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Salvador Ventura - One of the best experts on this subject based on the ideXlab platform.
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Bimolecular Fluorescence Complementation: Illuminating Cellular Protein Interactions
Current Molecular Medicine, 2011Co-Authors: Salvador VenturaAbstract:Abstract Many cellular processes depend on the establishment of selective stable or transient interactions between proteins. Therefore, the ability to identify and characterize these contacts in physiologically relevant environments is crucial to understanding the networks of contacts that allow the transmission and integration of biological information in living cells. Protein-fragment Complementation assays (PCA) have emerged as approaches that report on the proximity of two given proteins in the cell at a given location and time. In particular, Bimolecular Fluorescence Complementation (BIFC) allows noninvasive imaging of protein binding in living cells at high spatial resolution and without the requirement for exogenous substrates. In the present review, we discuss PCA and BIFC fundamentals, the implementation of BIFC assays and selected applications of BIFC in drug discovery, developmental studies or neurological disorders.
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Monitoring the interference of protein-protein interactions in vivo by Bimolecular Fluorescence Complementation: the DnaK case.
Proteomics, 2008Co-Authors: Montse Morell, Francesc X. Avilés, Patricia Czihal, Ralf Hoffmann, Laszlo Otvos, Salvador VenturaAbstract:Many cellular processes depend on protein-protein interactions. The identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Nevertheless, finding antagonists of protein interactions that work efficiently within the cell is a challenging task. Here, we describe the novel use of Bimolecular Fluorescence Complementation (BIFC) to detect compounds that block the interaction of target proteins in vivo. In the BIFC method, each interaction partner is fused to a complementary fragment of a fluorescent protein and interactions are detected by Fluorescence restoration after reporter reassembly. Here, we demonstrate that the inhibition of specific intracellular protein interactions results in a concomitant decrease in Fluorescence emission. We also show that integration of BIFC with flow cytometry might provide an effective means to detect interaction modulators by directly reading out changes in the reporter signal. The in vivo application of this approach is illustrated through monitoring the inhibition of the interaction between the Escherichia coli Hsp70 chaperone and a short peptidic substrate by pyrrhocoricin-derived antibacterial peptides.
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Study and selection of in vivo protein interactions by coupling Bimolecular Fluorescence Complementation and flow cytometry
Nature protocols, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Study and selection of in vivo protein interactions by coupling Bimolecular Fluorescence Complementation and flow cytometry
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detection of transient protein protein interactions by Bimolecular Fluorescence Complementation the abl sh3 case
Proteomics, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of Bimolecular Fluorescence Complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The Fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
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Detection of transient protein–protein interactions by Bimolecular Fluorescence Complementation: The Abl‐SH3 case
Proteomics, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of Bimolecular Fluorescence Complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The Fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
Montse Morell - One of the best experts on this subject based on the ideXlab platform.
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Monitoring the interference of protein-protein interactions in vivo by Bimolecular Fluorescence Complementation: the DnaK case.
Proteomics, 2008Co-Authors: Montse Morell, Francesc X. Avilés, Patricia Czihal, Ralf Hoffmann, Laszlo Otvos, Salvador VenturaAbstract:Many cellular processes depend on protein-protein interactions. The identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Nevertheless, finding antagonists of protein interactions that work efficiently within the cell is a challenging task. Here, we describe the novel use of Bimolecular Fluorescence Complementation (BIFC) to detect compounds that block the interaction of target proteins in vivo. In the BIFC method, each interaction partner is fused to a complementary fragment of a fluorescent protein and interactions are detected by Fluorescence restoration after reporter reassembly. Here, we demonstrate that the inhibition of specific intracellular protein interactions results in a concomitant decrease in Fluorescence emission. We also show that integration of BIFC with flow cytometry might provide an effective means to detect interaction modulators by directly reading out changes in the reporter signal. The in vivo application of this approach is illustrated through monitoring the inhibition of the interaction between the Escherichia coli Hsp70 chaperone and a short peptidic substrate by pyrrhocoricin-derived antibacterial peptides.
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Study and selection of in vivo protein interactions by coupling Bimolecular Fluorescence Complementation and flow cytometry
Nature protocols, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Study and selection of in vivo protein interactions by coupling Bimolecular Fluorescence Complementation and flow cytometry
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detection of transient protein protein interactions by Bimolecular Fluorescence Complementation the abl sh3 case
Proteomics, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of Bimolecular Fluorescence Complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The Fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
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Detection of transient protein–protein interactions by Bimolecular Fluorescence Complementation: The Abl‐SH3 case
Proteomics, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of Bimolecular Fluorescence Complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The Fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
David J Timson - One of the best experts on this subject based on the ideXlab platform.
