One of the best of these in terms of photostability and brightness may be the Emerald variant, but lack of a commercial source has limited its use. Several sources provide humanized green fluorescent protein variants that offer distinct advantages for fluorescence resonance energy transfer FRET experiments. Substitution of the phenylalanine residue at position 64 for leucine F64L ; GFP2 yields a mutant that retains the nanometer excitation peak and can be coupled as an effective partner for enhanced yellow fluorescent protein.
A variant of the S65C mutation normally substituting cysteine for serine having a peak excitation at nanometers has been introduced commercially as a more suitable FRET partner for enhanced blue fluorescent protein than the red-shifted enhanced green version.
Finally, a reef coral protein, termed ZsGreen1 and having an emission peak at nanometers, has been introduced as a substitute for enhanced green fluorescent protein. When expressed in mammalian cells, ZsGreen1 is very bright relative to EGFP, but has limited utility in producing fusion mutants and, similar to other reef coral proteins, has a tendency to form tetramers. The family of yellow fluorescent proteins was initiated after the crystal structure of green fluorescent protein revealed that threonine residue Thr was near the chromophore.
Mutation of this residue to tyrosine was introduced to stabilize the excited state dipole moment of the chromophore and resulted in a nanometer shift to longer wavelengths for both the excitation and emission spectra. Further refinements led to the development of the enhanced yellow fluorescent protein EYFP , which is one of the brightest and most widely used fluorescent proteins.
The brightness and fluorescence emission spectrum of enhanced yellow fluorescent protein combine to make this probe an excellent candidate for multicolor imaging experiments in fluorescence microscopy. Enhanced yellow fluorescent protein is also useful for energy transfer experiments when paired with enhanced cyan fluorescent protein ECFP or GFP2. However, yellow fluorescent protein presents some problems in that it is very sensitive to acidic pH and loses approximately 50 percent of its fluorescence at pH 6.
In addition, EYFP has also been demonstrated to be sensitive to chloride ions and photobleaches much more readily than the green fluorescent proteins. Continued development of fluorescent protein architecture for yellow emission has solved several of the problems with the yellow probes. The Citrine variant of yellow fluorescent protein is very bright relative to EYFP and has been demonstrated to be much more resistant to photobleaching, acidic pH, and other environmental effects.
Another derivative, named Venus , is the fastest maturing and one of the brightest yellow variants developed to date. The coral reef protein, ZsYellow1 , originally cloned from a Zoanthus species native to the Indian and Pacific oceans, produces true yellow emission and is ideal for multicolor applications.
Many of the more robust yellow fluorescent protein variants have been important for quantitative results in FRET studies and are potentially useful for other investigations as well.
Illustrated in Figure 3 are the absorption and emission spectral profiles for many of the commonly used and commercially available fluorescent proteins, which span the visible spectrum from cyan to far red. The variants derived from Aequorea victoria jellyfish, including enhanced cyan, green, and yellow fluorescent proteins, exhibit peak emission wavelengths ranging from to nanometers. Fluorescent proteins derived from coral reefs, DsRed2 and HcRed1 discussed below , emit longer wavelengths but suffer from oligomerization artifacts in mammalian cells.
The blue and cyan variants of green fluorescent protein resulted from direct modification of the tyrosine residue at position 66 Tyr66 in the native fluorophore see Figure 2. Conversion of this amino acid to histidine results in blue emission having a wavelength maxima at nanometers, whereas conversion to tryptamine results in a major fluorescence peak around nanometers along with a shoulder that peaks around nanometers. Both probes are only weakly fluorescent and require secondary mutations to increase folding efficiency and overall brightness.
Even with modifications, the enhanced versions in this class of fluorescent protein EBFP and ECFP are only about 25 to 40 percent as bright as enhanced green fluorescent protein.
In addition, excitation of blue and cyan fluorescent proteins is most efficient in spectral regions that are not commonly used, so specialized filter sets and laser sources are required. Despite the drawbacks of blue and cyan fluorescent proteins, the widespread interest in multicolor labeling and FRET has popularized their application in a number of investigations.
