designed to directly analyze dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling technique of adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in live analysis of adenovirus as compared to a single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX-mRFP1) and a green fluorescent minor core protein V (pV-EGFP), resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, the growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed approximately 150-fold reduced production of the viral progeny at 48 hours post-infection (h.p.i.) as compared to Ad5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and the nucleolus, respectively, at 18 h.p.i. These proteins were observed in the nucleus during the late stage of infection and the relocalization of the proteins was observed in an adenoviral replication-dependent manner. These results indicate that the simultaneous detection of adenovirus using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells, and monitoring viral spread, which will be required for complete evaluation of oncolytic adenoviruses. PMID:19853616

  20. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.

    PubMed

    Brakemann, Tanja; Stiel, Andre C; Weber, Gert; Andresen, Martin; Testa, Ilaria; Grotjohann, Tim; Leutenegger, Marcel; Plessmann, Uwe; Urlaub, Henning; Eggeling, Christian; Wahl, Markus C; Hell, Stefan W; Jakobs, Stefan

    2011-09-11

    Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

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

    NASA Astrophysics Data System (ADS)

    Zhang, Yi; Keegan, Gemma L.; Stranik, Ondrej; Brennan-Fournet, Margaret E.; McDonagh, Colette

    2015-07-01

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

  2. The use of fluorescence methods to monitor unfolding transitions in proteins.

    PubMed Central

    Eftink, M R

    1994-01-01

    This article discusses several strategies for the use steady-state and time-resolved fluorescence methods to monitor unfolding transitions in proteins. The assumptions and limitations of several methods are discussed. Simulations are presented to show that certain fluorescence observables directly track the population of states in an unfolding transition, whereas other observables skew the transition toward the dominant fluorescing species. Several examples are given, involving the unfolding of Staphylococcal aureus nuclease A, in which thermodynamic information is obtained for the temperature and denaturant induced transitions in this protein. PMID:8161701

  3. Real-time imaging of dynamic translocation of fluorescent proteins at synapses in living neurons.

    NASA Astrophysics Data System (ADS)

    De Koninck, Paul

    2003-02-01

    To understand the biology of living cells, such as the neurons in our brain, we focus on the molecular signaling interactions that proteins perform intracellularly. We have been studying the behavior of an enzyme, termed 'CaMKII', inside living neurons maintained in tissue culture. This enzyme plays a critical role in the control of synaptic transmission. Such role may involve the dynamic translocation of the enzyme at synaptic sites upon specific stimuli. To study this translocation, we use a cellular imaging technique that allows us to monitor the movement and targeting of CaMKII tagged by genetic engineering with a green fluorescent protein (GFP). We find that the enzyme translocates within seconds to synapses upon synaptic activation by neurotransmitter application. Our approach has lead to several key findings on the regulation of CaMKII translocation to the synapse and on its potential role in synaptic plasticity. However, several new advances in photonics and image analysis, which we will implement in our laboratory, will greatly help pushing the limits of our resolution of such type molecular event in living cells.

  4. Labeling cell surface GPIs and GPI-anchored proteins through cell metabolic engineering with artificial inositol derivatives**

    PubMed Central

    Guo, Zhongwu

    2015-01-01

    Protein GPI anchorage to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. This paper developed an effective strategy for metabolic engineering of cell surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins on live cells were then tagged with biotin via click reaction and with a fluorescent molecule. The strategy can be used to label GPI-anchored proteins with various tags for biological studies. PMID:26102235

  5. Molecular basis of the light-driven switching of the photochromic fluorescent protein Padron.

    PubMed

    Brakemann, Tanja; Weber, Gert; Andresen, Martin; Groenhof, Gerrit; Stiel, Andre C; Trowitzsch, Simon; Eggeling, Christian; Grubmüller, Helmut; Hell, Stefan W; Wahl, Markus C; Jakobs, Stefan

