INTRODUCTION

Detection of processes that occur in the body and a single cell plays a key role in the development of drugs and the therapy of various diseases [1]. The target of many drugs is initiation of apoptosis. Caspase-3 is well known for its role in specific cleavage of many cellular proteins during programmed cell death, as well as in the regulation of cellular homeostasis [2, 3]. The phenomenon of Förster resonance energy transfer within a pair of fluorescent proteins is widely used in FRET sensors to determine the activity of caspase-3, due to the possibility of their direct expression in a living cell [4, 5].

Förster resonance energy transfer (FRET) is the process of the interaction of two chromophores, in which a nonradiative transfer of the energy of the excited state from the donor to the acceptor occurs. This transfer results in a decrease in the intensity and the lifetime of the donor’s fluorescence. The fluorescence of the fluorescent acceptor is simultaneously excited during FRET. The most important parameters that determine the efficiency of the resonance transfer are:

(1) the degree of overlapping spectra of the fluorescence emission of the donor and the absorption of the acceptor;

(2) the distance between chromophores in a FRET pair, since the efficiency of the energy transfer is inversely proportional to the distance to the sixth power [6].

The use of sensors in which red fluorescent protein acts as a donor allows one to work in the spectral region with the lowest absorption and autofluorescence of animal tissues [7]. The use of nonfluorescent chromoproteins as an acceptor eliminates the need for spectral separation of the fluorescence of the donor and acceptor, which facilitates the detection of changes in FRET [8, 9]. The use of such sensors will allow one to detect caspase-3 activity not only spectrophotometrically, but also based on changes in the donor fluorescence lifetime. The detection of proteo-lysis is one of the most important applications of FRET sensors, since the role of various enzymes in molecular oncology during tumor progression is under careful study. One practical application of such sensors is the screening of new anticancer drugs aimed at activating apoptosis.

The aim of this work was to analyze the energy transfer efficiency in new FRET pairs of the red fluorescent protein TagRFP with chromoproteins by calculating overlap integrals, to obtain a monomeric sensor TagRFP-23-chromoprotein and to show the possibility of using the new fusion protein as a FRET sensor for apoptosis detection.

MATERIALS AND METHODS

Molecular cloning. DNA sequences of chromoproteins (gfasCP [10], spisCP [10], anm2CP [11], Ultramarine [12]) were synthesized by Synbio Technologies (China) and cloned into the pET29a (NdeI/SalI) vector (the plasmid was kindly provided by I.E. Granovsky, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences). The pTR23U-22b construct was obtained on the basis of the pTR23K construct created in [13] (based on the pET-22b vector, the plasmid was kindly provided by I.E. Granovsky): the gene of the KFP acceptor chromoprotein [14] was replaced with the gene of the Ultramarine acceptor protein [12] NcoI/SalI.

Expression in Escherichia coli, isolation and purification of proteins from cell lysate. Expression of chromoproteins and the biosensor was carried out in E. coli BL21(DE3) cells. The strain was first obtained in [15] and was kindly provided by Ivashina T.V. (Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences). At the end of expression, the cells were pelleted by centrifugation at 5000 g in a centrifuge (Optima XPN-100 Ultracentrifuge, Beckman Coulter, Germany), destroyed by ultrasound, and the lysate with the target protein was separated by centrifugation at 15 000 g (Optima XPN-100 Ultracentrifuge, Beckman Coulter, Germany). Purification of chromoproteins and the biosensor was carried out by chromatographic methods, as described for the SAASoti fluorescent protein [16]. For the new biosensor, the time and temperature of incubation of E. coli cells after the addition of the transcription inducer isopropyl-β-thiogalactoside (IPTG) were selected: 20 h at 20°С and 4 h at 37°С.

Determination of the oligomeric state. The oligomeric state was determined by gel filtration on a Superdex 200 100/20 GL carrier (GE Healthcare, Germany) as described earlier [17] using dynamic light scattering (DLS) on a DynaPro Titan molecular weight and particle size analyzer (Wyatt Technology Corporation, United States) at 25°С in 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 under 800 nm laser illumination in a 1.5 mm quartz cuvette (Hellma, Germany).