This is especially true for enhanced cyan fluorescent protein, which can be excited off-peak by an argon-ion laser using the nanometer spectral line and is significantly more resistant to photobleaching than the blue derivative. In contrast to other fluorescent proteins, there has not been a high level of interest for designing better probes in the blue region of the visible light spectrum, and a majority of the developmental research on fluorophores in this class has been focused on cyan variants.
Among the improved cyan fluorescent proteins that have been introduced, AmCyan1 and an enhanced cyan variant termed Cerulean show the most promise. Derived from the reef coral, Anemonia majano , the AmCyan1 fluorescent protein variant has been optimized with human codons to generate a high relative brightness level and resistance to photobleaching when compared to enhanced cyan fluorescent protein during mammalian expression.
On the downside, similar to most of the other reef coral proteins, this probe has a tendency to form tetramers. The Cerulean fluorescent probe was developed by site-directed mutagenesis of enhanced cyan fluorescent protein to yield a higher extinction coefficient and improved quantum yield. Cerulean is at least 2-fold brighter than enhanced cyan fluorescent protein and has been demonstrated to significantly increase the signal-to-noise ratio when coupled with yellow-emitting fluorescent proteins, such as Venus see Figure 4 , in FRET investigations.
A major goal of fluorescent protein development has become the construction of a red-emitting derivative that equals or exceeds the advanced properties of enhanced green fluorescent protein. Among the advantages of a suitable red fluorescent protein are the potential compatibility with existing confocal and widefield microscopes and their filter sets , along with an increased capacity to image entire animals, which are significantly more transparent to red light.
Because the construction of red-shifted mutants from the Aequorea victoria jellyfish green fluorescent protein beyond the yellow spectral region has proven largely unsuccessful, investigators have turned their search to the tropical reef corals.
The first coral-derived fluorescent protein to be extensively utilized was derived from Discosoma striata and is commonly referred to as DsRed. Once fully matured, the fluorescence emission spectrum of DsRed features a peak at nanometers whereas the excitation spectrum has a major peak at nanometers and a minor peak around nanometers. Several problems are associated with using DsRed, however. Maturation of DsRed fluorescence occurs slowly and proceeds through a time period when fluorescence emission is in the green region.
Termed the green state , this artifact has proven problematic for multiple labeling experiments with other green fluorescent proteins because of the spectral overlap. Furthermore, DsRed is an obligate tetramer and can form large protein aggregates in living cells. Although these features are inconsequential for the use of DsRed as a reporter of gene expression, the usefulness of DsRed as an epitope tag is severely limited. In contrast to the jellyfish fluorescent proteins, which have been successfully used to tag hundreds of proteins, DsRed conjugates have proven much less successful and are often toxic.
A few of the problems with DsRed fluorescent proteins have been overcome through mutagenesis. The second-generation DsRed, known as DsRed2 , contains several mutations at the peptide amino terminus that prevent formation of protein aggregates and reduce toxicity.
In addition, the fluorophore maturation time is reduced with these modifications. The DsRed2 protein still forms a tetramer, but it is more compatible with green fluorescent proteins in multiple labeling experiments due to the quicker maturation. Further reductions in maturation time have been realized with the third generation of DsRed mutants, which also display an increased brightness level in terms of peak cellular fluorescence.
Red fluorescence emission from DsRed-Express can be observed within an hour after expression, as compared to approximately six hours for DsRed2 and 11 hours for DsRed. A yeast-optimized variant, termed RedStar , has been developed that also has an improved maturation rate and increased brightness. The presence of a green state in DsRed-Express and RedStar is not apparent, rendering these fluorescent proteins the best choice in the orange-red spectral region for multiple labeling experiments.
