The reduction-oxidation sensitive

green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...

(roGFP) is a

engineered to be sensitive to changes in the local

redox Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate (chemistry), substrate change. Oxidation is the loss of Electron, electrons or an increase in the oxidation state, while reduction ...

environment. roGFPs are used as

-sensitive biosensors. In 2004, researchers in S. James Remington's lab at the University of Oregon constructed the first roGFPs by introducing two

cysteine Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, sometime ...

s into the beta barrel structure of GFP. The resulting engineered protein could exist in two different oxidation states (reduced

dithiol In organic chemistry, a dithiol is a type of organosulfur compound with two thiol () functional groups. Their properties are generally similar to those of monothiols in terms of solubility, odor, and volatility. They can be classified according ...

or oxidized disulfide), each with different

fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

properties. Originally, members of the Remington lab published six versions of roGFP, termed roGFP1-6 (see more structural details below). Different groups of researchers introduced cysteines at different locations in the GFP molecule, generally finding that

s introduced at the

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...

positions 147 and 204 produced the most robust results. roGFPs are often genetically encoded into cells for ''in-vivo'' imaging of redox potential. In cells, roGFPs can generally be modified by redox enzymes such as

glutaredoxin Glutaredoxins (also known as Thioltransferase) are small redox enzymes of approximately one hundred amino-acid residues that use glutathione as a cofactor. In humans this oxidation repair enzyme is also known to participate in many cellular functi ...

or thioredoxin. roGFP2 preferentially interacts with

glutaredoxins Glutaredoxins (also known as Thioltransferase) are small redox enzymes of approximately one hundred amino-acid residues that use glutathione as a cofactor. In humans this oxidation repair enzyme is also known to participate in many cellular functi ...

and therefore reports the cellular glutathione

redox potential Redox potential (also known as oxidation / reduction potential, ''ORP'', ''pe'', ''E_'', or E_) is a measure of the tendency of a chemical species to acquire electrons from or lose electrons to an electrode and thereby be reduced or oxidised respe ...

. Various attempts have been made to make roGFPs that are more amenable to live-cell imaging. Most notably, substituting three positively-charged amino acids adjacent to the disulfide in roGFP1 drastically improves the response rate of roGFPs to physiologically relevant changes in redox potential. The resulting roGFP variants, named roGFP1-R1 through roGFP1-R14, are much more suitable for live-cell imaging. The roGFP1-R12 variant has been used to monitor redox potential in bacteria and yeast, but also for studies of spatially-organized redox potential in live, multicellular organisms such as the model nematode

C. elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...

. In addition, roGFPs are used to investigate the topology of ER proteins, or to analyze the ROS production capacity of chemicals. One notable improvement to roGFPs occurred in 2008, when the specificity of roGFP2 for glutathione was 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 Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

to a cellular compartment, it is possible to measure the glutathione

in a specific cellular compartment in real-time and therefore provides major advantages compared to other invasive static methods e.g.

HPLC High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pa ...

. Given the variety of roGFPs, some effort has been made to benchmark their performance. For example, members of

Javier Apfeld Javier may refer to: Arts, entertainment, and media * Javier, in video game '' Advance Wars: Dual Strike'' * Javier Rios, a character in the Monsters, Inc. franchise. * ''Javier'' (album), a 2003 album by the American singer Javier Colon, known ...

's group published a method in 2020 describing the 'suitable ranges' of different roGFPs, determined by how sensitive each sensor is to experimental noise in different redox conditions.

Species of roGFP

See Kostyulk 2020 for a more comprehensive review of different redox sensors.

References

{{Reflist, 30em Biochemistry detection methods Biosensors Cell imaging Fluorescent proteins

Species of roGFP

See also

References