Measurement of spectral characteristics. The absorption spectra of chromoproteins and the biosensor were recorded on a Cary 60 spectrophotometer (Agilent, United States) at a constant temperature of 22°С in a 3 mm quartz cuvette (Hellma, Germany) in 20 mM Tris-HCl buffer with 150 mM NaCl, pH 7.4.

Fluorescence emission spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Varian, United States) at room temperature in a 3 mm quartz cuvette (Hellma) in 20 mM Tris-HCl buffer with 150 mM NaCl, pH 7.4.

Determination of the efficiency of TR-23-U fusion protein hydrolysis by caspase-3. Hydrolysis of the TR-23-U sensor with the PorcCasp3 WT caspase-3 (kindly provided by I.E. Granovsky) in 20 mM HEPES buffer, pH 7.4, with 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 1 mg/mL BSA at 37°C during the night.

Registration of the fluorescence lifetime. The fluorescence lifetime of TagRFP was recorded using a FluoTime 200 fluorescence spectrometer (PicoQuant, Germany) and analyzed using the FluoFit 4.2 software (PicoQuant, Germany).

Determination of fluorescence photoactivation kinetics. Fluorescence photoactivation was recorded for protein solutions with an optical density of 0.1 at the absorption maximum in 20 mM Tris-HCl buffer (pH 7.4), 150 mM NaCl using an installation assembled from a SpectrClaster spectrometer (Russia) and a Spectra X LED light source (Lumencor, United States) in a thermostated (25°C) microcuvette (Hellma, Germany), optical path 3 × 3 mm. Fluorescence was excited at a wavelength of 550 ± 15 nm with a power of 0.5 mW/cm2. The fluorescence intensity was recorded for KFP at a wavelength of 600 nm [14], for Ultramarine at 626 nm [12], and for anm2CP at 597 nm [11]; for gfasCP and spisCP the fluorescence maximum was not determined [10] and their fluorescence was completely absent in the experiment at wavelengths from 580 to 630 nm.

Calculation of overlap integrals and Förster radii. The overlap integrals were calculated using the a|e software, UV-Vis-IR Spectral Software 2.2 (FluorTools, www.fluortools.com) according to formula (1).

$$J\left( \lambda \right) \equiv \int\limits_0^\infty {{{\varepsilon }_{A}}\left( \lambda \right){{\lambda }^{4}}{{F}_{D}}\left( \lambda \right){\text{d}}\lambda } .$$
(1)

The Föster radii were calculated by formula (2):

$${{R}_{0}} = 0.211{{\left[ {\frac{{{{\kappa }^{2}}{{\phi }_{{\text{D}}}}J\left( \lambda \right)}}{{{{n}^{4}}}}} \right]}^{{{1 \mathord{\left/ {\vphantom {1 6}} \right. \kern-0em} 6}}}},$$
(2)

where κ2 is the coefficient describing the mutual orientation of the dipole moments of the donor and acceptor transitions and is equal to 2/3 for freely rotating dipoles; φD is the fluorescence quantum yield of the donor in the absence of an acceptor (φD (TagRFP) = 0.48); J(λ) is the overlap integral of the normalized spectra of the donor fluorescence and acceptor absorption; n is the refractive index of the solvent (it is equal to 1.33 for water).

RESULTS AND DISCUSSION

To create a red fluorescent protein–chromoprotein pair, it is necessary to choose a pair with the maximum overlap of the fluorescence emission spectra of the donor and the absorption of the acceptor. The bright red fluorescent protein TagRFP [18] (molecular brightness 48 000), which has already been successfully used as a donor in FRET sensors [13, 19], was chosen as a donor. Several chromoproteins with high values of molar absorption coefficients were selected for the role of a new nonfluorescent acceptor: Ultramarine [12], anm2CP [11], gfasCP [10], spisCP [10], whose spectral and physicochemical characteristics are given in Table 1.