Because these probes remain obligate tetramers, they are not the best choice for labeling proteins. Several additional red fluorescent proteins showing a considerable amount of promise have been isolated from the reef coral organisms. One of the first to be adapted for mammalian applications is HcRed1 , which was isolated from Heteractis crispa and is now commercially available.
HcRed1 was originally derived from a non-fluorescent chromoprotein that absorbs red light through mutagenesis to produce a weakly fluorescent obligate dimer having an absorption maximum at nanometers and an emission maximum of nanometers. Although the fluorescence emission spectrum of this protein is adequate for separation from DsRed, it tends to co-aggregate with DsRed and is far less bright.
An interesting HcRed construct containing two molecules in tandem has been produced to overcome dimerization that, in principle, occurs preferentially within the tandem pairing to produce a monomeric tag.
However, because the overall brightness of this twin protein has not yet been improved, it is not a good choice for routine applications in live-cell microscopy. In their natural states, most fluorescent proteins exist as dimers, tetramers, or higher order oligomers. Likewise, Aequorea victoria green fluorescent protein is thought to participate in a tetrameric complex with aequorin, but this phenomenon has only been observed at very high protein concentrations and the tendency of jellyfish fluorescent proteins to dimerize is generally very weak having a dissociation constant greater than micromolar.
Dimerization of fluorescent proteins has thus not generally been observed when they are expressed in mammalian systems.
However, when fluorescent proteins are targeted to specific cellular compartments, such as the plasma membrane, the localized protein concentration can theoretically become high enough to permit dimerization. This is a particular concern when conducting FRET experiments, which can yield complex data sets that are easily compromised by dimerization artifacts.
The construction of monomeric DsRed variants has proven to be a difficult task. More than 30 amino acid alterations to the structure were required for the creation of the first-generation monomeric DsRed protein termed RFP1. However, this derivative exhibits significantly reduced fluorescence emission compared to the native protein and photobleaches very quickly, rendering it much less useful then monomeric green and yellow fluorescent proteins.
Mutagenesis research efforts, including novel techniques such as somatic hypermutation, are continuing in the search for yellow, orange, red, and deep red fluorescent protein variants that further reduce the tendency of these potentially efficacious biological probes to self-associate while simultaneously pushing emission maxima towards longer wavelengths. Improved monomeric fluorescent proteins are being developed that have increased extinction coefficients, quantum yields, and photostability, although no single variant has yet been optimized by all criteria.
In addition, the expression problems with obligate tetrameric red fluorescent proteins are being overcome by the efforts to generate monomeric variants, which have yielded derivatives that are more compatible with biological function. Perhaps the most spectacular development on this front has been the introduction of a new harvest of fluorescent proteins derived from monomeric red fluorescent protein through directed mutagenesis targeting the Q66 and Y67 residues.
Named for fruits that reflect colors similar to the fluorescence emission spectral profile see Table 1 and Figure 5 , this cadre of monomeric fluorescent proteins exhibits maxima at wavelengths ranging from to nanometers.
Further extension of this class through iterative somatic hypermutation yielded fluorescent proteins with emission wavelengths up to nanometers. These new proteins essentially fill the gap between the most red-shifted jellyfish fluorescent proteins such as Venus , and the coral reef red fluorescent proteins. Although several of these new fluorescent proteins lack the brightness and stability necessary for many imaging experiments, their existence is encouraging as it suggests the eventuality of bright, stable, monomeric fluorescent proteins across the entire visible spectrum.
One of the most interesting developments in fluorescent protein research has been the application of these probes as molecular or optical highlighters see Table 2 , which change color or emission intensity as the result of external photon stimulation or the passage of time. As an example, a single point mutation to the native jellyfish peptide creates a photoactivatable version of green fluorescent protein known as PA-GFP that enables photoconversion of the excitation peak from ultraviolet to blue by illumination with light in the nanometer range.
Unconverted PA-GFP has an excitation peak similar in profile to that of the wild type protein approximately to nanometers. After photoconversion, the excitation peak at nanometers increases approximately fold. This event evokes very high contrast differences between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of molecular subpopulations within a cell.
