scholarly journals Subunit composition of an energy-coupling-factor-type biotin transporter analysed in living bacteria

2010 ◽  
Vol 431 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Friedrich Finkenwirth ◽  
Olivia Neubauer ◽  
Julia Gunzenhäuser ◽  
Janna Schoknecht ◽  
Silvia Scolari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Yellow Fluorescent Protein ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Supramolecular Organization ◽  
High Affinity ◽  
Donor Acceptor ◽  
Living Bacteria

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor–acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor–acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

scholarly journals Two Essential Arginine Residues in the T Components of Energy-Coupling Factor Transporters

2009 ◽  
Vol 191 (21) ◽  
pp. 6482-6488 ◽  
Author(s):  
Olivia Neubauer ◽  
Anja Alfandega ◽  
Janna Schoknecht ◽  
Ulrich Sternberg ◽  
Anne Pohlmann ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Transmembrane Protein ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Single Mutation ◽  
Modular Assembly ◽  
Double Mutation ◽  
Arginine Residues ◽  
Uptake Activity ◽  
Vitamin Uptake

ABSTRACT Energy-coupling factor (ECF) transporters, a recently discovered class of importers of micronutrients, are composed of a substrate-specific transmembrane component (S component) and a conserved energy-coupling module consisting of a transmembrane protein (T component) and pairs of ABC ATPases (A proteins). Based on utilization of a dedicated (subclass I) or shared (subclass II) energy-coupling module, ECF systems fall into two subclasses. The T components are the least-characterized proteins of ECF importers, and their function is essentially unknown. Using RcBioN and LmEcfT, the T units of the subclass I biotin transporter (RcBioMNY) of a gram-negative bacterium and of the subclass II folate, pantothenate, and riboflavin transporters of a lactic acid bacterium, respectively, we analyzed the role of two strongly conserved short motifs, each containing an arginine residue. Individual replacement of the two Arg residues in RcBioN reduced ATPase activity, an indicator of the transporter function, by two-thirds without affecting the modular assembly of the RcBioMNY complex. A double Arg-to-Glu replacement destroyed the complex and abolished ATPase activity. The corresponding single mutation in motif II of LmEcfT, as well as a double mutation, led to loss of the T unit from the subclass II ECF transporters and inactivated these systems. A single Arg-to-Glu replacement in motif I, however, abolished vitamin uptake activity without affecting assembly of the modules. Our results indicate that the conserved motif I in T components is essential for intramolecular signaling and, in cooperation with motif II, for subunit assembly of modular ECF transporters.

Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation

2011 ◽  
Vol 437 (3) ◽  
pp. 389-397 ◽  
Author(s):  
Bastian Thaa ◽  
Ilya Levental ◽  
Andreas Herrmann ◽  
Michael Veit
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Glutathione Transferase ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Yellow Fluorescent Protein ◽  
Cytoplasmic Tail ◽  
Proton Channel ◽  
Amphiphilic Helix ◽  
Cholesterol Binding

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [3H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2–GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.

scholarly journals Engineering yellow fluorescent protein probe for visualization of parallel DNA G-quadruplex

2018 ◽  
Vol 21 (3) ◽  
pp. 84-89 ◽  
Author(s):  
Tuom TT Truong ◽  
Trang PT Phan ◽  
Linh TT Le ◽  
Dung H Nguyen ◽  
Hoang D Nguyen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Biological Processes ◽  
Naked Eye ◽  
High Affinity ◽  
Quadruplex Structure ◽  
G Quadruplex

Introduction: The formation of G-quadruplex plays a key role in many biological processes. Therefore, visualization of G-quadruplex is highly essential for design of G-quadruplex-targeted small molecules (drugs). Herein, we report on an engineered fluorescent protein probe which was able to distinguish G-quadruplex topologies. Methods: The fluorescent protein probe was generated by genetically incorporating yellow fluorescent protein (YFP) to RNA helicase associated with AU-rich element (RHAU) peptide motif. Results: This probe could selectively bind and visualize parallel G-quadruplex structure (T95-2T) at high affinity (Kd~130 nM). Visualization of the parallel G-quadruplex by RHAU-YFP could be easily observed in vitro by using normal Gel Doc or the naked eye. Conclusion: The YFP probe could be encoded in cells to provide a powerful tool for detection of parallel G-quadruplexes both in vitro and in vivo.  