    2010-05-07

    Reversibly switchable fluorescent proteins can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with the light of two different wavelengths. The molecular basis of the switching process remains a controversial topic. Padron0.9 is a reversibly switchable fluorescent protein with "positive" switching characteristics, exhibiting excellent spectroscopic properties. Its chromophore is formed by the amino acids Cys-Tyr-Gly. We obtained high resolution x-ray structures of Padron0.9 in both the fluorescent and the nonfluorescent states and used the structural information for molecular dynamics simulations. We found that in Padron0.9 the chromophore undergoes a cis-trans isomerization upon photoswitching. The molecular dynamics simulations clarified the protonation states of the amino acid residues within the chromophore pocket that influence the protonation state of the chromophore. We conclude that a light driven cis-trans isomerization of the chromophore appears to be the fundamental switching mechanism in all photochromic fluorescent proteins known to date. Distinct absorption cross-sections for the switching wavelengths in the fluorescent and the nonfluorescent state are not essential for efficient photochromism in fluorescent proteins, although they may facilitate the switching process.

  6. Generation of a fluorescently labeled endogenous protein library in living human cells.

    PubMed

    Sigal, Alex; Danon, Tamar; Cohen, Ariel; Milo, Ron; Geva-Zatorsky, Naama; Lustig, Gila; Liron, Yuvalal; Alon, Uri; Perzov, Natalie

    2007-01-01

    We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100-200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3' rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

    Green fluorescent protein (GFP)-like pigments have been proposed to have beneficial effects on coral photobiology. Here, we investigated the relationships between green fluorescence, coral heating and tissue optics for the massive coral Dipsastraea sp. (previously Favia sp.). We used microsensors to measure tissue scalar irradiance and temperature along with hyperspectral imaging and combined imaging of variable chlorophyll fluorescence and green fluorescence. Green fluorescence correlated positively with coral heating and scalar irradiance enhancement at the tissue surface. Coral tissue heating saturated for maximal levels of green fluorescence. The action spectrum of coral surface heating revealed that heating was highest under red (peaking at 680 nm) irradiance. Scalar irradiance enhancement in coral tissue was highest when illuminated with blue light, but up to 62% (for the case of highest green fluorescence) of this photon enhancement was due to green fluorescence emission. We suggest that GFP-like pigments scatter the incident radiation, which enhances light absorption and heating of the coral. However, heating saturates, because intense light scattering reduces the vertical penetration depth through the tissue eventually leading to reduced light absorption at high fluorescent pigment density. We conclude that fluorescent pigments can have a central role in modulating coral light absorption and heating. PMID:27225857

  9. Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein.

    PubMed

    Hoesl, Michael Georg; Larregola, Maud; Cui, Haissi; Budisa, Nediljko

    2010-10-01

    Aequorea victoria green fluorescent protein and its widely used mutants enhanced green fluorescent protein and enhanced cyan fluorescent protein (ECFP) are ideal target proteins to study protein folding. The spectral signals of their chromophores are directly correlated with the folding status of the surrounding protein matrix. Previous studies revealed that tryptophan at position 57 (Trp57) plays a crucial role for the green fluorescent protein's structural and functional integrity. To precisely dissect its role in ECFP folding, we performed its substitution with the isosteric analogs 4-azatryptophan [(4-Aza)Trp] and 7-azatryptophan [(7-Aza)Trp]. Although Trp is moderately hydrophobic, these isosteric analogs are hydrophilic, which makes them an almost ideal tool to study the role of Trp57 in ECFP folding. We achieved high-level expression of both (4-Aza)Trp-ECFP and (7-Aza)Trp-ECFP. However, great portions (70-90%) of protein samples were insoluble and did not contain a maturated chromophore. All attempts to refold the insoluble protein fractions failed. Nevertheless, low amounts of fully labeled, soluble, chromophore containing fractions with altered spectral features were also isolated and identified. The most probable reason for the high yield of misfolding is the introduction of strong hydrophilicity at position 57 which strongly interferes with productive and efficient folding of ECFP. In addition, the results support a strong correlation between translational kinetics of non-canonical amino acids in the ribosome and in vivo folding of the related modified protein sequence.

  10. Application of cell-surface engineering for visualization of yeast in bread dough: development of a fluorescent bio-imaging technique in the mixing process of dough.

    PubMed

    Maeda, Tatsuro; Shiraga, Seizaburo; Araki, Tetsuya; Ueda, Mitsuyoshi; Yamada, Masaharu; Takeya, Koji; Sagara, Yasuyuki

    2009-07-01

    Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at -30 degrees C and sliced at 10 microm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.

