PEDS Advance Access originally published online on January 18, 2008
Protein Engineering Design and Selection 2008 21(2):101-106; doi:10.1093/protein/gzm075
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Red fluorescent protein variants with incorporated non-natural amino acid analogues
Department of Chemistry and Chemical Biology, Indiana University Purdue University, 402 N Blackford Street, LD326, Indianapolis, IN 46202, USA
1 To whom correspondence should be addressed. E-mail: deo{at}chem.iupui.edu
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Fluorescent proteins are important tools in biotechnology applications and biosensing. DsRed, a red fluorescent protein, has expanded the colors of fluorescent proteins beyond the more commonly used green fluorescent protein. Many genetic modifications have been performed on DsRed to overcome some of its drawbacks. These primarily focused on overcoming the oligomerization detrimental to DsRed activity, and the parasitic green fluorescence caused by the immature chromophore. One such variant, DsRed-monomer, has minimal green fluorescence and no oligomerization. A few traditional mutagenesis studies have been done with DsRed and its mutants to shift the fluorescence wavelengths creating additions to the pallet of fluorescent protein colors. We have explored incorporation of non-natural amino acid analogues into DsRed-Monomer, obtaining variants with differing emission properties. In this work, two such analogues of tyrosine have been incorporated into DsRed-Monomer: 3-amino-L-tyrosine and 3-fluoro-L-tyrosine. Tyrosine analogues were chosen due to the role of tyrosine in the formation and structure of the protein’s chromophore. The variants obtained in our study showed altered emission wavelengths and spectral characteristics. Our study demonstrates that incorporation of non-natural analogues into DsRed-Monomer is a viable approach to alter the spectral characteristics of the protein. We envision that this study will open up the door to non-natural mutagenesis studies with red fluorescent proteins and its mutants.
Keywords: DsRed-Monomer/fluorescent proteins/non-natural amino acid
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Introduction |
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Since the discovery of green fluorescent protein (GFP), many fluorescent proteins have been isolated, spectrally characterized and used as biochemical markers. DsRed, a red fluorescent protein isolated from Discosoma coral is one such protein (Matz et al., 1999a
,b; Shrestha and Deo, 2006). The fluorescence excitation and emission of DsRed, in the red region of the spectrum, is an attractive property for in vivo applications because of the low cellular autofluorescence in this region of the spectrum. However, less work has been reported on DsRed, than GFP (22% sequence identity), isolated from Aequorea vicotoria. DsRed is a naturally occurring tetramer in its native state (Baird et al., 2000; Heikal et al., 2000; Yarbrough et al., 2001). The chromophore of DsRed is made internally from three amino acids: Gln66, Tyr67 and Gly68 (Tubbs et al., 2005). The chromophore is formed in a three-step reaction which includes an autocatalytic cyclization reaction and two sequential dehydrogenations (Verkhusha and Lukyanov, 2004; Pakhomov et al., 2006). The cyclization and initial dehydrogenation create a short-lived and unstable green fluorescent chromophore (Cotlet et al., 2001). The red fluorescence emission seen for DsRed is believed to be caused by the second dehydrogenation reaction (Wall et al., 2000). This reaction extends the conjugated 
-system of the preceding green fluorescent structure. The use of DsRed is problematic for in vivo and in vitro studies due to its slow maturation and tendency to oligomerization (Campbell et al., 2002). Many mutations have been done on DsRed to eliminate these problems; subsequently, monomers with more rapid maturation times were generated (Campbell et al., 2002; Gavin, et al., 2002; Yanushevich et al., 2002; Shaner et al., 2004). DsRed monomers are a promising choice for in vivo studies due to their fluorescence in the red region which eliminates the poor tissue translucency seen with GFP. In addition, the monomeric forms of DsRed show a variety of characteristics which are desirable for biochemical studies: stability at physiological temperature and across a wide range of pH (5–12) and no photodegredation. We envision that the availability of variants of DsRed-Monomer with even farther red-shifted emission will prove beneficial in biochemical studies. Here, we describe exploratory studies in the construction and characterization of variants of DsRed-Monomer, with altered spectral properties by performing non-natural mutagenesis studies.