Table 1. The spectral and physicochemical characteristics of chromoproteins according to literature
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For the chosen chromoproteins, the values of the overlap integrals J(λ) of their absorption spectra with the fluorescence emission spectrum of the TagRFP red protein, quantitatively demonstrating the potential efficiency of the energy transfer in these pairs, were calculated. The Förster radii (R0) were also calculated for the resulting pairs, showing the distance at which the energy transfer efficiency will be 50% (Table 2).

Table 2. The theoretical values of the overlap integrals J(λ) and Förster radii R0 for TagRFP-chromoprotein pairs
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All four FRET pairs had high values of the overlap integrals and Förster radii, which makes them promising candidates for creating FRET sensors. It was also experimentally shown that the selected chromoproteins do not fluoresce when excited by powerful light fluxes at a wavelength of 530 nm, in contrast to the KFP protein previously used in the FRET sensor with TagRFP (Fig. 1) [14]. Thus, another advantage of the new FRET pairs is the absence of background fluorescence of the acceptor.

Fig. 1.
figure 1

The kinetic curves of fluorescence photoactivation (units) of chromoproteins under irradiation with the light with a wavelength of 530 nm (power 0.5 W/cm2): 1, Ultramarine; 2, anm2CP; 3, gfasCP; 4, spisCP; 5, KFP.

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All four chromoproteins were expressed in E. coli cells, purified, and their oligomeric state was characterized by the methods of gel filtration (GF) and dynamic light scattering (DLS). The results we obtained differed from the data given in the literature: anm2CP was present in solution as a dimer with an admixture of a tetrameric fraction, gfasCP and spisCP proteins were dimers, and Ultramarine was a monomer (Table 3). During the purification of chromoproteins by chromatographic methods, it was noted that their binding to carriers differed from other GFP-like proteins: the binding with a hydrophobic column was weaker, while with an anion exchange column it was stronger. Since weak ionic and hydrophobic interactions can occur during gel filtration, such data seem to be less reliable when determining the molecular weight than the results obtained by the DLS method. However, the DLS data were in sufficient agreement with the results of gel filtration, indicating the same oligomeric state of the isolated chromoproteins. The difference between the results and published data may be due to the difference in the methods used to determine the oligomeric state and concentrations of protein solutions. For gfasCP and spisCP, the oligomerization was determined in [10] by electrophoresis under seminative conditions [20]. For anm2CP, gel filtration was used; however, the carrier was different.

Table 3. The results of the oligomeric state characterization of chromoproteins by the methods of GF on the Superdex 200 carrier and DLS
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First of all, a FRET sensor based on TagRFP-23-Ultramarine (TR-23-U) monomeric proteins was obtained, in which 23 denotes a flexible linker of 23 amino acids containing the DEVD caspase-3 re-cognition site [13].

The conditions for optimal expression and maturation (folding and formation of the chromophore) of both proteins in the sensor were selected. After adding the transcription inducer IPTG, E. coli cells expressing the sensor were incubated in the LB medium at 20°C for 20 h for TagRFP maturation and then at 37°C for 4 h for Ultramarine maturation. The sensor was isolated and purified by successive hydrophobic and anion exchange chromatography as described previously [16]. At the stage of hydrophobic chromatography, the separation of the sample into two fractions was noted (Fig. 2a); during the elution of the fractions it was found that in the first fraction there was no absorption corresponding to the Ultramarine protein (586 nm). The absorption spectra of the fractions also showed that fraction 2 had a shoulder at 586 nm, while fraction 1 did not have it (Fig. 2b). Thus, the method of hydrophobic chromatography allowed us to separate the sensor with the mature Ultramarine protein from its fractions with the immature one.

Fig. 2.
figure 2

Elution profiles of the TR-23-U sensor during hydrophobic chromatography (a): 1, elution profile at 586 nm; 2, at a wavelength of 556 nm. Absorption spectra corresponding to the collected fractions (b): I and II.