  11. Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry.

    PubMed

    Gao, Xiaoye; Fang, Jie; Xue, Bin; Fu, Linglan; Li, Hongbin

    2016-09-12

    Constructing hydrogels from engineered proteins has attracted significant attention within the material sciences, owing to their myriad potential applications in biomedical engineering. Developing efficient methods to cross-link tailored protein building blocks into hydrogels with desirable mechanical, physical, and functional properties is of paramount importance. By making use of the recently developed SpyCatcher-SpyTag chemistry, we successfully engineered protein hydrogels on the basis of engineered tandem modular elastomeric proteins. Our resultant protein hydrogels are soft but stable, and show excellent biocompatibility. As the first step, we tested the use of these hydrogels as a drug carrier, as well as in encapsulating human lung fibroblast cells. Our results demonstrate the robustness of the SpyCatcher-SpyTag chemistry, even when the SpyTag (or SpyCatcher) is flanked by folded globular domains. These results demonstrate that SpyCatcher-SpyTag chemistry can be used to engineer protein hydrogels from tandem modular elastomeric proteins that can find applications in tissue engineering, in fundamental mechano-biological studies, and as a controlled drug release vehicle.

  12. Control of the blue fluorescent protein with advanced evolutionary pulse shaping

    SciTech Connect

    Tkaczyk, Eric R. Mauring, Koit; Tkaczyk, Alan H.; Krasnenko, Veera; Ye, Jing Yong; Baker, James R.; Norris, Theodore B.

    2008-11-28

    We demonstrate optical coherent control of the two-photon fluorescence of the blue fluorescent protein (BFP), which is of interest in investigations of protein-protein interactions. In addition to biological relevance, BFP represents an interesting target for coherent control from a chemical perspective due to its many components of highly nonexponential fluorescence decay and low quantum yield resulting from excited state isomerization. Using a genetic algorithm with a multiplicative (rather than ratiometric) fitness parameter, we are able to control the ratio of BFP fluorescence to second-harmonic generation without a considerable drop in the maximized signal. The importance of linear chirp and power-scaling on the discrimination process is investigated in detail.

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

    ERIC Educational Resources Information Center

    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. Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

    2013-02-01

    The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.

  15. Fluorescence origin of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) bound to proteins.

    PubMed

    Albani, J-R

    2009-05-01

    6,P-toluidinylnaphthalene-2-sulfonate (TNS) is a highly fluorescent molecule when dissolved in a low polarity medium or when bound to proteins. The aim of the present work is to explain origin of this fluorescence, to find out how the medium (solvent, protein matrix) affects fluorescence observables such as lifetimes and spectra and finally to put into evidence possible relation that exists between these observables and fluorophore structure. To achieve our goal we performed studies on TNS dissolved in ethanol, at high concentrations in water (aggregated form) and bound to proteins. Our experiments allowed us to find out that TNS in the three environments has different structures. Presence of three lifetimes observed in proteins and in water instead of one lifetime found in ethanol can be assigned to the high contact between TNS molecules. Our results are discussed in terms of solvent polarity and interaction within fluorophore molecules bound to proteins.

  16. Parallel Detection of Intrinsic Fluorescence from Peptides and Proteins for Quantification During Mass Spectrometric Analysis

    PubMed Central

    Russell, Jason D.; Hilger, Ryan T.; Ladror, Daniel T.; Tervo, Mark A.; Scalf, Mark; Shortreed, Michael R.; Coon, Joshua J.

    2011-01-01

    Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of the protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular, fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ~300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal was linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa. PMID:21314137

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

    PubMed

    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.

  18. Fluorescence-Detectino Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins

    SciTech Connect

    Kawate,T.; Gouaux, E.

    2006-01-01

    Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.

  19. A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk.

    PubMed

    Aoki, Takashi; Kazama, Hitoshi; Satoh, Marie; Mizuki, Kazuhiro; Watabe, Hiroyuki

    2005-09-01

    When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.

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

    PubMed

    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.

  1. Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution.

    PubMed

    Jones, Laurie J; Haugland, Richard P; Singer, Victoria L

    2003-04-01

    We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.

  2. Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1.

