scholarly journals Dynamic tuning of FRET in a green fluorescent protein biosensor

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw4988 ◽  
Author(s):  
Pablo Trigo-Mourino ◽  
Thomas Thestrup ◽  
Oliver Griesbeck ◽  
Christian Griesinger ◽  
Stefan Becker
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
X Ray ◽  
Dynamic Tuning ◽  
Green Fluorescent

Förster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with B02-dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.1 GHz. These structural data provide the unprecedented opportunity to calculate FRET from the x-ray structure and to compare it to experimental data in solution. We find that interdomain dynamics limits the FRET effect and show that a rigidification of the sensor further enhances FRET.

scholarly journals The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

2019 ◽  
Author(s):  
Jacob R. Pope ◽  
Rachel L. Johnson ◽  
W. David Jamieson ◽  
Harley L Worthy ◽  
Senthilkumar D. Kailasam ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Fluorescent Properties ◽  
Transition Dipole ◽  
Green Fluorescent

AbstractFluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ∼86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.Abstract Figure

scholarly journals Using green fluorescent protein to study intracellular signalling

2001 ◽  
Vol 170 (2) ◽  
pp. 297-306 ◽  
Author(s):  
JM Tavare ◽  
LM Fletcher ◽  
GI Welsh
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cell Signalling ◽  
Resonance Energy ◽  
Intracellular Signalling ◽  
Differential Sensitivity ◽  
Signalling Pathways ◽  
Green Fluorescent

Subcellular compartmentalisation of signalling molecules is an important phenomenon not only in defining how a signalling pathway is activated but also in influencing the desired physiological output of that pathway (e.g. cell growth or differentiation, regulation of metabolism, cytoskeletal changes etc.). Biochemical analyses of protein and lipid compartmentalisation by, for example, subcellular fractionation presents many technical difficulties. However, this aspect of cell signalling research has seen a major revolution thanks to the cloning and availability of a variety of mutant green fluorescent protein derivatives with distinct molecular properties. Mutants with increased brightness, altered excitation and emission maxima, altered stability and differential sensitivity to pH, are now in widespread use for following the trafficking and function of proteins in living cells and for monitoring the intracellular environment. In this review we focus on some of the recent developments in the use of green fluorescent proteins for studying intracellular signalling pathways often with special reference to the actions of insulin. We also discuss the future utility of these proteins to analyse protein--protein interactions in signalling pathways using fluorescence resonance energy transfer.

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

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
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.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

2001 ◽  
Vol 357 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan P. WAUD ◽  
Alexandra BERMÚDEZ FAJARDO ◽  
Thankiah SUDHAHARAN ◽  
Andrew R. TRIMBY ◽  
Jinny JEFFERY ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Donor And Acceptor ◽  
Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

scholarly journals Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

2016 ◽  
Vol 72 (12) ◽  
pp. 1298-1307 ◽  
Author(s):  
Damien Clavel ◽  
Guillaume Gotthard ◽  
David von Stetten ◽  
Daniele De Sanctis ◽  
Hélène Pasquier ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Directed Evolution ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
X Rays ◽  
Visible Absorption ◽  
X Ray ◽  
X Ray Crystallography ◽  
Green Fluorescent

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent proteinlanYFP fromBranchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures oflanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV–visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

Sign in / Sign up

Export Citation Format

Share Document