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Gel filtration and DLS methods showed that the chimeric protein behaves as a dimer in solution (GF: Vel = 15.5 mL, Mw = 32 kDa, DLS: R = 2.8 ± 0.2 nm, Mw = 37 ± 8 kDa); therefore, both proteins in the sensor are in the monomeric state and the sensor itself can be characterized as monomeric.

The properties of the new fusion protein as a FRET sensor were confirmed based on measurements of the lifetime and fluorescence intensity of TagRFP before and after incubation of the sensor with caspase-3 in vitro. The sensor was incubated with caspase-3 in a buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol; 1 mg/mL BSA) overnight to measure the maximum dynamic range of the fluorescent response of the sensor at 37°C. After incubation, the TagRFP fluorescence intensity increased by more than 2 times (Fig. 3), which indicated the effective hydrolysis of the sensor by caspase-3.

Fig. 3.
figure 3

Fluorescence emission spectra of TagRFP (fluorescence intensity; units) in the TR-23-U sensor before and after overnight incubation with caspase-3: 1, TagRFP fluorescence spectrum before incubation with caspase-3; 2, after incubation.

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For the characterization of the caspase-3 activity on mammalian cells, the donor fluorescence lifetime is a more indicative parameter, since this parameter does not depend on the protein concentration. The lifetime of TagRFP in the TR-23-U sensor is described by biexponential dependence (3):

$$I = {{I}_{1}}\exp \left( {{{-t} \mathord{\left/ {\vphantom {{-t} {{{\tau }_{1}}}}} \right. \kern-0em} {{{\tau }_{1}}}}} \right)-{{I}_{2}}\exp \left( {{{-t} \mathord{\left/ {\vphantom {{-t} {{{\tau }_{2}}}}} \right. \kern-0em} {{{\tau }_{2}}}}} \right) + c,$$
(3)

where I1, I2 are preexponential coefficients that characterize the ratio of sensor fractions with τ1 and τ2; τ1 = 2.4 ns corresponds to the fluorescence lifetime of free TagRFP, τ2 = 1.1 ns characterizes TagRFP in the pair with the Ultramarine acceptor, since the presence of FRET reduces the lifetime [19]; c is the background signal including residual fluorescence. The presence of a long-lived component for TR-23-U may indicate the presence of a protein conformation of the sensor with minimal FRET. The values of the parameters measured before and after incubation with caspase-3 are presented in Table 4. The ratio of the lifetime of free TagRFP and TagRFP-23-Ultramarine, in which FRET is realized, after overnight incubation with caspase-3 changed by more than 5.8 times (Table 4), and the value of I2 before and after, which corresponds to a fraction with a high FRET, decreased by 7 times.

Table 4. The fluorescence lifetime parameters of the TagRFP protein in the TR-23-U sensor before and after incubation with caspase-3
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Thus, four chromoproteins were expressed and isolated: Ultramarine, gfasCP, anm2CP, spisCP, which have high values of the overlap integrals and Förster radii with the TagRFP red fluorescent protein. Using the methods of GF and DLS, it was shown that Ultramarine is a monomer, gfasCP and spisCP are dimers, and anm2CP is dimer with an admixture of larger aggregates. The TagRFP-23-Ultramarine FRET sensor was created based on the TagRFP and Ultramarine proteins, which contains the caspase-3 recognition site in the linker. It was shown that the sensor is monomer. The new sensor is also a caspase-3 substrate. After incubation, the fluorescence intensity of TagRFP increased by more than 2 times, and the ratio of fluorescence lifetimes of free and bound TagRFP changed by 5.8 times towards free TagRFP. An increase in these two parameters indicates a disruption of FRET between TagRFP and Ultramarine, i.e., effective cleavage of the sensor by caspase-3, which will allow one to use the new TR-23-U fusion protein as a FRET sensor for caspase-3 to detect early stages of apoptosis.