Andrew J. Hirning: mRFP

=Red Fluorescent Protein=

Summary Information

 * 1) Paper1 pmid=15558047

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

Article Annotation
!Note: This paper was published in Nature Biotechnology in December of 2004. The full text cannot be accessed except with a subscription or in PDF form!

Table 1
The first table of this paper enumerates all of the proteins derived from the Discoma red fluorescent protein (DsRed), from the original monomeric red fluorescent protein (mRFP) to the red-shifted and blue-shifted variants. Most of the proteins created from the original red protein are monomers, due to the toxicity and assembly problems resulting from the use of the original tetrameric proteins in vivo. However, the variant dTomato exists as a dimer, and was genetically modified to be a linked, tandem dimer, known as tdTomato. This tandem dimer variant possesses the same fluorescent properties as the original, save that it double the intensity compared to their original DsRed protein. From the original mRFP, successive sets of mutations yielded mRFP1.1-1.4, which included the addition of GFP-like N and C termini to the protein. None of the final engineered proteins exhibited intensity better than 80% of the original, save tdTomato. However, tdTomato is actually two dimeric proteins joined to each other, so the actual intensity is only 80% of the original that the dTomato showed. Another quality quantified by the chart is the quantum efficiency of the protein. Quantum efficiency is the number of electrons excited per photon that hits the protein, and is usually expressed either as a decimal, or as electrons per thousand photons. None of the engineered proteins met the efficiency of the original DsRed, which posted an efficiency of 0.79. However, many of the proteins did post a quantum efficiency higher than EGFP, which has a quantum efficiency of 0.60. The final quality that is changed for the different engineered proteins is the maturation time, or the time required for the protein to fold and autocatalyze the formation of the fluorophore. All of the engineered proteins had a faster maturation time than the original DsRed, with the fastest Tm being that of the most red shifted protein, mCherry, which matures in only 15 minutes.

Figure 1
The first figure of this paper is a summary graphic of all of the variants of DsRed

Panel A
This panel shows the excitation wavelengths of the different DsRed derivatives color coded by protein. They are all excited between 540-590nm, with the exception of mHoneydew, which has an excitation maximum at 487/504nm.

Panel B
This panel shows the emission wavelengths of the different DsRed derivatives, again, color coded by protein. These are all between 550-610nm, again with the exception of mHoneydew, which has it's emission maximum at 537/562.

Panels C & D
These two panels are photos of the different derivative proteins in solution. Panel C is a picture of all of the proteins in white light. Panel D, on the other hand, is a composite of several images, each taken at a different excitation wavelength from 480 to 560nm.

Figure 2
The second figure in this paper is made up of two panels.

Panel A
The first panel is a sequence alignment of the protein sequences for the different derivatives of DsRed. The buried/internal residues in each protein are shaded gray. The mutations made to DsRed to get to mRFP1 are highlighted in blue. The mutations that were made to DsRed for each protein are highlighted in the sequence in a color corresponding to the color of the protein when it fluoresces. Several of the modified proteins had their carboxyl and amino terminals substituted for Enhanced Green Fluorescent Protein type termini to more easily allow for fusion to other proteins. The coloration really shows how the fluorophore evolved, including the residues surrounding the fluorophore.

Panel B
The second panel is a ‘genealogical’ tree of all of the different DsRed variants. This shows some of the same information as panel A, but in a different form, along with new information. The mutations that are key for each mutant are noted at the junctions of the tree. This view is more interesting because it includes the sequence in which the different variants were created, and it really gives one an idea of the different logical steps that allowed for the creation of all of these different mutants. The way that the different mutants evolved was interesting, with a special notice of the GFP termini. The forethought to use already successful terminal sequences was quite admirabe.

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