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https://www.readbyqxmd.com/read/29193968/structural-insight-into-the-photochemistry-of-split-green-fluorescent-proteins-a-unique-role-for-a-his-tag
#1
Alan Deng, Steven G Boxer
Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e. split GFP with a β-strand removed, that were found to behave differently in the presence of light...
December 1, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29192060/high-content-tripartite-split-gfp-cell-based-assays-to-screen-for-modulators-of-small-gtpase-activation
#2
Faten Koraïchi, Rémi Gence, Catherine Bouchenot, Sarah Grosjean, Isabelle Lajoie-Mazenc, Gilles Favre, Stéphanie Cabantous
The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications...
November 30, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/29158224/a-novel-fluorescent-reporter-detects-plastic-remodeling-of-mitochondria-er-contact-sites
#3
Zhaoying Yang, Xiaocui Zhao, Jiashen Xu, Weina Shang, Chao Tong
Mitochondria-ER contact sites (MERCs) enable communication between ER and mitochondria and serve as platforms for many cellular events, including autophagy. Nonetheless, the molecular organization of MERCs is not known, and there is no bona fide marker of these contact sites in mammalian cells. In this study, we designed a genetically encoded reporter using split super-folder GFP protein for labeling MERCs. We subsequently analyzed its distribution and dynamics during the cell cycle and under normal or stressful cellular conditions such as starvation, apoptosis, and ER stress...
November 20, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/29154784/tgf-%C3%AE-1-signaling-regulates-mouse-hepatic-stellate-cell-differentiation-via-the-jagged1-notch-pathway
#4
Yasen Aimaiti, Xin Jin, Wei Wang, Zixin Chen, Dewei Li
AIMS: We tested whether transforming growth factor β1 (TGF-β1) signaling plays an important role in hepatic stellate cell differentiation fate and investigated the role of Jagged1/Notch in this process. MATERIALS AND METHODS: TGF-β1 was overexpressed and transforming growth factor receptor 1 (TGF-β-R1) was knocked down by a lentiviral vector in mouse hepatic stellate cells (mHSCs). Transfection efficiency was assessed with immunofluorescence, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blotting...
November 14, 2017: Life Sciences
https://www.readbyqxmd.com/read/29108650/biophysical-characterization-of-genetically-encoded-voltage-sensor-asap1-dynamic-range-improvement
#5
Elizabeth E L Lee, Francisco Bezanilla
Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1...
November 21, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/29097689/marked-bias-towards-spontaneous-synaptic-inhibition-distinguishes-non-adapting-from-adapting-layer-5-pyramidal-neurons-in-the-barrel-cortex
#6
Ion R Popescu, Kathy Q Le, Rocío Palenzuela, Rebecca Voglewede, Ricardo Mostany
Pyramidal neuron subtypes differ in intrinsic electrophysiology properties and dendritic morphology. However, do different pyramidal neuron subtypes also receive synaptic inputs that are dissimilar in frequency and in excitation/inhibition balance? Unsupervised clustering of three intrinsic parameters that vary by cell subtype - the slow afterhyperpolarization, the sag, and the spike frequency adaptation - split layer 5 barrel cortex pyramidal neurons into two clusters: one of adapting cells and one of non-adapting cells, corresponding to previously described thin- and thick-tufted pyramidal neurons, respectively...
November 2, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29070802/split-gfp-technologies-to-structurally-characterize-and-quantify-functional-biomolecular-interactions-of-ftd-related-proteins
#7
Chiara Foglieni, Stéphanie Papin, Agnese Salvadè, Tariq Afroz, Sandra Pinton, Giona Pedrioli, Giorgio Ulrich, Magdalini Polymenidou, Paolo Paganetti
Protein multimerization in physiological and pathological conditions constitutes an intrinsic trait of proteins related to neurodegeneration. Recent evidence shows that TDP-43, a RNA-binding protein associated with frontotemporal dementia and amyotrophic lateral sclerosis, exists in a physiological and functional nuclear oligomeric form, whose destabilization may represent a prerequisite for misfolding, toxicity and subsequent pathological deposition. Here we show the parallel implementation of two split GFP technologies, the GFP bimolecular and trimolecular fluorescence complementation (biFC and triFC) in the context of TDP-43 self-assembly...
October 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29069446/6-4-pp-photolyase-encoded-by-atuvr3-is-localized-in-nuclei-chloroplasts-and-mitochondria-and-its-expression-is-down-regulated-by-light-in-a-photosynthesis-dependent-manner
#8
Agnieszka Katarzyna Banas, Pawel Hermanowicz, Olga Sztatelman, Justyna Labuz, Chhavi Aggarwal, Piotr Zglobicki, Dominika Jagiello-Flasinska, Wojciech Strzalka
Pyrimidine dimers are the most important DNA lesions induced by UVB irradiation. They can be repaired directly by photoreactivation or indirectly by the excision repair pathways. Photoreactivation is performed by photolyases, enzymes which bind to the dimers and use the energy of blue light or UVA to split bonds between adjacent pyrimidines. Arabidopsis thaliana has three known photolyases: AtPHR1, AtCRY3 and AtUVR3. Little is known about the cellular localization and regulation of AtUVR3 expression. We have found that its transcript level is down-regulated by light (red, blue or white) in a photosynthesis-dependent manner...