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The GAL genetic switch: visualisation of the interacting proteins by split-EGFP Bimolecular Fluorescence Complementation.
Journal of basic microbiology, 2011Co-Authors: Emma Barnard, David J TimsonAbstract:A split-EGFP Bimolecular Fluorescence Complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.
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Interactions between the budding yeast IQGAP homologue Iqg1p and its targets revealed by a split-EGFP Bimolecular Fluorescence Complementation assay.
Cell biology international, 2008Co-Authors: Sevvel Pathmanathan, Emma Barnard, David J TimsonAbstract:A split-EGFP based Bimolecular Fluorescence Complementation (BiFC) assay has been used to detect interactions between the Saccharomyces cerevisiae cytoskeletal scaffolding protein Iqg1p and three targets: myosin essential light chain (Mlc1p), calmodulin (Cmd1p) and the small GTPase Cdc42p. The format of the BiFC assay used ensures that the proteins are expressed at wild type levels thereby avoiding artefacts due to overexpression. This is the first direct in vivo detection of these interactions; in each case, the complex is localised to discrete regions of the yeast cytoplasm. The labelling with EGFP fragments results in changes in growth kinetics, cell size and budding frequency. This is partly due to the reassembled EGFP locking the complexes into essentially permanent interactions. The consequences of this for Iqg1p interactions and BiFC assays in general are discussed.
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Development and implementation of split-GFP-based Bimolecular Fluorescence Complementation (BiFC) assays in yeast.
Biochemical Society transactions, 2008Co-Authors: Emma Barnard, Neil V Mcferran, Alan Trudgett, John Nelson, David J TimsonAbstract:BiFC (Bimolecular Fluorescence Complementation) is a tool for investigating interactions between proteins. Non-fluorescent fragments of, for example, GFP (green fluorescent protein) are fused to the interacting partners. The interaction brings the fragments together, which then fold, reassemble and fluoresce. This process can be carried out in living cells and provides information both on the interaction and its subcellular location. We have developed a split-GFP-based BiFC assay for use in the budding yeast Saccharomyces cerevisiae in which the modifications are carried out at the genomic level, thus resulting in the tagged yeast proteins being expressed at wild-type levels. The system is capable of detecting interactions in all subcellular compartments tested (the cytoplasm, mitochondria and nucleus) and makes a valuable addition to techniques for the investigation of protein-protein interactions in this model organism.
Francesc X. Avilés - One of the best experts on this subject based on the ideXlab platform.
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Monitoring the interference of protein-protein interactions in vivo by Bimolecular Fluorescence Complementation: the DnaK case.
Proteomics, 2008Co-Authors: Montse Morell, Francesc X. Avilés, Patricia Czihal, Ralf Hoffmann, Laszlo Otvos, Salvador VenturaAbstract:Many cellular processes depend on protein-protein interactions. The identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Nevertheless, finding antagonists of protein interactions that work efficiently within the cell is a challenging task. Here, we describe the novel use of Bimolecular Fluorescence Complementation (BIFC) to detect compounds that block the interaction of target proteins in vivo. In the BIFC method, each interaction partner is fused to a complementary fragment of a fluorescent protein and interactions are detected by Fluorescence restoration after reporter reassembly. Here, we demonstrate that the inhibition of specific intracellular protein interactions results in a concomitant decrease in Fluorescence emission. We also show that integration of BIFC with flow cytometry might provide an effective means to detect interaction modulators by directly reading out changes in the reporter signal. The in vivo application of this approach is illustrated through monitoring the inhibition of the interaction between the Escherichia coli Hsp70 chaperone and a short peptidic substrate by pyrrhocoricin-derived antibacterial peptides.
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Study and selection of in vivo protein interactions by coupling Bimolecular Fluorescence Complementation and flow cytometry
Nature protocols, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Study and selection of in vivo protein interactions by coupling Bimolecular Fluorescence Complementation and flow cytometry
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detection of transient protein protein interactions by Bimolecular Fluorescence Complementation the abl sh3 case
Proteomics, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of Bimolecular Fluorescence Complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The Fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
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Detection of transient protein–protein interactions by Bimolecular Fluorescence Complementation: The Abl‐SH3 case
Proteomics, 2007Co-Authors: Montse Morell, Alba Espargaró, Francesc X. Avilés, Salvador VenturaAbstract:Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of Bimolecular Fluorescence Complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The Fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
Yutaka Kodama - One of the best experts on this subject based on the ideXlab platform.