    PubMed

    Brandner, Katrin; Sambade, Adrian; Boutant, Emmanuel; Didier, Pascal; Mély, Yves; Ritzenthaler, Christophe; Heinlein, Manfred

    2008-06-01

    The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.

  3. Whole-body multicolor spectrally resolved fluorescence imaging for development of target-specific optical contrast agents using genetically engineered probes

    NASA Astrophysics Data System (ADS)

    Kobayashi, Hisataka; Hama, Yukihiro; Koyama, Yoshinori; Barrett, Tristan; Urano, Yasuteru; Choyke, Peter L.

    2007-02-01

    Target-specific contrast agents are being developed for the molecular imaging of cancer. Optically detectable target-specific agents are promising for clinical applications because of their high sensitivity and specificity. Pre clinical testing is needed, however, to validate the actual sensitivity and specificity of these agents in animal models, and involves both conventional histology and immunohistochemistry, which requires large numbers of animals and samples with costly handling. However, a superior validation tool takes advantage of genetic engineering technology whereby cell lines are transfected with genes that induce the target cell to produce fluorescent proteins with characteristic emission spectra thus, identifying them as cancer cells. Multicolor fluorescence imaging of these genetically engineered probes can provide rapid validation of newly developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either express or not-express specific cell surface receptors. Various antibody-based or receptor ligand-based optical contrast agents with either green or near infrared fluorophores were developed to concurrently target and validate cancer cells and their positive and negative controls, such as β-D-galactose receptor, HER1 and HER2 in a single animal/organ. Spectrally resolved fluorescence multicolor imaging was used to detect separate fluorescent emission spectra from the exogenous agents and RFP. Therefore, using this in vivo imaging technique, we were able to demonstrate the sensitivity and specificity of the target-specific optical contrast agents, thus reducing the number of animals needed to conduct these experiments.

  4. Engineering Proteins for Thermostability with iRDP Web Server

    PubMed Central

    Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C. G.

    2015-01-01

    Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements. PMID:26436543

  5. Engineering Proteins for Thermostability with iRDP Web Server.

    PubMed

    Panigrahi, Priyabrata; Sule, Manas; Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C G

    2015-01-01

    Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

  6. Optical properties of green fluorescent proteins and their applications on virus infection

    NASA Astrophysics Data System (ADS)

    Lee, Ja-Yun; Kao, Chia-Yun; Chen, Ying-Ju; Wu, Tzong-Yuan; Hsu, I.-Jen

    2007-07-01

    Exogenous fluorescent agents such as green fluorescent protein (GFP) have been widely used as biological indicators in bioimaging techniques. Although GFP and its mutants have been used in many applications, their optical properties have not been completely investigated, especially when they are under various environmental conditions. In this research, we developed a spectrum-analyzing system to investigate the fluorescent properties of GFP in the environments of different temperatures. We found that the fluorescent spectrum of GFP consisted of two components that might come from the transitions between different electronic energy states where the quantum efficiencies of the two components varied with different temperature. This effect was expected to come from the thermal effect on the electron populations in the molecular energy states of GFP. Furthermore, GFP was used as fluorescent marker to monitor the infection process of cells by viruses with a dynamic spectral imaging system. The recombinant baculoviruses containing the red and green fluorescent protein gene that can simultaneously produce dual fluorescence were used as vectors in insect cells. The system was used to monitor the spatial distribution of fluorescent spectra of cells infected by virus during the process of infection.

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

    PubMed

    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.

  8. Application of portable in situ UV fluorescence sensors in natural and engineered aquatic systems.

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Rushworth, Cathy; Atrridge, John

