Superfolder mTurquoise2oxoptimized for the bacterial periplasm allows high efficiencyin vivoFRET of cell division antibiotic targets

AbstractFluorescent proteins (FP)s are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non-native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging or FRET. To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise 2 ox (sfTq2ox) and is able to withstand challenging, oxidizing environments. sfTq2oxhas improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide-bonds. The improved sfTq2oxhas the same spectroscopic properties as mTq2, i.e. high fluorescence lifetime and quantum yield. The sfTq2ox-mNeongreen FRET pair allows the detection of periplasmic protein-protein interactions with energy transfer rates exceeding 40 %. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way forin vivoantibiotic screening.

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Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Physiological Reviews ◽

10.1152/physrev.00038.2009 ◽

2010 ◽

Vol 90 (3) ◽

pp. 1103-1163 ◽

Cited By ~ 818

Author(s):

Dmitriy M. Chudakov ◽

Mikhail V. Matz ◽

Sergey Lukyanov ◽

Konstantin A. Lukyanov

Keyword(s):

Protein Interactions ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Visible Spectrum ◽

Living Cells ◽

Alternative Possibilities ◽

Structure Evolution ◽

Marine Animals

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

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Fluorescent proteins for in vivo imaging, where's the biliverdin?

Biochemical Society Transactions ◽

10.1042/bst20200444 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2657-2667

Author(s):

Felipe Montecinos-Franjola ◽

John Y. Lin ◽

Erik A. Rodriguez

Keyword(s):

Hydrogen Peroxide ◽

In Vivo Imaging ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Imaging ◽

Infrared Light ◽

Red Fluorescent Protein ◽

Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

Nuclear Receptor Signaling ◽

10.1621/nrs.04021 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04021 ◽

Cited By ~ 14

Author(s):

Kristen L. Koterba ◽

Brian G. Rowan

Keyword(s):

Energy Transfer ◽

Protein Interactions ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Renilla Luciferase ◽

Receptor Protein ◽

Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

Journal of Bacteriology ◽

10.1128/jb.00462-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6048-6059 ◽

Cited By ~ 17

Author(s):

Carine Robichon ◽

Glenn F. King ◽

Nathan W. Goehring ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Cell Division ◽

Protein Interactions ◽

Fluorescent Protein ◽

Bacterial Cell Division ◽

Protein Protein Interactions ◽

Positive Interaction ◽

E Coli ◽

Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

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Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

10.1101/367110 ◽

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.

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Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

International Journal of Molecular Sciences ◽

10.3390/ijms20163859 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3859 ◽

Cited By ~ 4

Author(s):

Michael Winkler ◽

Florian Wrensch ◽

Pascale Bosch ◽

Maike Knoth ◽

Michael Schindler ◽

...

Keyword(s):

Antiviral Activity ◽

Protein Interactions ◽

Viral Entry ◽

Cellular Localization ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

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Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Antibodies ◽

10.3390/antib7040043 ◽

2018 ◽

Vol 7 (4) ◽

pp. 43 ◽

Cited By ~ 2

Author(s):

Ekaterina Gorshkova ◽

Grigory Efimov ◽

Ksenia Ermakova ◽

Ekaterina Vasilenko ◽

Diana Yuzhakova ◽

...

Keyword(s):

Autoimmune Diseases ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Humanized Mice ◽

Whole Body ◽

Whole Body Imaging ◽

Theranostic Agent ◽

Variable Heavy

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1513094113 ◽

2015 ◽

Vol 113 (3) ◽

pp. 497-502 ◽

Cited By ~ 81

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.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Biomolecules ◽

10.3390/biom10010142 ◽

2020 ◽

Vol 10 (1) ◽

pp. 142 ◽

Cited By ~ 1

Author(s):

Arman Kulyyassov ◽

Vasily Ogryzko

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Fluorescent Protein ◽

Quantitative Estimation ◽

Multiple Reaction Monitoring ◽

Negative Control ◽

Protein Protein Interactions ◽

Close Proximity ◽

Green Fluorescent

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

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