Rational mutagenesis has been a method of choice so far to create variants of proteins with altered properties and to study structure–function relationships (Noren et al., 1989; Baird et al., 2000). In this method, canonical amino acids were inserted into specific positions within the protein. For example, site-directed mutagenesis performed on mRFP1, a monomeric DsRed variant, resulted in the creation of variants with shifted excitation and emission wavelength maxima of up to 18 nm. These shifts have been attributed to altered electrostatic interactions with the chromophore of the protein (Shu et al., 2006). However, incorporation of non-natural amino acids offers an alternative method, allowing for the addition of novel functional groups into proteins (Noren et al., 1989; Hohsaka et al., 2001; Hohsaka and Sisido, 2002; Link et al., 2003; Liu et al., 2007). For example, site-specific incorporation of non-natural amino acids has been used to create mutants of GFP (Taki et al., 2001; Hyun Bae et al., 2003; Wang et al., 2003; Kajihara et al., 2005) which show a variety of fluorescence shifts and properties. Specifically, incorporation of Trp analogues into GFP, using a method of forced biosynthetic incorporation, has produced a number of ‘gold’-shifted fluorescent proteins (Hyun Bae et al., 2003). In addition, mutations at the Tyr66 position of the GFP chromophore created mutants with no fluorescence or blue-shifted emission maximum depending on the analogue (Wang et al., 2003; Kajihara et al., 2005). In addition, incorporation of aromatic natural and non-natural amino acids into the residue 66 of GFP has shown a variety of emission shifts (Wang et al., 2003). The fluorescence shifts observed for these mutants suggest the possibility of obtaining alternative, and biochemically appropriate, fluorescent proteins by incorporating non-natural amino acid analogues into DsRed-Monomer.
Although incorporation of non-natural amino acids has been successfully demonstrated in GFP, such studies have not previously been done on DsRed, its mutants or any red fluorescent proteins. In this article, we describe the incorporation of two tyrosine analogues into DsRed-Monomer, creating variants with non-natural amino acid residues within the chromophore of the protein. The position of the Tyr67 residue as part of the DsRed chromophore prompted the use of tyrosine analogues in our study to shift the observed fluorescence of the protein. The non-natural amino acid analogues were incorporated into the protein by a system of forced biosynthetic incorporation (Brooks et al., 1998), a simpler and quicker method than modified tRNA-based site-specific genetic incorporation. Hyun Bae et al. (Hyun Bae et al., 2003) showed that the forced biosynthetic incorporation could be used for the overall incorporation of amino acid analogues into GFP. However, further studies showed that incorporation of non-natural amino acids into any position outside of the chromophore had no effect on the spectral properties of GFP (Hyun Bae et al., 2003). On the basis of this, we selected fluoro and amino analogues of tyrosine for our study since the Tyr at the 67 position in DsRed is part of the tripeptide which forms the chromophore (Yarbrough et al., 2001). Furthermore, an X-ray crystal structure study of DsRed has shown that the other Tyr residues within the protein do not interact with the chromophore and therefore should not affect the spectral properties of the protein (Yarbrough et al., 2001). The rational behind the selection of a fluoro analogue is that the fluoro group has a significantly different pKa than the hydroxy functional group and has been previously used to alter the pKa of functional groups within the active site of enzymes, changing their catalytic properties (Brooks et al., 1998; Minks et al., 2000). The addition of fluorinated amino acid analogues has also been found to affect the conjugation of the -system of the chromophore and to cause limited changes in the bond lengths within such systems. In addition, this analogue may promote the formation of additional hydrogen bonds within the system (Brooks et al., 1998; Minks et al., 2000). In contrast, the amino group should primarily affect the electrostatic charge within the aromatic ring of the tyrosine and allow either a different pattern of hydrogen bonding with surrounding amino acid residues or contribute to the conjugation of the chromophore.