    2016-04-01

    Natural organic matter (NOM) is ubiquitous throughout aquatic systems. This heterogeneous mixture of organic matter is central for aquatic ecosystems and, both local and global, biogeochemical cycling. Improvements in technology and data analysis has allowed for advances in the understanding and characterisation of aquatic organic matter. However, much of the technological expansions have focussed on benchtop instruments. In recent years, there has been interest in the continued development of portable in situ sensors for monitoring NOM characteristics within a wide range of applications, spanning both natural and engineered systems. The UviLux (Chelsea Technologies Group Ltd., UK) is an in situ portable UV fluorescence sensor that can be configured to monitor a range of NOM in aquatic systems, as well as anthropogenic inputs such as polycyclic aromatic hydrocarbons (PAH) and optical brighteners. Here we will focus on the use of the Tryptophan and CDOM UviLux sensors across a variety of applications in both natural systems, such as rivers and leachate into groundwater, and engineered systems, including drinking water and waste water treatment. Recent work has focused on standardising the fluorescence output across the UviLux range of sensors, reporting data in quinine sulphate units (QSU), which enables the output from two different fluorometers to be directly compared both to each other, and to bench-top data. A key advantage of deploying multiple sensors is the ability to fingerprint the fluorescence, by providing, for example, a Tryptophan/CDOM ratio. From the data collected, the ratio of the different fluorescence regions has been shown to provide more robust in situ data and help identify true temporal variations and patterns across multiple applications and sampling locations.

  9. Osteoconductivity of Complex Biomaterials Assayed by Fluorescent-Engineered Osteoblast-like Cells.

    PubMed

    Manfrini, Marco; Mazzoni, Elisa; Barbanti-Brodano, Giovanni; Nocini, Pierfrancesco; D'agostino, Antonio; Trombelli, Leonardo; Tognon, Mauro

    2015-04-01

    Biomaterials employed for the bone regeneration can be assayed for specific features such as osteoconductivity and gene expression. In this study, the composite HA/collagen/chondroitin-sulfate biomaterial was investigated using an engineered human cell line, named Saos-eGFP. This cell line, a green fluorescent engineered human osteoblast-like cell, was employed as a cellular model for the in vitro study of biomaterial characteristics. The cytotoxicity was indirectly evaluated by fluorescence detection, osteoconductivity was assayed both by fluorescence and electron microscope analysis as well as cell morphology, whereas the RT-PCR technique was employed to assay gene expression. Saos-eGFP cells viability detection after 24 and 96 h of incubation showed that biomaterial enables the adhesion and proliferation of seeded cells as well as that of the plastic surface, the control. Fluorescence and scanning electron microscopy (SEM) analyses indicated that Saos-eGFP cells were homogeneously distributed on the HA granule surfaces, exhibiting cytoplasmic bridges, and were localized on the collagen-chondroitin sulfate extra-cellular matrix. An expression analysis of specific genes encoding for differentiation markers, showed that biomaterial assayed did not alter the osteogenic pathway of the Saos-eGFP cell line. Our assays confirm the cytocompatibility of this biomaterial, suggesting an osteoconductive capacity mediated by its chemical contents. We showed that the Saos-eGFP cellular model is suitable for in vitro biomaterial assays, and more specifically for assessing osteoconductivity. This result suggests that the cytocompatibility and osteoconductive features of the biomaterial assayed as bone substitute, could have a positive downstream effect on implant osteo-integration.

  10. Fluorescent probe for high-throughput screening of membrane protein expression

    PubMed Central

    Backmark, A E; Olivier, N; Snijder, A; Gordon, E; Dekker, N; Ferguson, A D

    2013-01-01

    Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture. PMID:23776061

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

    PubMed

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

    2011-05-01

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

  12. Assessing the Utility of Photoswitchable Fluorescent Proteins for Tracking Intercellular Protein Movement in the Arabidopsis Root

    PubMed Central

    MacRae-Crerar, Aurora; Gallagher, Kimberly L.

    2011-01-01

    One way in which cells communicate is through the direct transfer of proteins. In plants, many of these proteins are transcription factors, which are made by one cell type and traffic into another. In order to understand how this movement occurs and its role in development, we would like to track this movement in live, intact plants in real-time. Here we examine the utility of the photoconvertible proteins, Dendra2 and (to a lesser extent) EosFP as tags for studying intracellular and intercellular protein movement in the Arabidopsis root. To this end, we made fusions between Dendra2 and six mobile transcription factors. Our results show that Dendra2 is an effective tool for studying protein movement between plant cells. Interestingly, we found that Dendra2 could not simply be swapped into existing constructs that had originally contained GFP. Most of the fusions made in this way failed to produce a fluorescent fusion. In addition we found that the optimal settings for photoconversion of Dendra2 in stably transformed roots were different from what has been published for photoconversion in transient assays in plants or in animal cells. By modifying the confocal setting, we were able to photoconvert Dendra2 in all cell layers in the root. However the efficiency of photoconversion was affected by the position of the cell layer within the root, with more internal tissues requiring more energy. By examining the Dendra2 fusions, we confirmed the mobility of the SHORT-ROOT (SHR) and CAPRICE (CPC) transcription factors between cells and we further discovered that SHR movement in stele and CPC movement in the epidermis are non-directional. PMID:22132108