October 24, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/28991414/in-situ-spatial-complementation-of-aptamer-mediated-recognition-enables-live-cell-imaging-of-native-rna-transcripts-in-real-time
#9
Zejun Wang, Yao Luo, Xiaodong Xie, Xingjie Hu, Haiyun Song, Yun Zhao, Jiye Shi, Lihua Wang, Gennadi Glinsky, Nan Chen, Ratnesh Lal, Chunhai Fan
Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their functions and reveals novel mechanisms at the single-cell resolution. Here, we report the development of an aptamer-initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, nascent mRNA molecules recruited split Broccoli and brought the two fragments to the spatial proximity, which in-situ formed a fluorophore-binding site and turned on fluorescence...
October 9, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28977453/enrichment-of-dynamic-chromosomal-crosslinks-drive-phase-separation-of-the-nucleolus
#10
Caitlin Hult, David Adalsteinsson, Paula A Vasquez, Josh Lawrimore, Maggie Bennett, Alyssa York, Diana Cook, Elaine Yeh, Mark Gregory Forest, Kerry Bloom
Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology...
November 2, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28956769/epstein-barr-virus-fusion-with-epithelial-cells-triggered-by-gb-is-restricted-by-a-gl-glycosylation-site
#11
Britta S Möhl, Jia Chen, Seo Jin Park, Theodore S Jardetzky, Richard Longnecker
Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells...
December 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28945234/a-homodimer-interface-without-base-pairs-in-an-rna-mimic-of-red-fluorescent-protein
#12
Katherine Deigan Warner, Ljiljana Sjekloća, Wenjiao Song, Grigory S Filonov, Samie R Jaffrey, Adrian R Ferré-D'Amaré
Corn, a 28-nucleotide RNA, increases yellow fluorescence of its cognate ligand 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) by >400-fold. Corn was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, in which six unpaired adenosines break overall two-fold symmetry, lacks any intermolecular base pairs...
November 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/28842906/application-of-split-gfp-reassembly-assay-to-study-myogenesis-and-myofusion-in-vitro
#13
Manami Kodaka, Xiaoyin Xu, Zeyu Yang, Junichi Maruyama, Yutaka Hata
Green fluorescent protein (GFP) is composed of 11 β-strands, and loses GFP signals, when divided into the N-terminal ten β-strands (GFP1-10) and the C-terminal last β-strand (GFP11). However, when GFP1-10 and GFP11 encounter, they reassemble into the fluorescent GFP. We expressed GFP1-10 and blasticidin resistance gene product-fused GFP11 (BSR-GFP11) in C2C12 cells. Both the cell lines do not show GFP but when they undergo myogenesis and myofusion, GFP1-10 and BSR-GFP11 form the fluorescent complex in multi-nuclear myotubes, so that GFP signals reflect myogenesis and myofusion...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28767038/microtubule-dependent-ribosome-localization-in-c-elegans-neurons
#14
Kentaro Noma, Alexandr Goncharov, Mark H Ellisman, Yishi Jin
Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level...
August 2, 2017: ELife
https://www.readbyqxmd.com/read/28621789/display-of-functional-proteins-on-supramolecular-peptide-nanofibrils-using-a-split-protein-strategy
#15
John T M DiMaio, Danielle M Raymond, Bradley L Nilsson
The display of functional proteins on self-assembled peptide nanofibrils is challenging since the steric bulk of proteins attached to simple self-assembling peptides often impedes incorporation into nanofibrils. Herein is described a split-protein strategy to tether functional proteins to preassembled peptide nanofibrils. In this strategy, a short affinity motif peptide derived from a split protein system is appended to a self-assembly motif (the amphipathic Ac-(FKFE)2-NH2 peptide) to form an affinity-assembly fusion peptide...
June 27, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28619883/spatiotemporal-monitoring-of-pseudomonas-syringae-effectors-via-type-iii-secretion-using-split-fluorescent-protein-fragments
#16
Eunsook Park, Hye-Young Lee, Jongchan Woo, Doil Choi, Savithramma P Dinesh-Kumar
Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells...
July 2017: Plant Cell
https://www.readbyqxmd.com/read/28619616/activity-independent-screening-of-secreted-proteins-using-split-gfp
#17
Andreas Knapp, Myriam Ripphahn, Kristina Volkenborn, Pia Skoczinski, Karl-Erich Jaeger
The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay...
June 12, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28551989/optimized-fluorescence-complementation-platform-for-visualizing-salmonella-effector-proteins-reveals-distinctly-different-intracellular-niches-in-different-cell-types
#18
Alexandra M Young, Michael Minson, Sarah E McQuate, Amy E Palmer
The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG, and SlrP, that have never before been visualized over time during infection of live cells...
June 9, 2017: ACS Infectious Diseases
https://www.readbyqxmd.com/read/28470605/split-gfp-complementation-as-reporter-of-membrane-protein-expression-and-stability-in-e-coli-a-tool-to-engineer-stability-in-a-lat-transporter
#19
Ekaitz Errasti-Murugarren, Arturo Rodríguez-Banqueri, José Luis Vázquez-Ibar
Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28391244/introducing-inducible-fluorescent-split-cholesterol-oxidase-to-mammalian-cells
#20
Konstantin G Chernov, Maarit Neuvonen, Ivonne Brock, Elina Ikonen, Vladislav V Verkhusha
Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments...
May 26, 2017: Journal of Biological Chemistry
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