scholarly journals Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers

2021 ◽  
Vol 22 (24) ◽  
pp. 13631
Author(s):  
Spase Stojanov ◽  
Tina Vida Plavec ◽  
Julijana Kristl ◽  
Špela Zupančič ◽  
Aleš Berlec
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Electrospun Nanofibers ◽  
Resolving Power ◽  
Lactobacillus Species ◽  
Vaginal Lactobacilli ◽  
Lactobacillus Jensenii ◽  
Poly Ethylene ◽  
Green Fluorescent ◽  
Infrared Fluorescent Protein

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

2018 ◽  
Author(s):  
Clémence Roggo ◽  
Jan Roelof van der Meer
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bacterial Chemotaxis ◽  
Ph Sensitive ◽  
Flagellar Motors ◽  
Receptor Interactions ◽  
Split Egfp ◽  
Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

2003 ◽  
Vol 284 (5) ◽  
pp. H1647-H1654 ◽  
Author(s):  
Jean-Philippe Fortin ◽  
Johanne Bouthillier ◽  
François Marceau
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Brefeldin A ◽  
Metabolic Inhibitors ◽  
Maximal Effect ◽  
Hek 293 ◽  
B1 Receptors ◽  
Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

scholarly journals Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

2015 ◽  
Vol 113 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Marie-Aude Plamont ◽  
Emmanuelle Billon-Denis ◽  
Sylvie Maurin ◽  
Carole Gauron ◽  
Frederico M. Pimenta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Activation Mechanism ◽  
Photoactive Yellow Protein ◽  
Reversible Binding ◽  
A Cell ◽  
Rapid Labeling ◽  
Green Fluorescent

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.

Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent-protein genes

2020 ◽  
Author(s):  
Noriyuki Satoh ◽  
Koji Kinjo ◽  
Kohei Shintaku ◽  
Daisuke Kezuka ◽  
Hiroo Ishimori ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Biological Significance ◽  
Gene Expression Profiles ◽  
Gfp Expression ◽  
Red Fluorescent Proteins ◽  
Color Morphs ◽  
Acropora Tenuis ◽  
Green Fluorescent

ABSTRACTCorals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow-green (G) and purple (P) forms. The tips of axial coral polyps exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, the N and P morphs experienced bleaching, in which some N morphs died while P morphs recovered. In contrast, G morphs successfully withstood the stress. Symbiotic dinoflagellates are essential symbiotic partners of scleractinian corals. Photosynthetic activity of symbionts was reduced in July in N and P morphs; however, the three color-morphs host similar sets of Clade-C zoothanthellae, suggesting that beaching of N and P morphs cannot be attributed to differences in symbiont clades. The decoded Acropora tenuis genome includes five genes for green fluorescent proteins (GFP), two for cyan fluorescent proteins (CFP), three for red fluorescent proteins (RFP), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles demonstrated that (a) expression of CFP and REP was quite low in all three morphs, (b) P morphs expressed higher levels of ChrP, (c) both N and G morphs expressed GFP highly, and (d) GFP expression was reduced in N morphs, compared to G morphs, which maintained higher levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of Acropora tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs.

scholarly journals N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 802
Author(s):  
Oksana V. Nekrasova ◽  
Alexandra L. Primak ◽  
Anastasia A. Ignatova ◽  
Valery N. Novoseletsky ◽  
Olga V. Geras’kina ◽  
...  
Keyword(s):  
Binding Site ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Hek293 Cells ◽  
Kv1.3 Channel ◽  
C Terminus ◽  
Peptide Toxins ◽  
Fluorescent Ligands ◽  
Voltage Gated Channels ◽  
Green Fluorescent

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

scholarly journals Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins

2004 ◽  
Vol 70 (12) ◽  
pp. 7530-7538 ◽  
Author(s):  
Christopher J. Reuter ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Specific Inhibitor ◽  
Haloferax Volcanii ◽  
Reporter System ◽  
Reporter Protein ◽  
Protein Variant ◽  
Fluorescent Reporter ◽  
New Genes ◽  
Green Fluorescent

ABSTRACT Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin β-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ∼40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

scholarly journals 1.8 Å bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants

2007 ◽  
Vol 402 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Andre C. Stiel ◽  
Simon Trowitzsch ◽  
Gert Weber ◽  
Martin Andresen ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Blue Light ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Uv Light ◽  
Structural Features ◽  
Trans Isomerization ◽  
Fluorescent State ◽  
Green Fluorescent ◽  
New Applications ◽  
Key Events

RSFPs (reversibly switchable fluorescent proteins) may be repeatedly converted between a fluorescent and a non-fluorescent state by irradiation and have attracted widespread interest for many new applications. The RSFP Dronpa may be switched with blue light from a fluorescent state into a non-fluorescent state, and back again with UV light. To obtain insight into the underlying molecular mechanism of this switching, we have determined the crystal structure of the fluorescent equilibrium state of Dronpa. Its bicyclic chromophore is formed spontaneously from the Cys62–Tyr63–Gly64 tripeptide. In the fluorescent state, it adopts a slightly non-coplanar cis conformation within the interior of a typical GFP (green fluorescent protein) β-can fold. Dronpa shares some structural features with asFP595, another RSFP whose chromophore has previously been demonstrated to undergo a cis–trans isomerization upon photoswitching. Based on the structural comparison with asFP595, we have generated new Dronpa variants with an up to more than 1000-fold accelerated switching behaviour. The mutations which were introduced at position Val157 or Met159 apparently reduce the steric hindrance for a cis–trans isomerization of the chromophore, thus lowering the energy barrier for the blue light-driven on-to-off transition. The findings reported in the present study support the view that a cis–trans isomerization is one of the key events common to the switching mechanism in RSFPs.

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