Browse
Submit Data
Databases
API
Help

Dataset Information

61 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A photostable monomeric superfolder green fluorescent protein.

ABSTRACT: The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N- and C-terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general-purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.

SUBMITTER: Valbuena FM

PROVIDER: S-EPMC7508230 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A photostable monomeric superfolder green fluorescent protein.

Valbuena Fernando M FM Fitzgerald Ivy I Strack Rita L RL Andruska Neal N Smith Luke L Glick Benjamin S BS

Traffic (Copenhagen, Denmark) 20200616 8

The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP ...[more]

PMID: 32415747

Similar Datasets

A highly photostable and bright green fluorescent protein.

Project description:The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

| S-EPMC9287174 | biostudies-literature

Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant.

Project description:Aims: Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). Results: SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo. Innovation: This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. Conclusion: Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1-18.

| S-EPMC9293687 | biostudies-literature

Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

Project description:Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

| S-EPMC4815332 | biostudies-literature

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.

Project description:We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

| S-EPMC3811051 | biostudies-literature

A Superfolder Green Fluorescent Protein-Based Biosensor Allows Monitoring of Chloride in the Endoplasmic Reticulum.

Project description:Though the concentration of chloride has been measured in the cytoplasm and in secretory granules of live cells, it cannot be measured within the endoplasmic reticulum (ER) due to poor fluorescence of existing biosensors. We developed a fluorescent biosensor composed of a chloride-sensitive superfolder GFP and long Stokes-shifted mKate2 for simultaneous chloride and pH measurements that retained fluorescence in the ER lumen. Using this sensor, we showed that the chloride concentration in the ER is significantly lower than that in the cytosol. This improved biosensor enables dynamic measurement of chloride in the ER and may be useful in other environments where protein folding is challenging.

| S-EPMC9425558 | biostudies-literature

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

Project description: Not available

| S-EPMC11040664 | biostudies-literature

Green monomeric photosensitizing fluorescent protein for photo-inducible protein inactivation and cell ablation.

Project description:BackgroundPhotosensitizing fluorescent proteins, which generate reactive oxygen species (ROS) upon light irradiation, are useful for spatiotemporal protein inactivation and cell ablation. They give us clues about protein function, intracellular signaling pathways and intercellular interactions. Since ROS generation of a photosensitizer is specifically controlled by certain excitation wavelengths, utilizing colour variants of photosensitizing protein would allow multi-spatiotemporal control of inactivation. To expand the colour palette of photosensitizing protein, here we developed SuperNova Green from its red predecessor, SuperNova.ResultsSuperNova Green is able to produce ROS spatiotemporally upon blue light irradiation. Based on protein characterization, SuperNova Green produces insignificant amounts of singlet oxygen and predominantly produces superoxide and its derivatives. We utilized SuperNova Green to specifically inactivate the pleckstrin homology domain of phospholipase C-δ1 and to ablate cancer cells in vitro. As a proof of concept for multi-spatiotemporal control of inactivation, we demonstrate that SuperNova Green can be used with its red variant, SuperNova, to perform independent protein inactivation or cell ablation studies in a spatiotemporal manner by selective light irradiation.ConclusionDevelopment of SuperNova Green has expanded the photosensitizing protein toolbox to optogenetically control protein inactivation and cell ablation.

| S-EPMC5928576 | biostudies-literature

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein.

Project description:The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

| S-EPMC8329867 | biostudies-literature

A new bright green-emitting fluorescent protein--engineered monomeric and dimeric forms.

Project description:Fluorescent proteins have become essential tools in molecular and biological applications. Here, we present a novel fluorescent protein isolated from warm water coral, Cyphastrea microphthalma. The protein, which we named vivid Verde fluorescent protein (VFP), matures readily at 37 degrees C and emits bright green light. Further characterizations revealed that VFP has a tendency to form dimers. By creating a homology model of VFP, based on the structure of the red fluorescent protein, DsRed, we were able to make mutations that alter the protein's oligomerization state. We present two proteins, mVFP and mVFP1, that are both exclusively monomeric, and one protein, dVFP, which is dimeric. We characterized the spectroscopic properties of VFP and its variants in comparison with enhanced green fluorescent protein (EGFP), a widely used variant of GFP. All the VFP variants are at least twice as bright as EGFP. Finally, we demonstrated the effectiveness of the VFP variants in both in vitro and in vivo detection applications.

| S-EPMC2855763 | biostudies-literature

A bright and photostable photoconvertible fluorescent protein.

Project description:Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the approximately 10-nm localization precision of its parent.

| S-EPMC2745648 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data