RoGFP
Encyclopedia
The reduction-oxidation sensitive green fluorescent protein
(roGFP) is a redox
sensitive biosensor
. Two cysteine
s were introduced into the beta barrel structure of the GFP, the oxidation state (reduced dithiol or the oxidized disulfide
) of the engineered thiols determines the fluorescence
properties of the sensor. Originally different roGFP versions were presented, for in vivo imaging of reducing compartments such as the cytosol
roGFP2, with cysteine
s introduced at the amino acid
positions 147 and 204, turned out to be the most suitable version. roGFP2 preferentially interacts with glutaredoxins and therefore reports the cellular glutathione
redox potential. The specificity of roGFP2 for glutathione
is further increased by linking it to the human glutaredoxin 1 (Grx1).
By expressing the Grx1-roGFP fusion sensors in the organism of interest and/or targeting the protein
to a cellular compartment allows to measure the glutathione
redox potential in a specific cellular compartment at real-time and therefore provides major advantages compared to other invasive static methods e.g. HPLC
.
In addition, roGFPs are used to investigate the topology of ER
proteins, or to analyze the ROS
production capacity of chemicals.
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(roGFP) is a redox
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
sensitive biosensor
Biosensor
A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.It consists of 3 parts:* the sensitive biological element A biosensor is an analytical device for the detection of an analyte that combines a biological...
. Two cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
s were introduced into the beta barrel structure of the GFP, the oxidation state (reduced dithiol or the oxidized disulfide
Disulfide
In chemistry, a disulfide usually refers to the structural unit composed of a linked pair of sulfur atoms. Disulfide usually refer to a chemical compound that contains a disulfide bond, such as diphenyl disulfide, C6H5S-SC6H5....
) of the engineered thiols determines the fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
properties of the sensor. Originally different roGFP versions were presented, for in vivo imaging of reducing compartments such as the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
roGFP2, with cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
s introduced at the amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
positions 147 and 204, turned out to be the most suitable version. roGFP2 preferentially interacts with glutaredoxins and therefore reports the cellular glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
redox potential. The specificity of roGFP2 for glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
is further increased by linking it to the human glutaredoxin 1 (Grx1).
By expressing the Grx1-roGFP fusion sensors in the organism of interest and/or targeting the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
to a cellular compartment allows to measure the glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
redox potential in a specific cellular compartment at real-time and therefore provides major advantages compared to other invasive static methods e.g. HPLC
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
.
In addition, roGFPs are used to investigate the topology of ER
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
proteins, or to analyze the ROS
Reactive oxygen species
Reactive oxygen species are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons....
production capacity of chemicals.