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Expression of DsRed-Monomer with incorporated non-natural amino acids
The plasmid DsRed-Monomer was obtained from Clontech. The gene for DsRed-Monomer was digested with BamHI and EcoRI and cloned into pRSetB (Invitrogen, Carlsbad, CA, USA) to construct the plasmid pSKD1. M9 minimal media (Neidhardt et al., 1974; Lu et al., 1976) containing 100 µg ml–1 ampicillin was prepared without tyrosine, tryptophan and phenalalanine. A 5 ml sample of the minimal media was inoculated with Escherichia coli containing the plasmid pSKD1 and incubated overnight in a shaker at 37°C. The culture was transferred to a 200 ml sample of the minimal media and grown to an OD420 of 1.00 and induced with isopropyl-β-D-thiogalactoside (IPTG, 0.5 mM final concentration, from Sigma). Tryptophan, phenalalanine and either tyrosine or the appropriate tyrosine analogue (1 mM final concentration of each amino acid, from Spectrum Chemicals) (Brooks et al., 1998) were also added at that time. The cultures were grown for a further 5 h with shaking, 250 r.p.m., at 37°C and collected by centrifugation, 7000 r.p.m., for 30 min at 4°C. The pellet was dissolved in the PBS-binding buffer (100 mM Na2PO3, 50 mM NaCl, pH 7.0) and sonicated for 5 min to lyse the cells.
Purification of DsRed-Monomer and non-natural mutants
The protein was purified using an Ni Sepharose high-performance affinity column charged with copper (Rahimi et al., 2007). A volume of 1.5 ml of the Ni Sepharose high-performance beads was centrifuged and the storage ethanol was poured off. The beads were resuspended in sterile water and applied to the column. A volume of 2 ml of the stripping buffer (0.02 M NaPO3, 0.5 M NaCl, 0.05 M EDTA, pH 7.4) was then applied to the column. The column was rotated overnight to fully remove the Ni from the beads. The column was washed with multiple column volumes of sterile water, and 1.5 ml of copper sulfate (0.1 M) solution was applied. The column was rotated for at least 2 h to assure full binding of the Cu2+ ions to the beads. The column was washed with up to 10 column volumes of PBS-binding buffer to remove all unbound copper. A volume of 1.5 mL aliquots of the crude protein were applied to the column, with the column being rotated for 2 h between additions. The column was again washed with up to 10 column volumes of PBS-binding buffer to remove anything not bound to the copper immobilized column. Wash buffers (50 mM NaPO3, 300 mM NaCl, pH 8.0, containing 1.0–3.0 mM imadizole) were used to wash the column. The protein was eluted using an elution buffer containing 10.0 mM imidazole (50 mM NaPO3, 300 mM NaCl, pH 8.0, containing 10.0 mM imidazole). The purified protein was collected and dialyzed to remove the imidazole, using PBS buffer (50 mM Na2PO3, 50 mM NaCl, pH 8.0).
Determination of protein purity and concentration
Protein purity was determined by running an SDS–PAGE assay with 12.5% acrylamide gel at 150 V for 90 min at room temperature. Coomassie Brilliant Blue R-250 solution was used to stain the gels overnight and destained for at least 2 h. Protein concentration was determined by Bradford assay.
Spectral analysis of purified DsRed-Monomer with incorporated non-natural amino acid and comparison with native DsRed-monomer
Mass spectrometry
Mass spectrometry analysis was performed by following the protocol of Gross et al. (Gross et al., 2000). Approximately 2 nmol of purified protein was lyophilized and dissolved in 15 µl of sterilized water, 3 µl of guanidinium chloride (6 M) was added and the solution heated to 80°C until it turned yellow. The solution was cooled to room temperature and 1 µl of HCl (0.33 M) was added followed by 10 µl of LysC endoprotease (0.05 µg/µl in 100 mM Tris, pH 9.2). The digestion proceeded for 22 h at 37°C and was quenched with trifluoroacetic acid (0.1% vol/vol) (Gross et al., 2000). The mass spectra of each of the digested proteins were recorded on an Agilent 1100 Series LC/MSD, from Agilent Technologies (Santa Clara, CA, USA), by direct injection, passing through the LC column.