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A novel orange-colored Bimolecular Fluorescence Complementation (BiFC) assay using monomeric Kusabira-Orange protein.
BioTechniques, 2018Co-Authors: Yuta Fujii, Ayaka Yoshimura, Yutaka KodamaAbstract:The Bimolecular Fluorescence Complementation (BiFC) assay was developed as a tool for the visualization of protein–protein interactions in living cells. To date, many types of BiFC systems with dis...
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Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein–Protein Interaction: How to Calculate Signal-to-Noise Ratio
Methods in cell biology, 2013Co-Authors: Yutaka KodamaAbstract:Bimolecular Fluorescence Complementation (BiFC) is a technique to visualize protein-protein interactions in living cells, and has been widely used in various model organisms. The principle of the BiFC assay is based on the reconstitution of an intact fluorescent protein. The two non-fluorescent fragments are fused to proteins of interest that may interact. If the two proteins interact, the two non-fluorescent fragments are brought together to reconstitute an intact fluorescent protein. The purpose of this protocol is to calculate signal-to-noise (S/N) ratio in the Bimolecular Fluorescence Complementation (BiFC) assay and to provide a semi-quantitative analysis of protein-protein interaction (PPI) in living cells.
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Bimolecular Fluorescence Complementation bifc analysis of protein protein interaction how to calculate signal to noise ratio
Methods in Cell Biology, 2013Co-Authors: Yutaka KodamaAbstract:Bimolecular Fluorescence Complementation (BiFC) is a technique to visualize protein-protein interactions in living cells, and has been widely used in various model organisms. The principle of the BiFC assay is based on the reconstitution of an intact fluorescent protein. The two non-fluorescent fragments are fused to proteins of interest that may interact. If the two proteins interact, the two non-fluorescent fragments are brought together to reconstitute an intact fluorescent protein. The purpose of this protocol is to calculate signal-to-noise (S/N) ratio in the Bimolecular Fluorescence Complementation (BiFC) assay and to provide a semi-quantitative analysis of protein-protein interaction (PPI) in living cells.
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Bimolecular Fluorescence Complementation (BiFC): A 5-year update and future perspectives
BioTechniques, 2012Co-Authors: Yutaka KodamaAbstract:Over the past decade, Bimolecular Fluorescence Complementation (BiFC) has emerged as a key technique to visualize protein-protein interactions in a variety of model organisms. The BiFC assay is based on reconstitution of an intact fluorescent protein when two complementary non-fluorescent fragments are brought together by a pair of interacting proteins. While the originally reported BiFC method has enabled the study of many protein-protein interactions, increasing demands to visualize protein-protein interactions under various physiological conditions have not only prompted a series of recent BiFC technology improvements, but also stimulated interest in developing completely new approaches. Here we review current BiFC technology, focusing on the development and improvement of BiFC systems, the understanding of split sites in fluorescent proteins, and enhancements in the signal-to-noise ratio. In addition, we provide perspectives on possible future improvements of the technique.
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A bright green-colored Bimolecular Fluorescence Complementation assay in living plant cells
Plant Biotechnology, 2011Co-Authors: Yutaka KodamaAbstract:Protein-protein interactions are important for various biological and cellular processes. To analyze protein- protein interactions in living cells, the Bimolecular Fluorescence Complementation (BiFC) assay, based on structural Complementation of two non-fluorescent N- and C-terminal fragments from a fluorescent protein, has been developed and widely used in various research fields. Here I report a bright green-colored BiFC assay in living plant cells by using the N- terminal fragment (GN) of green fluorescent protein-S65T (GFP-S65T) and the C-terminal fragment (CC) of cyan fluorescent protein (CFP), but not GN and the C-terminal fragment (GC) of GFP. Fluorescence intensity of the GN/CC- based BiFC was 7-fold higher than that of the GN/GC-based BiFC. The emission spectrum of the GN/CC-based BiFC in planta was identical to that of full-length GFP-S65T. Ala163 residue within the CC fragment was found to be responsible for the improvement of the BiFC efficiency. These findings provide a BiFC method for in vivo protein-protein interaction studies to many GFP users in various research fields.