scholarly journals Aequorea’s secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000936
Author(s):  
Gerard G. Lambert ◽  
Hadrien Depernet ◽  
Guillaume Gotthard ◽  
Darrin T. Schultz ◽  
Isabelle Navizet ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Polypeptide Chain ◽  
De Novo ◽  
Fluorescent Proteins ◽  
Transcriptome Assembly ◽  
X Ray ◽  
X Ray Crystallography ◽  
Green Fluorescent ◽  
New Generation

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

scholarly journals Aequorea victoria’s secrets

2019 ◽  
Author(s):  
Gerard G. Lambert ◽  
Hadrien Depernet ◽  
Guillaume Gotthard ◽  
Darrin T. Schultz ◽  
Isabelle Navizet ◽  
...  
Keyword(s):  
Blue Light ◽  
Fluorescent Protein ◽  
Polypeptide Chain ◽  
De Novo ◽  
Transcriptome Assembly ◽  
X Ray ◽  
Aequorea Victoria ◽  
X Ray Crystallography ◽  
New Generation

Using mRNA-Seq and de novo transcriptome assembly, we identified, cloned and characterized nine previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from avGFP. Among these FPs are the brightest GFP homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including two that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

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.

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 Next-Generation Fluorogen-Based Reporters and Biosensors for Advanced Bioimaging

2019 ◽  
Vol 20 (24) ◽  
pp. 6142 ◽  
Author(s):  
Tiphaine Péresse ◽  
Arnaud Gautier
Keyword(s):  
Green Fluorescent Protein ◽  
Temporal Resolution ◽  
Fluorescent Probes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Systems ◽  
Next Generation ◽  
Fluorescent Reporters ◽  
Green Fluorescent ◽  
New Generation

Our ability to observe biochemical events with high spatial and temporal resolution is essential for understanding the functioning of living systems. Intrinsically fluorescent proteins such as the green fluorescent protein (GFP) have revolutionized the way biologists study cells and organisms. The fluorescence toolbox has been recently extended with new fluorescent reporters composed of a genetically encoded tag that binds endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activates their fluorescence. This review presents the toolbox of fluorogen-based reporters and biosensors available to biologists. Various applications are detailed to illustrate the possible uses and opportunities offered by this new generation of fluorescent probes and sensors for advanced bioimaging.

scholarly journals Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

2021 ◽  
Vol 22 (4) ◽  
pp. 2127
Author(s):  
Jakub Suchodolski ◽  
Anna Krasowska
Keyword(s):  
Candida Albicans ◽  
Multidrug Resistance ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Pathogenic Fungus ◽  
Serum Levels ◽  
Efflux Transporters ◽  
Fluconazole Resistance ◽  
Green Fluorescent

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

scholarly journals Nematode Infection Triggers the de Novo Formation of Unloading Phloem That Allows Macromolecular Trafficking of Green Fluorescent Protein into Syncytia

2005 ◽  
Vol 138 (1) ◽  
pp. 383-392 ◽  
Author(s):  
Stefan Hoth ◽  
Alexander Schneidereit ◽  
Christian Lauterbach ◽  
Joachim Scholz-Starke ◽  
Norbert Sauer
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Nematode Infection ◽  
Macromolecular Trafficking ◽  
Green Fluorescent

scholarly journals X-ray crystallographic studies on the hydrogen isotope effects of green fluorescent protein at sub-ångström resolutions

2019 ◽  
Vol 75 (12) ◽  
pp. 1096-1106 ◽  
Author(s):  
Yang Tai ◽  
Kiyofumi Takaba ◽  
Yuya Hanazono ◽  
Hoang-Anh Dao ◽  
Kunio Miki ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Active Site ◽  
Hydrogen Isotope ◽  
Fluorescent Protein ◽  
Isotope Effects ◽  
Protonation State ◽  
X Ray ◽  
Two Samples ◽  
Deuterated Proteins ◽  
Green Fluorescent

Hydrogen atoms are critical to the nature and properties of proteins, and thus deuteration has the potential to influence protein function. In fact, it has been reported that some deuterated proteins show different physical and chemical properties to their protiated counterparts. Consequently, it is important to investigate protonation states around the active site when using deuterated proteins. Here, hydrogen isotope effects on the S65T/F99S/M153T/V163A variant of green fluorescent protein (GFP), in which the deprotonated B form is dominant at pH 8.5, were investigated. The pH/pD dependence of the absorption and fluorescence spectra indicates that the protonation state of the chromophore is the same in protiated GFP in H2O and protiated GFP in D2O at pH/pD 8.5, while the pK a of the chromophore became higher in D2O. Indeed, X-ray crystallographic analyses at sub-ångström resolution revealed no apparent changes in the protonation state of the chromophore between the two samples. However, detailed comparisons of the hydrogen OMIT maps revealed that the protonation state of His148 in the vicinity of the chromophore differed between the two samples. This indicates that protonation states around the active site should be carefully adjusted to be the same as those of the protiated protein when neutron crystallographic analyses of proteins are performed.

Sign in / Sign up

Export Citation Format

Share Document