Monitoring Protein Interactions in the Living Cell Through the Fluorescence Decays of the Cyan Fluorescent Protein

ChemPhysChem ◽  
2006 ◽  
Vol 7 (7) ◽  
pp. 1442-1454 ◽  
Author(s):  
Regis Grailhe ◽  
Fabienne Merola ◽  
Jacqueline Ridard ◽  
Stephen Couvignou ◽  
Chantal Le Poupon ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Cyan Fluorescent Protein

scholarly journals Cover Picture: Monitoring Protein Interactions in the Living Cell Through the Fluorescence Decays of the Cyan Fluorescent Protein (ChemPhysChem 7/2006)

ChemPhysChem ◽  
2006 ◽  
Vol 7 (7) ◽  
pp. 1393-1393
Author(s):  
Regis Grailhe ◽  
Fabienne Merola ◽  
Jacqueline Ridard ◽  
Stephen Couvignou ◽  
Chantal Le Poupon ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Cyan Fluorescent Protein

Living-Cell MicroRNA Imaging with Self-Assembling Fragments of Fluorescent Protein-Mimic RNA Aptamer

ACS Sensors ◽  
2021 ◽  
Author(s):  
Yu Gu ◽  
Li-Juan Huang ◽  
Wei Zhao ◽  
Ting-Ting Zhang ◽  
Mei-Rong Cui ◽  
...  
Keyword(s):  
Living Cell ◽  
Fluorescent Protein ◽  
Rna Aptamer ◽  
Self Assembling

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

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
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.

scholarly journals 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

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
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.

Ultrafast Proton Shuttling in Psammocora Cyan Fluorescent Protein

2013 ◽  
Vol 117 (38) ◽  
pp. 11134-11143 ◽  
Author(s):  
John T. M. Kennis ◽  
Ivo H. M. van Stokkum ◽  
Dayna S. Peterson ◽  
Anjali Pandit ◽  
Rebekka M. Wachter
Keyword(s):  
Fluorescent Protein ◽  
Cyan Fluorescent Protein ◽  
Proton Shuttling

scholarly journals Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

2009 ◽  
Vol 204 (3) ◽  
pp. 275-285 ◽  
Author(s):  
Akiko Katoh ◽  
Hiroaki Fujihara ◽  
Toyoaki Ohbuchi ◽  
Tatsushi Onaka ◽  
W Scott Young ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusion Gene ◽  
Plasma Osmolality ◽  
Cyan Fluorescent Protein ◽  
Posterior Pituitary ◽  
Protein Fusion ◽  
Transgenic Rats ◽  
Salt Loading ◽  
Wide Range ◽  
Enhanced Cyan Fluorescent Protein

We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt–eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt–eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.

scholarly journals Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays

2012 ◽  
Vol 3 (3) ◽  
pp. e288-e288 ◽  
Author(s):  
C Wong ◽  
D J Anderson ◽  
E F Lee ◽  
W D Fairlie ◽  
M J C Ludlam
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Direct Visualization ◽  
Family Protein

scholarly journals High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

2021 ◽  
pp. 247255522098504
Author(s):  
Jeffrey R. Simard ◽  
Linda Lee ◽  
Ellen Vieux ◽  
Reina Improgo ◽  
Trang Tieu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rapid Development ◽  
Dependent Manner ◽  
Western Blots ◽  
Advantages And Disadvantages ◽  
Degradation Rates

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

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.

Cyan Fluorescent Protein Carries a Constitutive Mutation That Prevents Its Dimerization

Biochemistry ◽  
2011 ◽  
Vol 50 (4) ◽  
pp. 437-439 ◽  
Author(s):  
Agathe Espagne ◽  
Marie Erard ◽  
Karine Madiona ◽  
Valérie Derrien ◽  
Gabriella Jonasson ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cyan Fluorescent Protein ◽  
Constitutive Mutation
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