scholarly journals GFP fusion protein with embedded foreign peptide

2019 ◽  
Author(s):  
K.A. Glukhova ◽  
V.G. Klyashtorny ◽  
B.S. Melnik
Keyword(s):  
Green Fluorescent Protein ◽  
Tertiary Structure ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Chimeric Protein ◽  
Bound Water ◽  
Alpha Helix ◽  
Point Of View ◽  
Foreign Peptide ◽  
Green Fluorescent

AbstractFrom the point of view structural biology and protein engineering the green fluorescent protein (GFP) is an exceptionally attracting object. The tertiary structure of GFP is quite unique: it reminds a “cylinder” or a “barrel” consisting of beta-layers that contains an alpha-helix inside. The “barrel” is a special container for an alpha-helix serving to protect the latter from the influence of the surroundings. Therefore a reasonable question arises whether the “barrel” can function as a container for preservation and isolation of other peptides. The alpha-helix itself contains hydrophilic amino acids, whereas inside the barrel there are many molecules of bound water. We supposed that the central alpha-helix of green fluorescent protein could be substituted for foreign peptide. In this study we checked the possibility for creation of such a system on base of GFP, where the toxic peptide is isolated from the environment inside the protein. The modification of green fluorescent protein was carried out. An antimicrobial peptide was inserted into the central alpha-helix. The results of our experiments show that such a chimeric protein is compact, soluble and non-toxic for the producing cell culture, but its structure is destabilized. The obtained data show that the idea of use of green fluorescent proteins as a «container» for storing foreign peptides could be realized.

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

ACS Nano ◽  
2015 ◽  
Vol 9 (10) ◽  
pp. 9528-9541 ◽  
Author(s):  
Sam Duwé ◽  
Elke De Zitter ◽  
Vincent Gielen ◽  
Benjamien Moeyaert ◽  
Wim Vandenberg ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

scholarly journals Relationship between Acropora millepora juvenile fluorescence and composition of newly established Symbiodinium assemblage

2018 ◽  
Author(s):  
KM Quigley ◽  
ME Strader ◽  
MV Matz
Keyword(s):  
Life Cycle ◽  
Green Fluorescent Protein ◽  
Coral Reefs ◽  
Deep Sequencing ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Biological Interaction ◽  
Acropora Millepora ◽  
Continuous Trait ◽  
Green Fluorescent

AbstractCoral-dinoflagellate symbiosis is the key biological interaction enabling existence of modern-type coral reefs, but the mechanisms regulating initial host–symbiont attraction, recognition and symbiont proliferation thus far remain largely unclear. A common reef-building coral, Acropora millepora, displays conspicuous fluorescent polymorphism during all phases of its life cycle, due to the differential expression of fluorescent proteins (FPs) of the green fluorescent protein family. In this study, we examine whether fluorescent variation in young coral juveniles exposed to natural sediments is associated with the uptake of disparate Symbiodinium assemblages determined using ITS-2 deep sequencing. We found that Symbiodinium assemblages varied significantly when redness values varied, specifically in regards to abundances of clades A and C. Whether fluorescence was quantified as a categorical or continuous trait, clade A was found at higher abundances in redder juveniles. These preliminary results suggest juvenile fluorescence may be associated with Symbiodinium uptake, potentially acting as either as an attractant to ecologically specific types or as a mechanism to modulate the internal light environment to control Symbiodinium physiology within the host.

Against the NEER principle: The third type of photochromism for GFP chromophore derivatives

2021 ◽  
Author(s):  
Jun-Wei Liao ◽  
Robert Sung ◽  
Kuangsen Sung
Keyword(s):  
Excited State ◽  
Green Fluorescent Protein ◽  
Proton Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
The Third ◽  
Excited State Proton Transfer ◽  
Gfp Chromophore ◽  
Green Fluorescent

Photochromism is the heart of photochromic fluorescent proteins. Excited-state proton transfer (ESPT) is the major photochromism for green fluorescent protein (GFP) and Z-E photoisomerization through τ-torsion is the major photochromism...

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein

1999 ◽  
Vol 112 (11) ◽  
pp. 1709-1719 ◽  
Author(s):  
C. Ostlund ◽  
J. Ellenberg ◽  
E. Hallberg ◽  
J. Lippincott-Schwartz ◽  
H.J. Worman
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Muscular Dystrophy ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Transmembrane Segment ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Green Fluorescent

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.

scholarly journals Coronavirus Spike Glycoprotein, Extended at the Carboxy Terminus with Green Fluorescent Protein, Is Assembly Competent

2004 ◽  
Vol 78 (14) ◽  
pp. 7369-7378 ◽  
Author(s):  
Berend Jan Bosch ◽  
Cornelis A. M. de Haan ◽  
Peter J. M. Rottier
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Viral Envelope ◽  
Wild Type ◽  
Virus Propagation ◽  
S Protein ◽  
Protein Functions ◽  
Geometrical Constraints ◽  
Green Fluorescent

ABSTRACT Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.

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 A photoactivatable green-fluorescent protein from the phylum Ctenophora

2009 ◽  
Vol 277 (1685) ◽  
pp. 1155-1160 ◽  
Author(s):  
Steven H. D. Haddock ◽  
Nadia Mastroianni ◽  
Lynne M. Christianson
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Optical Probes ◽  
Fluorescent Emission ◽  
The Family ◽  
Fluorescent State ◽  
Green Fluorescent

Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the x YG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals.

scholarly journals Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

2010 ◽  
Vol 76 (17) ◽  
pp. 5990-5994 ◽  
Author(s):  
Thomas Drepper ◽  
Robert Huber ◽  
Achim Heck ◽  
Franco Circolone ◽  
Anne-Kathrin Hillmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Flavin Mononucleotide ◽  
Fluorescence Signal ◽  
Reporter Protein ◽  
Reporter Proteins ◽  
Green Fluorescent

ABSTRACT Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

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.

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