Fluorescence spectra A volume of 200 µl (0.1 µM) of each of the non-natural mutants and the native DsRed-Monomer was placed into a microtiter plate, and excitation and emission scans of the protein were obtained at room temperature using a Cary Eclipse Fluorescence Microtiter Plate Reader from Varian Inc. (Walnut Creek, CA, USA).
UV–visible spectra A volume of 1 ml (0.1 µM) of all the mutants and the native protein was placed in a cuvette, and the absorption spectra of the proteins were recorded at room temperature using an Agilent 8453 UV–visible spectrometer from Agilent Technologies (Santa Clara, CA, USA).
CD spectra Purified non-natural mutants and the native protein (250 µl, 0.1 µM) were placed in a 0.2 cm cell, and the adsorption spectrum was obtained at room temperature using a Jasco Corporation spectropolarimeter (Tokyo, Japan).
Temperature and pH stability Fluorescence spectra were recorded at 4°C, room temperature (25°C) and 37°C for both the native protein and the two variants. Each protein was incubated at the respective temperature for at least 2 h before readings were taken. The pH of the proteins was adjusted from 4 to 11, with MOPS buffer, in increments of 1 pH unit, and the fluorescence emission of each protein was recorded.
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Fluorescent protein variants with differing spectral characteristics are highly desirable in biotechnology and cell biology studies. To obtain these variants, a number of approaches including random mutagenesis, directed evolution and non-natural mutagenesis have been successfully used. Previously, in these studies, GFP was primarily used as the scaffold. The availability of the red fluorescent protein, DsRed, provides a unique opportunity to further extend the spectral diversity of FPs. Thus far, only mutagenesis of naturally available amino acids has been attempted with DsRed. Here, we studied incorporation of non-natural amino acid analogues into DsRed-Monomer to construct variants that yield alterations in the spectral characteristics of the protein. In our study, DsRed-Monomer and its variants with non-natural amino acids were expressed in minimal salt media using the method of forced biosynthetic incorporation. In this method, the non-natural analogue is added to the culture media at a stage right before the protein expression. This method was chosen for this study, instead of directed incorporation of non-natural amino acids because the latter method is complicated and the aim of this study was initial demonstration of amenability of DsRed to non-natural mutagenesis.
The plasmid pSKD1 was transformed into E. Coli cells and the proteins were expressed. During expression, the time for the initial observation of color and the time to reach the maximum fluorescence for the mutants were comparable with the native DsRed-Monomer. This indicates no change in the folding ability of the proteins upon mutation. Furthermore, we did not observe any formation of protein aggregates in the mutants as well as in the native DsRed-Monomer, implying no effect on the solubility of the proteins (Fig. 1). The proteins were purified using an immobilized copper column. DsRed-Monomer has a unique affinity for copper, which has been explored by our group. This affinity has allowed for the creation of a simple and efficient purification system (Rahimi et al., 2007). Therefore, we utilized a copper immobilized column for purification in this study. The purified proteins were run on an SDS–PAGE gel to verify the purity (Fig. 1). The non-natural variants of DsRed-Monomer were analyzed by spectroscopic and spectrometry techniques, and the characteristics were compared with native DsRed-Monomer prepared in the same manner.
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The mass spectra were collected for the DsRed-Monomer and for the non-natural mutants. After LysC digestion, a peptide that included the chromophore of the protein, of mass 2185, was seen for the native DsRed-Monomer as expected (Gross et al., 2000
). The spectra of the non-natural variants showed the expected increases in mass, compared with the native protein (Table I). A mass increase of approximately 15 was seen for the peptide of the amino mutant (expected 2200, found 2199.6) compared with the chromophoric peptide obtained with the native protein. This increase coincides with the addition of an amino group onto the aromatic ring of tyrosine, within the chromophore. The fluoro mutant showed a mass increase of approximately 18 (expected 2204, found 2205) compared with the native protein, which corresponds to the addition of fluorine onto the tyrosine ring. The mass analysis confirms the presence of non-natural amino acid analogues in the peptide forming the chromophore. Furthermore, the mass spectra of the mutants did not show any peak corresponding to the native DsRed-Monomer, suggesting the full incorporation of the non-natural analogue into the protein.
