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Fluorescent proteins (FPs) are powerful and specific marker tools for cellular imaging, and their range of applications is continuously expanding. Red fluorescent proteins (RFPs) are of particular interest as they extend the color palette for multi-channel and fluorescence resonance energy transfer (FRET) imaging, and the reduced scattering of long-wavelength light makes them attractive as markers for deep tissue imaging. Compared to variants of the classical green fluorescent protein (GFP), a performance gap has been noticed for natural RFPs after isolation from marine invertebrates.
An overview of RFPs
In 1999, six new FPs were cloned from nonbioluminescent Anthozoa species. One of the proteins, named drFP583, differed from GFP in its spectral properties, demonstrating a red fluorescence. The drFP583 protein, whose gene was optimized for expression in mammalian cells, became the first commercially available RFP, named DsRed for Discosoma sp. Currently, the majority of RFPs have been isolated and cloned from Anthozoa species living in the Indo-Pacific region. Later on, the race was on to succeed in monomerization and improvement of wild-type RFPs in order to produce new probes suitable for multicolor imaging of cellular proteins and FRET pairs with emission in the longer wavelength region. Many desirable changes to the physical and biochemical properties of FPs have been achieved through the intense molecular evolution. The increasing brightness, photostability, maturation efficiency, pH stability, and minimizing cytotoxicity dramatically improved the utilization of RFPs for live-cell microscopy.
RFPs fall into two families, including DsRed (pink background) and Kaede (green background) (Figure 1). The figure shows how the chromophore structures of these RFPs arose by engineering (dotted arrows) or evolution (solid arrows). DsRed and DsRed-like RFPs are the prototype members of DsRed family. These prototypes can produce yellow-emitting, orange-emitting, and far-red-emitting RFPs; RFPs with large Stokes shifts; and photomodulatable RFPs. It should be noted that these chromophores’ spectral properties are often determined by the equilibrium between the protonated and deprotonated states of the phenol hydroxyl group. Nevertheless, because chromophore development is catalyzed by the whole protein, further spectral diversity can arise from the protein cavity which holds the chromophore.
Figure 1. Chemical transformations of the chromophores in RFPs.
The main application areas of red fluorescent proteins
Fluorescent proteins are invaluable tools widely applied to study of different biological systems. The application of GFP-like proteins in biotechnology, biochemistry and cytology allowed not only imaging cell dynamics in a novel way, but also accelerated development of new techniques in microscopy. The creation of RFPs broadens the palette of fluorescent proteins making possible multicolor microscopy. The GFP-like mutant, recombinant RFPs developed recently differ from the wild-type precursors in quantum yield, stability, the character of the absorption spectra, fluorescence excitation and emission.
An advantageous feature of the GFP-like proteins that can form chromophore without any co-factors other than molecular oxygen is their high stabilities. Moreover, both N- and C-termini of fluorescent proteins are accessible for fusion, which allows coupling of a fluorescent protein with the target protein. The GFP- like proteins are generally non-toxic for cells. Compared to green proteins, the main advantage of RFPs is that the level of autofluorescence is much lower in the red spectral range and lesser scattering of the longer wavelength light. The three main subjects of FP application are cell, protein and organelle labelling.
Red fluorescent proteins as reporter markers
The fluorescent protein gene located under control of a certain gene promoter allows studying temporal and spatial expression of the gene through measuring the fluorescence signal in living cells and tissues. The oligomeric state of an expressed fluorescent protein does not matter for its application as a marker of gene expression. Slow chromophore posttranslational maturation and stability of the expressed FP are the main factors that limit the use of FPs in studies of fast activation of transcription.
Fluorescent proteins are most commonly used as markers for protein labelling. Such fusion proteins enable the analysis of localization and dynamics of proteins, organelles and even cells in living organisms, studies of protein ± protein interactions and determination of their biological function. Protein localization requires that a fluorescent protein, to be used as a marker, was present in the monomeric state and possessed high brightness. The tendency of the fluorescent protein to oligomerization affects strongly the possibility of localization of a protein under study, especially if it is an oligomer itself. The efficiency of fluorescent protein maturation is also very important since the complete folding of fusion proteins often prevents inclusion bodies formation. In addition, TagRFP, mKate, mCherry, mKO, etc. are the proteins of choice for fusion.
Protein engineering in combination with high-throughput manners of cell sorting significantly extended color variety of fluorescent proteins and improved such physicochemical characteristics of the proteins as maturation, photostability, brightness, etc. The first attempts to improve wild-type RFPs resulted in monomerization because the oligomeric state limited their application greatly. Subsequently, optimization of physicochemical characteristics of monomeric FPs has been performed. Fluorescent proteins became indispensable markers for studies in biotechnology, biochemistry and cell biology. FPs enable visualization of dynamic processes in cells in a new way. Development of photoactivatable and photo-switchable fluorescent proteins for super-resolution microscopy of cell structures became an important step in the development of fluorescent protein technology.