scholarly journals Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

2018 ◽  
Vol 72 (4) ◽  
pp. 252-258
Author(s):  
Laura Hippe ◽  
Šimons Svirskis ◽  
Modra Murovska ◽  
Mārtiņš Kālis
Keyword(s):  
Energy Transfer ◽  
Calcium Release ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Data Interpretation ◽  
Wide Field ◽  
Measurement Quality ◽  
Donor Acceptor ◽  
Molecule Interactions ◽  
Separate Molecule

Abstract The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.

scholarly journals A Novel Class of Modular Transporters for Vitamins in Prokaryotes

2008 ◽  
Vol 191 (1) ◽  
pp. 42-51 ◽  
Author(s):  
Dmitry A. Rodionov ◽  
Peter Hebbeln ◽  
Aymerick Eudes ◽  
Josy ter Beek ◽  
Irina A. Rodionova ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Transmembrane Protein ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Human Pathogens ◽  
Gram Positive Bacteria ◽  
New Class ◽  
Vitamin Uptake ◽  
Genome Context ◽  
Nucleotide Binding Proteins

ABSTRACT The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.

scholarly journals Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses

2006 ◽  
Vol 174 (1) ◽  
pp. 153-161 ◽  
Author(s):  
Bebhinn Treanor ◽  
Peter M.P. Lanigan ◽  
Sunil Kumar ◽  
Chris Dunsby ◽  
Ian Munro ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Killer Cell ◽  
Inhibitory Synapses ◽  
Homogeneous Distribution ◽  
Target Cells ◽  
Supramolecular Organization ◽  
Immune Synapse

We report the supramolecular organization of killer Ig–like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein–tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein–tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.

scholarly journals Quaternary Structure and Functional Unit of Energy Coupling Factor (ECF)-type Transporters

2010 ◽  
Vol 286 (7) ◽  
pp. 5471-5475 ◽  
Author(s):  
Josy ter Beek ◽  
Ria H. Duurkens ◽  
Guus B. Erkens ◽  
Dirk Jan Slotboom
Keyword(s):  
Quaternary Structure ◽  
Functional Unit ◽  
Energy Coupling ◽  
Coupling Factor

scholarly journals Transcriptional and Biochemical Characterization of Cytosolic Pyruvate Kinases in Arabidopsis thaliana

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 353
Author(s):  
Sabine Wulfert ◽  
Sören Schilasky ◽  
Stephan Krueger
Keyword(s):  
Arabidopsis Thaliana ◽  
Pyruvate Kinase ◽  
Expression Pattern ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Quaternary Structure ◽  
Tricarboxylic Acid Cycle ◽  
Yellow Fluorescent Protein ◽  
Allosteric Effectors ◽  
Pyruvate Kinases

Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the Arabidopsis thaliana genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter β-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in Arabidopsis thaliana, by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.

Discovery of Antibacterial Agents Inhibiting the Energy-Coupling Factor (ECF) Transporters by Structure-Based Virtual Screening

2020 ◽  
Author(s):  
Eleonora Diamanti ◽  
Inda Setyawati ◽  
Spyridon Bousis ◽  
leticia mojas ◽  
lotteke Swier ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Antibacterial Agents ◽  
Antimicrobial Agents ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Design Synthesis ◽  
Screening Design ◽  
Starting Point ◽  
Wide Range ◽  
Vitamin Uptake

Here, we report on the virtual screening, design, synthesis and structure–activity relationships (SARs) of the first class of selective, antibacterial agents against the energy-coupling factor (ECF) transporters. The ECF transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. Because of their central role in the metabolism of bacteria and their absence in humans, ECF transporters are novel potential antimicrobial targets to tackle infection. The hit compound’s metabolic and plasma stability, the potency (20, MIC Streptococcus pneumoniae = 2 µg/mL), the absence of cytotoxicity and a lack of resistance development under the conditions tested here suggest that this scaffold may represent a promising starting point for the development of novel antimicrobial agents with an unprecedented mechanism of action.<br>

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