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The fluorescence excitation and emission spectra of both purified DsRed-Monomer and the purified non-natural mutants were recorded. The incorporated analogues showed no effect on the excitation spectra of the protein, showing the expected excitation maximum wavelength at 556 nm. However, variations in peak intensity were seen, the amino mutant showed the strongest fluorescence excitation intensity with the fluoro mutant intermediate and DsRed-Monomer the weakest. Spectral shifts were observed in the emission spectra. The fluoro tyrosine-incorporated mutant showed a distinct blue shift of 12 nm (
max = 591 nm) compared with DsRed-Monomer (max = 603 nm), whereas the amino tyrosine-incorporated mutant showed a red shift of 12 nm (max = 615 nm) (Fig. 2). These opposite shifts in emission wavelength maximum were expected since the two analogues would have an opposite effect on the conjugated electron system of the chromophore. The addition of the amino group will lead to a more expanded -conjugated chromophore system (Kajihara et al., 2005), at the aromatic ring, because of the electron donation of this group. We expect that this will stabilize the conjugation in the chromophore leading to the observed red shift in the emission spectra. The fluoro addition, however, would withdraw electrons from the conjugated system of the aromatic ring and chromophore in general. This reduction in the aromaticity of the Tyr phenyl ring may cause destabilization of the conjugation leading to the observed blue shift in emission. The absence of any secondary peaks or shoulders indicates that 100% incorporation of the non-natural amino acid of interest into the protein occurred. Any partial incorporation could have also given peaks or shoulders similar to the native protein, which were not observed. These results were obtained from a number of different expressions, and demonstrate the reproducibility of these observations. Emission spectra were recorded using the same concentration of all three proteins (data not shown); these spectra showed that the amino mutant gave the most intense fluorescence followed by the fluoro mutant and native DsRed-Monomer giving the lowest intensity emission at their respective emission wavelength maxima. Further studies showed that temperature insensitivity (between 4 and 37°C) of the protein’s fluorescence was maintained for the mutants compared with native DsRed. The pH dependence of the fluorescence intensity of native DsRed-Monomer, the amino variant and the fluoro variant was studied. The fluorescence intensity was found to be unaffected between pH 5 and 11. This is consistent with the results obtained by Baird et al. that showed negligible dependence, of DsRed fluorescence intensity, on pH (Baird et al., 2000).
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The UV–visible spectra of the purified DsRed-Monomer and the purified non-natural mutants were recorded from 400 to 650 nm to further explore the spectral properties of these mutants. It has previously been reported (Verkhusha et al., 2001
; Campbell et al., 2002) that the UV absorption spectra of a monomeric DsRed, mRFP1, showed a strong peak at 556 nm corresponding to the red fluorescent species. The spectra also showed two additional peaks at 
480 and 520 nm (Matz et al., 1999a). The peak at 480 nm has previously been attributed to a green-emitting intermediate for native (oligomerized) DsRed (Baird et al., 2000; Cotlet et al., 2001). The UV–visible spectra of DsRed-Monomer and its non-natural mutants (Fig. 3) showed that the chromophore of all the proteins was absorbed strongly at the expected 556 nm and showed two additional peaks at 480 and 520 nm. An excitation study performed at 480 nm yielded only minimal fluorescence, on the order of 20% of that seen at 591 nm. The same pattern of peaks was seen for all three proteins; however, the intensity of the absorbance peaks varied. Of the three proteins, the fluoro variant showed the most pronounced peak at 480 nm. Within the spectra of the fluoro mutant, the peak at 480 nm appeared more intense than the peak at 556 nm corresponding to the red fluorescence species. This suggested that incorporation of the fluoro group may hinder the conjugation necessary for the red chromophore formation, locking the chromophore at an intermediate stage. This also may account for the decrease in fluorescence emission, at 591 nm, seen for this protein compared with the amino mutant. The amino variant showed the most pronounced peak at 556 nm corresponding to emission from the major red fluorescent species. The 556 nm peak of the amino mutant was also of highest absorbance units among the three proteins. This coincides with the fluorescence emission spectra of the proteins in which the amino mutant showed the most intense fluorescence emission. This variant, also, showed an absorbance intermediate to both native DsRed-Monomer and the fluoro variant, at 480 nm. The native DsRed showed the lowest absorbance at both 480 and 556 nm; however, the shoulder at 520 nm was more pronounced for this protein than for either of the variants. This shoulder was almost undetectable for the two variants. These fluorescence and absorbance observations further indicate that the position and electron density on Tyr67 are crucial for red chromophore formation in addition to the amino acids (Gln66 and Gly68) necessary for the extended conjugation observed in DsRed. Further, Tyr67 can potentially be targeted to generate variants with far red-shifted emission wavelengths.
