mCherry is a red fluorescent protein (RFP), classified as a luminescent protein, that belongs to a group of fluorescent protein chromophores. mCherry is a part of the mFruits protein family, which is a family of mRFPs, monomeric red fluorescent proteins. mCherry’s amino acid sequence positions it taxonomically in the green fluorescent protein, GFP, superfamily of proteins; these proteins all have fluorescent and bioluminescent functions and includes proteins such as DsRed, GFP, mStrawberry, and mOrange. Specifically, mCherry is known as being derived from the protein DsRed, which was originally found in Discosoma species, most often Discosoma sea anemones.
Function and Uses
The official biological process of mCherry is bioluminescence and this process allows mCherry to aid in the generation of precursor metabolites and gene expression analytics. mCherry is most commonly used as a fluorescent reporter, acting as a labeled tag for genes, cells, or organelles of interest. The emissive and fluorescent properties allow this protein to function as an intracellular probe, used especially for viewing constitutive gene expression. mCherry is a very valuable protein due to its function as a fluorescent tag, allowing different cell components to be viewed in a variety of scenarios, such as analyzing gene expression, genome editing, and identifying species of microorganisms. mCherry is also used for protein study and research, as it is a common spectroscopic label that can identify the mobility, localization, and interactions of different proteins in tissues, cells, and organelles. mCherry, along with being used to analyze gene expression, is also commonly used to analyze the specific functions of these genes within the cell. mCherry is used for long-term studies, both in vivo and in vitro, since it has high photostability, which makes it a very common protein to use, especially in the fields of biotechnology. Also, mCherry is among one of the most popular mFruit proteins used for cell biology research due to its high photostability, maturation properties, and tagging tolerance.
Structure
The gene responsible for mCherry is 711 base pairs and is oftentimes inserted and transcribed from plasmids to obtain a sizable quantity for the tagging and probing of cellular components. mCherry is a monomeric protein that contains 236 amino acids and has a mass of 26.7 kDa, making it a low molecular weight protein that often folds faster than tetrameric proteins, such as its parent DsRed. The amino acids with the highest count in the mCherry primary sequence are lysine, glycine, and glutamate, each of which are in mCherry’s primary structure 24 times, each composing 10% of the entire sequence. The amino acid with the least number of residues in the primary sequence of mCherry is alanine, with only 11 residues, making up only 5% of the protein’s sequence. The total atom count in mCherry is 2121 and the whole protein is composed of only one polypeptide chain, thus leading to a tertiary structure once fully folded. The complete three-dimensional structure of mCherry has been determined by x-ray diffraction which gave an experimental resolution of 1.36 angstroms. mCherry is composed of only three alpha helices with a beta barrel that is made up of 13 beta sheets. Thus, the protein is composed mostly of beta sheets which surround the chromophore (ligand) and central helix as a barrel-like structure, which shields the chromophore and central helix from the cytosolic environment. The central helix is composed of the only three alpha helices present in the protein’s structure.
The chromophore-binding domain, the functional area of mCherry that is responsible for binding the chromophore that gives it its color, is due mainly to 3 residues – tyrosine72, glycine73, and methionine71. Once mCherry is translated in the cell on a ribosome, these chromophore-binding amino acids are modified with imidazoline groups via post-translational modification. The red emission is produced by the generation of an acylimine linkage in the backbone of the polypeptide during a second oxidation step that occurs due to illumination. The chromophore environment is then indirectly modified to produce the red emission shift that can be seen with fluorescence spectroscopy or microscopy. This indirect modification includes the movement of the charged lysine70 residue and the protonation of the glutamine215 residue, both of which alter the distribution of the electron-density in the chromophore, thus causing the red emission.
As mentioned above, the ligand for mCherry is the chromophore CH6, which is a popular chromophore among red fluorescent proteins. This molecule, which is characterized as a methionine, tyrosine, and glycine chromophore, has a formula of C16H19N3O4S, with a molecular weight of 349.9 grams/mol, and is the molecule responsible for giving mCherry its red color and fluorescent properties. The CH6 chromophore is similar to the ligand of DsRed and related mFruit fluorescent proteins; the relationship is known by the similarities in the extension of the pi-system of GFP’s chromophore, specifically the extension of another N-acylimine group, between the chromophore present in mCherry and the parental DsRed. The chromophore binds to mCherry via L-peptide linkages and is structurally supported by noncovalent interactions within the central helix. Two alpha helices in the central helix core are bound to the chromophore via L-peptide linkages, producing two of the alpha helices with the chromophore in between them all within the beta barrel. Serine69 on one alpha helix forms a peptide linkage with the chromophore at a carboxyl carbon. This same carboxyl carbon forms another peptide linkage with the second alpha helix on the amino acid residue phenylalanine65. Due to these binding locations and residues, in mCherry, the imidazoline ring and the phenolate rings of the CH6 chromophore, when bound to the polypeptide in its fully folded three-dimensional structure, have tilt and twist angles of 11 and 14 degrees, respectively.
The functional domains seen in both alpha helices and beta sheets present in the mCherry protein are related to those found in GFP-like, or fluorescent, proteins. Thus, the folds that these domains undertake are common in most GFP-like proteins, especially those that have a fluorescent or luminescent function. Likewise, the domains present only in the beta barrel portion of the protein are also homologous to the domains found in GFP and GFP-related proteins, such as mStrawberry and mOrange. These proteins contain a similar structure to mCherry, which means they also contain a beta barrel that composes most of their three-dimensional structure with the same domains that are present in mCherry’s beta barrel. The beta barrel of mCherry, since it is closely related to the beta barrel of other mFruits, and is a derivative of DsRed, contains a weakness between beta sheet 7 and beta sheet 10, as is seen with all other mFruit beta barrels, originating in the DsRed fluorescent protein. This weakness stems from parental DsRed being a tetrameric protein and causes an increase in permeability to oxygen.
Visualization
mCherry is visible under UV light, making it a photoprotein, which aids in its ease of detection. mCherry emits light between 550 and 650 nm and absorbs light between 540 and 590 nm and uses pi-electron conjugation to produce a red emission and absorption. The quantum yield, as explained by the non-planarity of the chromophore, which, in mCherry, is extended, is 0.22. mCherry is constitutively fluorescent, meaning it can be visible, in at least some degree, at any time by use of the UV spectra. mCherry is most often visualized via fluorescence spectroscopy or fluorescence microscopy. This protein exists in multiple brightness states, which means that mCherry’s visual emission fluctuates in amplitude. These states are useful since their brightness is not dark, like it is with mCherry’s relative, the fluorescent protein mRFP1, but instead has long-lived, two states with differing values in brightness, which include bright and dim states.