Illustrated in Figure 6 a is a transfected living mammalian cell containing PA-GFP in the cytoplasm being imaged with nanometer argon-ion laser excitation before Figure 6 a and after Figure 6 d photoconversion with a nanometer blue diode laser.
Other fluorescent proteins can also be employed as optical highlighters. Three-photon excitation at less than nanometers of DsRed fluorescent protein is capable of converting the normally red fluorescence to green.
This effect is likely due to selective photobleaching of the red chromophores in DsRed, resulting in observable fluorescence from the green state. Zacharias, David A. Tsien, Roger Y. Buckley, Anthony M. Kumagai, Akiko, et al. Thorn, Kurt. Cranfill, Paula J. Hink, Mark A. Lotze, Jonathan, et al. Chatterjee, Abhishek, et al. Hilaire, Mary Rose, et al. Querido, Emmanuelle, and Pascal Chartrand. Chen, Baohui, et al. Chernov, Konstantin G. Takai, Akira, et al. Iwano, Satoshi, et al.
Adjobo-Hermans, Merel JW, et al. Mastop, Marieke, et al. Additional Resources on the Addgene Blog. Topics: Fluorescent Proteins , Fluorescent Proteins Add Comment. Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues. GFP needs oxygen Chromophore formation requires a fully folded beta-barrel structure, followed by an intramolecular reaction that generates the chromophore Tsien, GFP fluorescence overlaps with autofluorescence The cyan light that is used to excite GFP, may also excite several components that are naturally present in cells or media.
Summary GFP is an all-round useful genetically encoded probe, but there are limitations. Many thanks to our guest blogger, Joachim Goedhart! References 1. Sharing science just got easier Subscribe to our blog. Follow Addgene on Social.
The total radioactivity in bands corresponding to GFP or its tagged variants was calculated by volume integration using the built-in software after background correction. The GFP half-life was calculated by linear regression analysis of log total radioactivity per band against time. The integrity of the fusion proteins produced was determined both by fluorescence microscopy and by immunoprecipitation.
Visually, all cell lines displayed green fluorescence, which indicated that they were expressing the GFP fusions although with intensity levels and distribution patterns specific for each construct described below. Several additional bands, 39 kDa or larger, were also observed even in the parental untransfected LA-9 control lane and represent non-GFP proteins with affinity for the polyclonal antibody.
While one of these bands co-migrates with the CDB—GFP fusion protein, the relative intensity of the bands indicates that this was an unrelated band and did not result from contamination of the parental cell line, which was consistently non-fluorescent. Immunoprecipitated samples of pulse-chase labelled protein extracts were taken over a 48 h period to determine the stabilities of each of the fusion proteins Figure 2. As predicted from the structure, CAGG—GFP was a stable protein whose destruction followed first-order kinetics with a half-life of approximately 26 h.
The fusion protein containing the C-terminal mouse ODC PEST sequence was also degraded with first-order kinetics, but at an increased rate, resulting in a reduced half-life of 9.
This represents a significant destabilization of the protein of 2. This observation was quantitatively confirmed by subsequent FACS analysis and indicates that the steady-state levels of the protein are the same. This was not the case for CDB fusion proteins that showed highly variable levels of expression between individual cells from unsynchronized cultures of the same clonal line.
The left-hand peak in this profile corresponded to cells undergoing mitosis which, nevertheless, showed minimal levels of fluorescence 2—3 times brighter than the non-fluorescent parental LA-9 cells Figure 3B. During this period of the cell cycle, many proteins are being degraded by the 26S proteosome and so complete removal of the excessive amounts of GFP produced may be hindered by saturation of the degradation machinery. The cyclin destruction box produced the most significant effect on protein stability, as the CDB—GFP fusion protein had a half-life of 5.