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From the UV and fluorescence data, the extinction coefficients and quantum yields of DsRed-Monomer and each of the mutants were determined (Table I). The quantum yield for all three proteins was calculated using DsRed-Express with a known quantum yield as the standard and by using the following equation (Povrozin and Terpetschnig
):
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where Q represents the quantum yield, I the intensity of the fluorescence, OD the optical density and n the refractive index. The values with subscript R correspond to the reference protein, DsRed-Express. This protein was chosen as the reference because it is the tetrameric form of DsRed. For DsRed-Monomer, a surprisingly low quantum yield of 0.05 was calculated. The quantum yield of the two mutants appeared to be closer to the value of 0.4, reported for DsRed-Express. The quantum yields calculated here show that the variants obtained have improved brightness compared with the DsRed-Monomer. This is an interesting observation since increases in quantum yield is obtained for both amino and fluoro variants. This indicates that the increase in quantum yield is not due to the presence of the fluoro or amino group on the Tyr. The quantum yield of DsRed-Monomer has been observed to decrease when compared with DsRed-Tetramer (Campbell et al., 2002). This is attributed to the loss of shielding of the chromophore in the monomer. Therefore, we speculate that the presence of the amino or fluoro group on the Tyr, which can increase the bulkiness of the Tyr, may improve the shielding of the chromophore in the mutated DsRed-Monomer, leading to the improved quantum yield observed here.
The far UV CD spectra of native DsRed-Monomer and the non-natural variants were recorded (Fig. 4). This information was submitted for k2D analysis. These spectra showed that the overall secondary structure characteristics of the protein were maintained after the incorporation of the tyrosine analogues.
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The incorporation of non-natural amino acid residues into the chromophore of DsRed-Monomer caused a shift in the fluorescence emission observed. The fluoro variant showed a 12 nm blue shift and the amino variant a 12 nm red shift in fluorescence emission. The incorporation of these tyrosine analogues was verified by mass analysis of the peptide, containing the chromophore. These variants also displayed improvements in their quantum yields and fluorescence emission intensities compared with the unmodified DsRed-Monomer. In addition, the absorption spectra of all three showed the same pattern of peaks, with variations in relative intensities. The incorporation of these tyrosine analogues into the chromophore of DsRed-Monomer appears to have no effect on the overall structure of the variants. The studies performed by us demonstrate that the spectral properties of red fluorescent protein can be altered by non-natural mutagenesis. The variants produced in this article can be purified and used as labels for in vitro multicolor applications. Furthermore, the studies performed show that the Tyr in the chromophore can be manipulated to produce variants of DsRed-Monomer with unique properties. For in vivo applications, variants can be produced using modified tRNAs that code for non-natural amino acids.
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This work was supported by the IUPUI Research Support Fund Grant, and E.H. thanks IUPUI Honors Fellowship Program.
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Edited by Andrew Bradbury
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Received June 14, 2007; revised November 5, 2007; accepted November 19, 2007.
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