  13. The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos.

    PubMed

    Amsterdam, A; Lin, S; Hopkins, N

    1995-09-01

    The green fluorescent protein (GFP) from the cnidarian Aequorea victoria is capable of producing fluorescence without an exogenously added substrate. Here we demonstrate that a cDNA for GFP driven by a Xenopus elongation factor 1 alpha enhancer-promoter can confer fluorescence upon live zebrafish embryos, either as an injected plasmid or as a transgene after passage through the germline. When injected into zebrafish embryos at the one-cell stage, this construct starts to express detectable GFP after about 4 hr of development at 28 degrees C, about 1 hr after the midblastula transition. Fluorescence can be observed in cells of many tissue types in the embryo for at least 3 weeks after injection. We used three different expression constructs, each employing a modified ef1 alpha enhancer-promoter, to generate 12 transgenic lines. Eight of the 12 lines, including 5 of 5 derived from one construct with an intron, express detectable fluorescence in the F1 and, where tested, in the F2 generation. Most expressing lines showed very similar expression patterns. Generally, fluorescence is not seen in the transgenic embryos before 20 hr postfertilization, at which point it appears uniformly throughout the embryo. Fluorescence is most visible between 24-36 hr, and it becomes less visible after this, except that in many lines strong fluorescence remains visible in the eye for at least 5 days. A single inherited copy of the transgene is sufficient to produce detectable fluorescence in hemizygous F1 and F2 embryos.

  14. Bimolecular fluorescence complementation (BiFC) assay for protein-protein interaction in onion cells using the helios gene gun.

    PubMed

    Hollender, Courtney A; Liu, Zhongchi

    2010-06-12

    Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay(1) allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)(2) and the visualization of their interactions mediated by BiFC in onion epidermal cells.

  15. Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein.

    PubMed

    Yoshizawa, Susumu; Karatani, Hajime; Wada, Minoru; Kogure, Kazuhiro

    2012-04-01

    Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490 nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max)  ≈ 490 to λ(max)  ≈ 476 nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472 nm, whereas other Vibrio strains emit light with a peak at around 482 nm. Therefore, we investigated the mechanism underlying this blue shift in V. azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V. azureus.

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

    PubMed

    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.

  17. A visible-light-excited fluorescence method for imaging protein crystals without added dyes.

    PubMed

    Lukk, Tiit; Gillilan, Richard E; Szebenyi, Doletha M E; Zipfel, Warren R

    2016-02-01

    Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visible-light-inducible autofluorescence serves as a complementary method to bright-field microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the non-damaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays.

  18. A visible-light-excited fluorescence method for imaging protein crystals without added dyes

    PubMed Central

    Lukk, Tiit; Gillilan, Richard E.; Szebenyi, Doletha M. E.; Zipfel, Warren R.

    2016-01-01

    Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visible-light-inducible autofluorescence serves as a complementary method to bright-field microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the non-damaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays. PMID:26937240

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

    PubMed

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

    2015-05-15

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

  20. Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida

    PubMed Central

    2012-01-01

    Background Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida. However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes. Results In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence. Conclusions To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments. PMID:23095243

  1. Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

    NASA Astrophysics Data System (ADS)

    Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

    2009-11-01

    The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants ( Ka) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02 × 10 7 and 2.07 × 10 4 L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S 0 → S 1 transition of esculin ( λexmax≈340 nm) appears, which is similar to the λemmax of BSA and HSA. The critical distance ( R0) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

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

    ERIC Educational Resources Information Center

    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.