However, this rate was not constant during this time, as demonstrated when synchronized, rather than unsynchronized, cultures were examined Figure 3A. In this case, the majority of GFP turnover was seen to occur as cells entered mitosis, during which the GFP half-life was 4. Outside the mitotic phase, the protein remained predominantly stable. Thus, the figure of 5. Thus, addition of the PEST region does not appear to make a significant difference to fluorescence when protein levels are non-limiting; at lower concentrations, its effect is more evident.
The most prominent consequence of fusing the GFP sequence to the cyclin destruction box was highlighted through a time-dependent analysis of fluorescence development and decay in cells from synchronized culture Figure 3A and F.
The FACS profiles of these cells immediately after removal of the synthesis block showed similar distributions of fluorescence intensities, with wild-type GFP being slightly higher Figure 3C and E. However, over a period of 26 h, corresponding to approximately one and a half cell cycles for LA-9 cells, the population profiles differed dramatically. Inevitably, below this threshold some cells were included as non-fluorescent despite expressing significant levels of GFP.
Fluorescence levels increased subsequently as cells progressed through G1 and S phase before a second decrease occurred as cells entered their second mitotic division. These dynamic changes in CDB—GFP fluorescence with cell cycle progression are illustrated in Figure 3F , in which each cell of a synchronized pair undergoes mitosis resulting in reductions in fluorescence levels. At the four-cell stage, once the cells are in interphase, fluorescence levels have returned to a maximum and are comparable between all cells.
Although it is not possible from these data to establish the exact timing of these changes with respect to the cell cycle, the pattern is consistent with that reported for normal cyclin B1 accumulation and degradation Glotzer et al. The most notable impact of green fluorescent protein technology to date in cell biology has been its use in fusion proteins for monitoring a specific protein's localization, trafficking and processing Corbett et al.
For these purposes, GFP already has been engineered to be brighter and to exhibit a wider range of excitation and extinction frequencies, which are amenable to double labelling studies Tsien, However, further development of GFP will undoubtedly take the direction of utilizing GFP per se as an indicator of cellular state, as opposed merely to conferring fluorescence on previously non-fluorescent proteins of interest.
It is to this class of modification that the GFP variants described in this work belong. The reduction in GFP stability seen in the mODC PEST-tagged variant, while significant, still places GFP at the higher end of stability compared with more traditional reporters of gene activity such as luciferase which has a half-life of approximately 3 h Thompson et al. However, as most enzymatic methods involve cell disruption, this improvement in using GFP non-invasively with a shorter half-life will be suitable for many applications.
In particular, for monitoring chromosome transmission, where expression of GFP to readily detectable levels is not problematic, the shorter half-life means that parentally derived cytoplasmic GFP is removed quickly, allowing the measurement of de novo GFP synthesis from the daughter cells.
GFP may yet be a viable alternative for in vivo gene expression studies if the destabilization can be improved to reduce the half-life further. However, the reported half-life of this variant is 2 h and further reports of a 1 h variant d1GFP are based on fluorescence measurements, not biochemical purification.
The discrepancy between those claims and the results presented here has been investigated to distinguish between cell differences, experimental protocols and intrinsic properties of the GFP variants used.
We have confirmed the increased rate of degradation of the d1GFP variant, compared with the GFP—PEST described here data not shown , and using the more accurate method of pulse-chase labelling we have determined its half-life to be 50 min in human HT cells. Consequently, the presence of a PEST motif within a protein merely implies a propensity to instability and cannot indicate the magnitude of the degradation rate even when close to or identical with the consensus PEST sequence.
While the patterns of protein degradation for the cyclin—GFP fusion proteins are more complex than the simple linear profiles obtained with the PEST fusions, these variants are potentially useful as non-invasive indicators of cell-cycle status.
The FACS profile of unsynchronized cells expressing these proteins Figure 3D is an indirect measurement of the proportion of cells in defined stages of the cell cycle. Although more detailed investigation of the correlation between fluorescence levels and cellular status is necessary, the lower peak of the fluorescence distribution corresponded broadly to mitotic and early G1 cells.
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