  3. Reactive oxygen species in photochemistry of the red fluorescent protein "Killer Red".

    PubMed

    Vegh, Russell B; Solntsev, Kyril M; Kuimova, Marina K; Cho, Soohee; Liang, Yue; Loo, Bernard L W; Tolbert, Laren M; Bommarius, Andreas S

    2011-05-07

    The fluorescent protein aptly named "Killer Red" (KRed) is capable of killing transfected cells and inactivating fused proteins upon exposure to visible light in the presence of oxygen. We have investigated the source of the bioactive species through a variety of photophysical and photochemical techniques. Our results indicate a Type I (electron transfer mediated) photosensitizing mechanism.

  4. Clinical translation of controlled protein delivery systems for tissue engineering

    PubMed Central

    Spiller, Kara L.; Vunjak-Novakovic, Gordana

    2013-01-01

    Strategies that utilize controlled release of drugs and proteins for tissue engineering have enormous potential to regenerate damaged organs and tissues. The multiple advantages of controlled release strategies merit overcoming the significant challenges to translation, including high costs and long, difficult regulatory pathways. This review highlights the potential of controlled release of proteins for tissue engineering and regenerative medicine. We specifically discuss treatment modalities that have reached preclinical and clinical trials, with emphasis on controlled release systems for bone tissue engineering, the most advanced application with several products already in clinic. Possible strategies to address translational and regulatory concerns are also discussed. PMID:25787736

  5. GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

    PubMed Central

    Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

    2008-01-01

    Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

  6. Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications

    PubMed Central

    Zhou, Xin X.; Lin, Michael Z.

    2013-01-01

    Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. In the last ten years, many new RSFPs have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties. This review will describe research on the mechanisms of reversible photoswitching and recent applications using RSFPs. While cis-trans isomerization of the chromophore is believed to be the general mechanism for most RSFPs, structural studies reveal diversity in the details of photoswitching mechanisms, including different effects of protonation, chromophore planarity, and pocket flexibility. Applications of RSFPs include new types of live-cell superresolution imaging, tracking of protein movements and interactions, information storage, and optical control of protein activity. PMID:23876529

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.

    PubMed

    Cody, C W; Prasher, D C; Westler, W M; Prendergast, F G; Ward, W W

    1993-02-09

    The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.

  9. An arsenic fluorescent compound as a novel probe to study arsenic-binding proteins.

    PubMed

    Femia, A Lis; Temprana, C Facundo; Santos, Javier; Carbajal, María Laura; Amor, María Silvia; Grasselli, Mariano; Alonso, Silvia Del V

    2012-12-01

    Arsenic-binding proteins are under continuous research. Their identification and the elucidation of arsenic/protein interaction mechanisms are important because the biological effects of these complexes may be related not only to arsenic but also to the arsenic/protein structure. Although many proteins bearing a CXXC motif have been found to bind arsenic in vivo, new tools are necessary to identify new arsenic targets and allow research on protein/arsenic complexes. In this work, we analyzed the performance of the fluorescent compound APAO-FITC (synthesized from p-aminophenylarsenoxide, APAO, and fluorescein isothiocyanate, FITC) in arsenic/protein binding assays using thioredoxin 1 (Trx) as an arsenic-binding protein model. The Trx-APAO-FITC complex was studied through different spectroscopic techniques involving UV-Vis, fluorescence, atomic absorption, infrared and circular dichroism. Our results show that APAO-FITC binds efficiently and specifically to the Trx binding site, labeling the protein fluorescently, without altering its structure and activity. In summary, we were able to study a protein/arsenic complex model, using APAO-FITC as a labeling probe. The use of APAO-FITC in the identification of different protein and cell targets, as well as in in vivo biodistribution studies, conformational studies of arsenic-binding proteins, and studies for the design of drug delivery systems for arsenic anti-cancer therapies, is highly promising.

  10. Time-resolved fluorescence immunoassay for C-reactive protein using colloidal semiconducting nanoparticles.

    PubMed

    Härmä, Harri; Toivonen, Juha; Soini, Juhani T; Hänninen, Pekka; Parak, Wolfgang J

    2011-01-01

    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.

  11. Intrinsic fluorescence excitation-emission matrix spectral features of cottonseed protein fractions and the effects of denaturants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation-emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, an...

  12. Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study.

    PubMed

    Taskent-Sezgin, Humeyra; Chung, Juah; Patsalo, Vadim; Miyake-Stoner, Shigeki J; Miller, Andrew M; Brewer, Scott H; Mehl, Ryan A; Green, David F; Raleigh, Daniel P; Carrico, Isaac

    2009-09-29

    The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.

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

    PubMed

    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.

  14. Protein design in systems metabolic engineering for industrial strain development.

    PubMed

    Chen, Zhen; Zeng, An-Ping

    2013-05-01

    Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering.

  15. Construction of cell-based neurotransmitter fluorescently engineered reporters (CNiFERs) for optical detection of neurotransmitters in vivo

    PubMed Central

    Lacin, Emre; Muller, Arnaud; Fernando, Marian; Kleinfeld, David; Slesinger, Paul A

    2016-01-01

    Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL – activation of the receptor leads to an increase in the FRET signal. Because a CNiFER clone utilizes the native receptor for a particular neurotransmitter (e.g. D2R for dopamine), it has nanomolar sensitivity and a temporal response of seconds. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred micrometers, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER. PMID:27214050

  16. Engineering chimaeric proteins from fold fragments: 'hopeful monsters' in protein design.

    PubMed

    Höcker, Birte

    2013-10-01

    Modern highly complex proteins evolved from much simpler and less specialized subunits. The same concept can be applied in protein engineering to construct new well-folded proteins. Hybrid proteins or chimaeras can be built from contemporary protein fragments through illegitimate recombination. Even parts from different globular folds can be fitted together using rational design methodologies. Furthermore, intrinsic functional properties encoded in the fold fragments allow rapid adaptation of the new proteins and thus provide interesting starting scaffolds for further redesign.

  17. Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.

    PubMed

    Branchini, Bruce R; Rosenberg, Justin C; Ablamsky, Danielle M; Taylor, Kelsey P; Southworth, Tara L; Linder, Samantha J

    2011-07-15

    We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.

  18. A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II.

    PubMed

    Ruan, Kangcheng; Li, Jiong; Liang, Ruqiang; Xu, Chunhe; Yu, Yong; Lange, Reinhard; Balny, Claude

    2002-04-26

    An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Dipolar relaxation within the protein matrix of the green fluorescent protein: a red edge excitation shift study.

    PubMed

    Haldar, Sourav; Chattopadhyay, Amitabha

    2007-12-27

    The fluorophore in green fluorescent protein (GFP) is localized in a highly constrained environment, protected from the bulk solvent by the barrel-shaped protein matrix. We have used the wavelength-selective fluorescence approach (red edge excitation shift, REES) to monitor solvent (environment) dynamics around the fluorophore in enhanced green fluorescent protein (EGFP) under various conditions. Our results show that EGFP displays REES in buffer and glycerol, i.e., the fluorescence emission maxima exhibit a progressive shift toward the red edge, as the excitation wavelength is shifted toward the red edge of the absorption spectrum. Interestingly, EGFP displays REES when incorporated in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), independent of the hydration state. We interpret the observed REES to the constrained environment experienced by the EGFP fluorophore in the rigid protein matrix, rather than to the dynamics of the bulk solvent. These results are supported by the temperature dependence of REES and characteristic wavelength-dependent changes in fluorescence anisotropy.

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

    PubMed Central

    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

  2. Phagocytic mechanism of live neutrophilic granulocyte with green fluorescent protein gene

    NASA Astrophysics Data System (ADS)

    Zeng, Shaoqun; Chen, Weiguo; Zhou, Wei; Luo, Qingming; Zhang, Zhi

    1999-09-01

    The whole process of neutrophilic granulocyte phagocytosis of E.Coli BL21-expressed green fluorescent protein gene was recorded with CCD coupled fluorescence microscopy in a real-time. This process contains of three stages: adhesion, ingestion, and exocytosis. The properties of each stages of phagocytosis were analyzed. The method opened new perspective in continuously observing the specific and non-specific immunity mechanism on alive cell.

  3. Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

  4. Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

    PubMed Central

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

    2010-01-01

    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. PMID:20161802

  5. Ultrasensitive reporter protein detection in genetically engineered bacteria.

    PubMed

    Wells, Mona; Gösch, Michael; Rigler, Rudolf; Harms, Hauke; Lasser, Theo; van der Meer, Jan Roelof

    2005-05-01

    We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.

  6. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    NASA Astrophysics Data System (ADS)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya;