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Electric field effects on fluorescence of the green fluorescent protein
Chemical Physics Letters 457 (2008) 408–412
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Electric field effects on fluorescence of the green fluorescent protein Takakazu Nakabayashi a, Masataka Kinjo a,b, Nobuhiro Ohta a,* a b
Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 060-0812, Japan Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan
a r t i c l e
i n f o
Article history: Received 26 February 2008 In final form 7 April 2008 Available online 10 April 2008
a b s t r a c t External electric field effects on state energy and photoexcitation dynamics have been examined for a mutant of UV-excited green fluorescent protein (GFPuv5) in a PVA film. The electrofluorescence spectrum of GFPuv5 is reproduced by a linear combination between the fluorescence spectrum and its second derivative spectrum, indicating the field-induced fluorescence quenching and the difference in electric dipole moment between the fluorescent state and the ground state. The direct measurements of the field-induced change in fluorescence decay show that the field-induced quenching results from the field-induced increase in the rate of the non-radiative process from the fluorescent state. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction The green fluorescent protein (GFP) from Aequorea victoria has become an invaluable tool in molecular and cell biology [1,2]. GFP has been used in many applications as a fluorescent marker for gene expression and protein localization in cells and organisms. Various kinds of mutants have also been developed for adding useful characters such as shifted fluorescence wavelength and enhanced fluorescence intensity [3,4]. The chromophore of GFP that is responsible for absorption and fluorescence in the visible region is formed via the biosynthesis from the polypeptide backbone in the absence of any external cofactor [5,6]. The chromophore of GFP consists of a p-hydroxybenzylideneimidazolidinone group (see Fig. 1) that exists in either the neutral phenol form or the anionic phenolate form [5,6]. These two forms exhibit distinct absorbance and fluorescence characteristics. The chromophore is rigidly encapsulated inside a cylinder of b-sheets and is highly protected from bulk medium and fluorescence quenching agents [6–8]. The main non-radiative pathway for the photoexcited chromophore is considered to be the relaxation of the structure of the chromophore [9,10]. The large steric hindrance due to the protein cavity interferes with the non-radiative transition of the chromophore, resulting in the increase in fluorescence quantum yield [9,10]. The protein environment surrounding the chromophore is also responsible for other fluorescence properties of GFP such as fluorescence wavelength, relative contributions between the neutral and anionic chromophores [3,4]. The strong GFP fluorescence has been used for in vivo imaging of cells and organisms; however, fluorescence intensity in itself depends on a variety of biophysical and experimental factors such as excitation intensity fluctuations and photobleaching, and thus is * Corresponding author. E-mail address: [email protected] (N. Ohta). 0009-2614/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2008.04.018
difficult to be analyzed quantitatively. To overcome these problems, imaging of fluorescence lifetime has been proposed [11–13] because fluorescence lifetime is independent of photobleaching, excitation power, and other factors that limit intensity measurements. The fluorescence lifetime images of GFP could also be used for probing cellular environments [11] and analyzing fluorescence resonance energy transfer [12]. However, environmental factors that affect the fluorescence decay profile of GFP have not been fully understood [11]. In the present study, we have focused on the external electric field effects on the photoexcitation dynamics of the GFP chromophore; how the excitation dynamics of the fluorescent state of the GFP chromophore is affected by an electric field. As mentioned above, the GFP chromophore is rigidly embedded in the protein cavity and is surrounded by both apolar and polar residues of a polypeptide chain [6–8]. These charged residues produce a strong electric field inside the protein cavity, which may affect the photoexcitation dynamics of the GFP chromophore [14,15]. Charged and polar groups within protein structures have been reported to produce an electric field of 1–80 MV cm1 for embedded molecules [16–18]. Thus it is conceivable that the electric field produced by the protein cavity is one of the vital factors that control the rate of the non-radiative process of the GFP chromophore. To investigate the electric field effects on the photoexcitation dynamics of the GFP chromophore, we prepared a poly(vinyl alcohol) (PVA) film in which GFP is isotropically distributed and examined the external electric field effects on fluorescence spectrum and fluorescence decay profile of the GFP chromophore [19–21]. Since the molecular structure of the GFP chromophore remains unchanged in PVA, there is fundamental importance in investigating external electric field effects on the fluorescence of GFP-doped films to understand effects of the surrounding environment on the photoexcitation dynamics of the GFP chromophore. We also evaluated the difference in electric dipole moment between the
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Fig. 1. Schematic structure of the p-hydroxybenzylidene-imidazolidinone group of the chromophore of GFP.
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cipitation of GFPuv5. Then the maximum of the absorbance of GFPuv5 in PVA at 490 nm was in the order of 103 at the thickness of 9000 Å. The mixture was then cast on an indium-tinoxide (ITO)-coated quartz substrate by a spin coating method. A semitransparent aluminum (Al) film was deposited on the polymer film by vacuum vapor deposition. The ITO and Al films were used as electrodes. 3. Results and discussion
ground state and the fluorescent state of the GFP chromophore from the analysis of the field-induced change in fluorescence spectrum. 2. Experimental All the measurements were performed under vacuum conditions at room temperature. Plots of the electric field-induced change in fluorescence intensity as a function of wavelength, i.e., the so-called electrofluorescence spectrum, was obtained using electric field modulation spectroscopy with the same apparatus as reported previously [19,21]. A sinusoidal ac voltage was applied to a sample with a modulation frequency of 40 Hz. Field-induced change in fluorescence intensity was detected with a lock-in amplifier at the second harmonic of the modulation frequency. A field-free component of the fluorescence intensity was simultaneously observed. Applied field strength was evaluated from the applied voltage divided by the thickness of a film. Hereafter, electrofluorescence spectrum is abbreviated as E–F spectrum, and applied electric field is denoted by F. Measurements of the field-induced change in fluorescence decay were carried out using a single-photon counting system combined with a pulse generator supplying a bipolar square wave [20]. The second harmonic of the output from a mode-locked Ti:sapphire laser (Spectra Physics, Tsunami) was used for the excitation, and the repetition rate of the laser pulse was selected to be 5.8 MHz with a pulse picker (Conoptics, 350-160). Fluorescence from the sample was dispersed with a monochromator (Nikon, G-250) and detected with a microchannel-plate photomultiplier (Hamamatsu, R3809U-52). Color glass filters were also used to eliminate scattered excitation light. Fluorescence decays were obtained with a multichannel pulse height analyzer (SEIKO EG&G, 7700). The instrumental response function had a pulse width of 60 ps (full-width at a half-maximum). Applied voltage was a repetition of rectangular waves of positive, zero, negative, and zero bias. The time duration of each bias was 30 ms, but the first 2 ms was a deadtime to exclude an overshooting effect of applied field just after the change in applied voltage. Four different decays were collected, corresponding to positive, zero, negative, and zero sample bias, respectively. In the present study, GFPuv5, which is a UV-excited GFP variant, was used for the preparation of the polymer film because it exhibits fluorescence brighter than those of wild-type GFP (wtGFP) and enhanced GFP (EGFP) [22–25]. The GFPuv5 has the GFPuv (F99S/ M153T/V163A), the EGFP (F64L/S65T), the S208L, and the I167T mutations relative to wtGFP [23,24] that do not affect the molecular structure of the chromophore. The crystal structure of GFPuv indicates that all the three mutations are present on the surface of the cylinder of b-sheets [26]. PVA (molecular mass 124 000– 186 000) was obtained from Sigma–Aldrich and purified several times by precipitation with water and methanol. A 0.4-ml portion of the aqueous solution (5 ml total) containing PVA (88 mg) was mixed with 50 ll of ca. 100 lM GFP buffer solution and stirred for several minutes at room temperature. Organic reagents are generally used for precipitation of proteins, so that the concentration of GFPuv5 was adjusted to be as low as possible to avoid pre-
3.1. Optical properties in a PVA film The optical properties of GFP in a PVA polymer film are firstly examined by measuring fluorescence spectra, fluorescence excitation spectra, and fluorescence decay profiles. As mentioned in Section 1, the GFP chromophore in the ground state exists as equilibrium between the neutral and anionic forms (see Fig. 1), the magnitude of which depends on mutation and physiological parameters. Thus the absorption spectra of wtGFP and its mutants are generally characterized by two bands around 400 and 480 nm, which arise from the neutral and anionic forms of the chromophore, respectively [3,22,27–31]. Fluorescence emission mostly comes from the anionic chromophore. The absorption of GFPuv5 is dominated by the one of the anionic chromophore with a peak at 490 nm, and the fluorescence intensity is stronger than those of other GFP mutants [22–24]. Fig. 2a shows the fluorescence spectra of GFPuv5 in a PVA film and in phosphate-buffered saline (PBS) solution at pH 7.3 excited at 440 nm. It is clearly seen that the fluorescence spectra are identical between PVA and PBS, although the fluorescence intensity seems to be lower in PVA. The fluorescence spectra of GFPuv5 exhibit a strong peak at 513 nm and a shoulder around 540 nm [22,23], which roughly mirrors the absorption spectrum of the anionic chromophore. Such a fluorescence spectrum is also observed for wtGFP, EGFP and other GFP mutants [3,22,27–31]. Fig. 2b shows the fluorescence excitation spectra of GFPuv5 in a PVA film and in PBS solution with 540-nm detection. The excitation spectrum of GFPuv5 is very similar in shape to those of EGFP and S65T, indicating that the absorption spectrum of GFPuv5 is also very similar in shape to those of EGFP and S65T [3,22,28,30,31].
Fig. 2. (a) Fluorescence spectra of GFPuv5 in a PVA film (solid line) and in PBS solution (dotted line) excited at 440 nm. (b) Excitation spectra of GFPuv5 in a PVA film (solid line) and in PBS solution (dotted line) with 540-nm detection. (c) Fluorescence decay profiles of GFPuv5 in a PVA film (solid line) and in PBS solution (dotted line). Excitation and fluorescence wavelengths were 440 and 515 nm, respectively. Fluorescence is normalized to unity in every case.
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It is found that the excitation spectrum in PVA is almost the same in shape as that in PBS, indicating that the dominance of the anionic form as well as the molecular structure of the chromophore remains unchanged in a PVA film. From the measurements of the fluorescence and fluorescence excitation spectra, no clear difference was observed between PVA polymer film and buffer solution. However, a significant difference was observed in the fluorescence decay. Fig. 2c shows the fluorescence decay profiles of GFPuv5 observed at 515 nm in a PVA film and in PBS solution. The fluorescence of GFPuv5 in PBS shows a bi-exponential decay with the average fluorescence lifetime of 2.62 ns, which is within the range reported for the fluorescence lifetimes of other GFP families [11,25,29–32]. However, the average fluorescence lifetime of GFPuv5 becomes shorter in PVA to 432 ps (the detailed analysis is given later). It is known that the fluorescence of GFP is completely quenched by denaturation [33,34], indicating that the fluorescence lifetime of GFP becomes drastically shorter during denaturation. If the denaturation completely occurs, therefore, no fluorescence is detected. The short lifetime of fluorescence in PVA, which is equivalent to the low fluorescence quantum yield, may indicate that small conformational changes in protein structure occur in PVA, which weakens the hindrance to the structural relaxation of the excited chromophore. It is also conceivable that the chromophore is not shielded completely from PVA by the change in protein structure, and the interaction between the chromophore and PVA should be very small because both the fluorescence and excitation spectra remain unchanged in a PVA film. To summarize, the fluorescence lifetime of the chromophore of GFPuv5 is shorter in PVA than in solution, which is probably due to the protein folding not in the native conformation [33,34], but the molecular structure of the chromophore of GFPuv5 with the anionic form remains unchanged in a PVA film. Then, it is possible to examine the electric field effects on photoexcitation dynamics of the GFP chromophore at the fluorescent state by using GFPuv5doped polymer films. It is conceivable that the results are very useful to discuss the local fields produced by the surroundings of the GFP chromophore. 3.2. Electric field effect on fluorescence spectrum Fig. 3a shows the E–F spectrum of GFPuv5 in a PVA film with a field strength of 0.5 MV cm1, together with the fluorescence spectrum simultaneously observed. We could not obtain the reliable electroabsorption spectrum of GFPuv5 because of the low concen-
Fig. 3. (a) Electrofluorescence spectrum of GFPuv5 in a PVA film (thick solid line) and the fluorescence spectrum (thin solid line) simultaneously observed. The simulated spectrum is shown by a dotted line. Excitation wavelength was 440 nm and applied field strength was 0.5 MV cm1. (b) The first (dotted line) and second (solid line) derivatives of the fluorescence spectrum.
trations of GFPuv5 in PVA, but the absorption spectrum was essentially the same in shape as the excitation spectrum shown in Fig. 2b. In the present study, therefore, excitation wavelength was chosen to be 440 nm because the field-induced change in absorption intensity is negligible at 440 nm for S65T [35]. Note that the absorption spectra of GFPuv5 and S65T are nearly the same [3,35]. The first and second derivatives of the fluorescence spectrum are shown in Fig. 3b. A field-induced quenching of the fluorescence is clearly observed in the E–F spectrum. The dominance of the field-induced fluorescence quenching was also confirmed at the excitation wavelength of 478 nm. The E–F spectrum is well reproduced by a linear combination of the zeroth and second derivatives of the fluorescence spectrum (see Fig. 3a). In the presence of F, each energy level of GFPuv5 is shifted, depending on electric dipole moment (l) and molecular polarizability (a) of the state concerned. When the magnitude of l or a in the excited state is different from the one in the ground state, the fluorescence spectrum is shifted since the magnitudes of the level shift in both states are different from each other. For an isotropic distribution of chromophores in rigid matrices such as PVA, the E–F spectrum (DIF(m)) is given by the following equation [15,36]: " # 2 3 3 2 3 dfI F ðmÞ=m g 3 d fI F ðmÞ=m g þ Cm DIF ðmÞ ¼ ðf FÞ AIF ðmÞ þ Bm ð1Þ dm dm2 where f is the internal field factor. The coefficient A corresponds to the field-induced change in fluorescence quantum yield. B and C correspond to the spectral shift and spectral broadening resulting from the differences in molecular polarizability (Da) and in electric dipole moment (Dl), respectively, between the ground state and the excited state, which are given as follows: D a=2 þ ðDam D aÞð3 cos2 v 1Þ=10 hc 2 2 2 ½5 þ ð3 cos n 1Þð3 cos v 1Þ C ¼ jDlj 2 30h c2
B¼
ð2Þ ð3Þ
D a denotes the trace of Da; Dam is the diagonal component of Da with respect to the direction of the transition dipole moment; v is the angle between the direction of F and the electric vector of the light; and n is the angle between the direction of Dl and the transition dipole moment. It is worth mentioning that the first-order term in F, i.e., any odd term, becomes zero when this term is integrated over the full space in a randomly distributed system; however, the second-order term in F, i.e., any even term, can give the nonzero value even when this term is integrated over the full space. The magnitude of the field-induced change in fluorescence intensity is proportional to the square of the applied field strength, as expected from Eq. (1). The zeroth derivative component in the E–F spectrum indicates that the fluorescence of GFPuv5 is quenched in the presence of F. The magnitude of the quenching is 0.1% with a field strength of 0.5 MV cm1. The second derivative component in the E–F spectrum arises from |Dl| following the transition from the fluorescent state to the ground state (see Eqs. (1) and (3)). The |Dl| value between the fluorescent state and the ground state of the anionic chromophore is evaluated to be 3D by assuming that the angle-dependent term in Eq. (3) is negligible and f = 1. The angle-dependent term induces the uncertainty of ±1D for the evaluated |Dl| value. It is difficult to determine the f value precisely, thus |Dl| is evaluated using f = 1 or the unit of D/ f in a variety of cases [17,27,37]. The magnitude of Dl for the fluorescence is smaller than those for the absorption of wtGFP and the S65T mutant (7D) [27,35]. This may arise from the geometrical relaxation from the Franck-Condon state to the fluorescent state [10]. The effect of the medium on |Dl| may also have to be considered. It is computed that the anionic form of the p-hydroxybenzylidene-imidazolidinone structure (see Fig. 1) has the |Dl| value of
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1–2 D for the Franck-Condon transition [10,38]. The first derivative component is negligibly small in the E–F spectrum, indicating that the magnitude of Da is very small following the transition. 3.3. Electric field effect on fluorescence decay profile From the steady state measurements, it is difficult to confirm whether the field-induced change in fluorescence intensity is ascribed to a change in fluorescence lifetime or a change in initial population of the fluorescent component. The field-induced change in fluorescence intensity resulting from the Stark shift of the absorption spectrum can be examined by measuring the field-induced change in fluorescence decay. This is because the field-induced change in absorption intensity only affects the initial population of the fluorescent component. In the present study, therefore, we have measured the external electric field effects on fluorescence decay profile of GFPuv5 in a PVA film. Fig. 4a shows the fluorescence decay of GFPuv5 in a PVA film in the absence of F. Excitation and monitoring wavelengths were 440 and 515 nm, respectively. The difference between the decays observed at zero field (I0(t)) and at 0.7 MV cm1 (IF(t)), i.e., IF(t) I0(t), referred to as DIf(t), is shown in Fig. 4b. The DIf(t) value is negative during the full decay, showing that the fluorescence is quenched by F in the whole time region. This result agrees with the one obtained from the E–F spectrum. The time dependence of DIf(t) is different from that of I0(t), indicating that the fluorescence lifetime of GFPuv5 is influenced by F. If the fluorescence lifetime is independent of F, the intensity ratio between the two decays observed in the presence and absence of F should remain constant over the whole time region. As shown in Fig. 4c, however, the IF(t)/I0(t) value is not constant but decreases with time. These results clearly indicate that the fluorescence lifetime of GFPuv5 is reduced in the presence of F. It should also be noted that the IF(t)/I0(t) value is almost unity just after photoexcitation, indicating that the field-induced change in the initial population of the fluorescent component is very small. Thus, the field-induced change in fluorescence intensity originating from the Stark shift of the absorption spectrum is confirmed to be negligible at 440 nm excitation. GFPuv5 in PVA exhibits a multi-exponential fluorescence decay, which probably arises from inhomogeneous environments in a polymer film [20,21,39,40]. The lifetime change could be evaluated from the profiles of I0(t), DIf(t), and IF(t)/I0(t) by assuming a tri-exponential P decay, i.e., i Ai expðt=si Þ, where Ai and si denote the pre-exponen-
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tial factor and the fluorescence lifetime of component i, respectively. The profiles of DIf(t) and IF(t)/I0(t) are very sensitive to the parameters of a tri-exponential decay [20,21,39]. By simulating all the three time profile of I0(t), DIf(t) and IF(t)/I0(t), the field-induced change in lifetime and pre-exponential factor could be evaluated with an error as small as 0.05% [20,21,39]. The lifetime and the pre-exponential factor of each time component in the absence of F are evaluated to be as follows: s1 = 239.3 ps, s2 = 861.9 ps, s3 = 2498.7 ps, F1 = 0.6995, F2 = 0.2971, F3 = 0.0034. The corresponding values in the presence of 0.7 MV cm1 are determined to be as follows: s1 = 238.4 ps, s2 = 859.8 ps, s3 = 2498.2 ps, F1 = 0.7000, F2 = 0.2966, F3 P = 0.0034. The average fluorescence lifetime, defined by i Ai si = P i Ai , is determined to be 432 ps at zero field and 430 ps at 0.7 MV cm1. This result indicates that the field-induced quenching of the fluorescence in Fig. 3 results from the decrease in the fluorescence lifetime. The observed field-induced decrease in the fluorescence lifetime can be attributed to the field-induced acceleration of the rate of the non-radiative process from the fluorescent state. It has been shown that the presence of F induces significant changes in charge transfer (CT) dynamics of intramolecular as well as intermolecular donor–acceptor systems, while internal conversion including no CT process such as radiationless decay of pyrene and N-ethylcarbazole is not affected by F [15]. It is therefore concluded that the non-radiative process of the GFP chromophore includes significant CT process. The synthetic GFP chromophore derivative does not exhibit fluorescence in liquid ethanol but fluoresces when it is confined in an ethanol matrix at 77 K, indicating that the conformational relaxation is the main non-radiative pathway of the GFP chromophore [9]. Ab initio theoretical calculations suggest that the critical non-radiative pathway of the GFP chromophore is the twisting between the two rings of the chromophore, resulting in the formation of a non-radiative twisted intermediate state with CT character [10]. We recently showed that the trans–cis photoisomerization processes of diphenylpolyenes are accelerated by F, which arises from CT character of the twisted intermediate of the conformational relaxation [21]. It is thus concluded that the field-induced enhancement of the non-radiative process of the GFP chromophore comes from the CT character of the intermediate of the non-radiative process. It should be noted that the field-induced acceleration of the non-radiative rate of the chromophore should occur in normal GFP because the molecular structure of the chromophore itself remains constant in buffer solution and in PVA (see Fig. 2). Since the magnitude of the change in fluorescence yield in a randomly distributed system is proportional to the square of applied field strength (see Eq. (1)), the field-induced change in fluorescence lifetime (DsF) is written as follows: DsF/sF = A(fF)2, where sF is the fluorescence lifetime at zero field and A is the coefficient of Eq. (1). The magnitude of the field-induced decrease in fluorescence lifetime at 0.7 MV cm1 is 0.5%. Then, DsF of the GFP chromophore can be qualitatively estimated using the following equation: DsF = 0.01sF|F|2, where F is in MV cm1. By measuring the change in fluorescence lifetime, therefore, we can evaluate the change in magnitude of the electric field applied to the embedded chromophore. The obtained result is also applicable to the native condition because no specific interaction or relaxation is assumed in the present study.
4. Conclusions
Fig. 4. (a) Fluorescence decay (solid line) of GFPuv5 in a PVA film at zero field. (b) The difference between the decays at 0.7 MV cm1 and at zero field (solid line). (c) The ratio of the decay at 0.7 MV cm1 relative to that at zero field (solid line). The simulated curve is shown in each panel by a dotted line. Excitation and monitoring wavelengths were 440 and 515 nm, respectively.
External electric field effects on fluorescence spectrum and fluorescence decay profile of GFPuv5 in a PVA film have been measured. The E–F spectrum of GFPuv5 is reproduced by a linear combination between the fluorescence spectrum and its second derivative spectrum, indicating both the field-induced quenching
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of the fluorescence and the change of |Dl| following the transition. The measurements of the field-induced change in fluorescence decay profile indicate that the field-induced decrease in fluorescence intensity results from the field-induced increase in rate of the nonradiative process of the GFP chromophore. The enhancement of the non-radiative process in the presence of F may arise from the CT character of the twisted intermediate on the way of the non-radiative process. The present study indicates that the local electric field applied to the GFP chromophore is one of the significant factors that determine the fluorescence lifetime of GFPs. Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research in Priority Area ‘Molecular Nano Dynamics’ from the Ministry of Education, Culture, Sports, Science, and Technology in Japan. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
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Electric field effects on fluorescence of the green fluorescent protein Takakazu Nakabayashi a, Masataka Kinjo a,b, Nobuhiro Ohta a,* a b
Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo 060-0812, Japan Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan
a r t i c l e
i n f o
Article history: Received 26 February 2008 In final form 7 April 2008 Available online 10 April 2008
a b s t r a c t External electric field effects on state energy and photoexcitation dynamics have been examined for a mutant of UV-excited green fluorescent protein (GFPuv5) in a PVA film. The electrofluorescence spectrum of GFPuv5 is reproduced by a linear combination between the fluorescence spectrum and its second derivative spectrum, indicating the field-induced fluorescence quenching and the difference in electric dipole moment between the fluorescent state and the ground state. The direct measurements of the field-induced change in fluorescence decay show that the field-induced quenching results from the field-induced increase in the rate of the non-radiative process from the fluorescent state. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction The green fluorescent protein (GFP) from Aequorea victoria has become an invaluable tool in molecular and cell biology [1,2]. GFP has been used in many applications as a fluorescent marker for gene expression and protein localization in cells and organisms. Various kinds of mutants have also been developed for adding useful characters such as shifted fluorescence wavelength and enhanced fluorescence intensity [3,4]. The chromophore of GFP that is responsible for absorption and fluorescence in the visible region is formed via the biosynthesis from the polypeptide backbone in the absence of any external cofactor [5,6]. The chromophore of GFP consists of a p-hydroxybenzylideneimidazolidinone group (see Fig. 1) that exists in either the neutral phenol form or the anionic phenolate form [5,6]. These two forms exhibit distinct absorbance and fluorescence characteristics. The chromophore is rigidly encapsulated inside a cylinder of b-sheets and is highly protected from bulk medium and fluorescence quenching agents [6–8]. The main non-radiative pathway for the photoexcited chromophore is considered to be the relaxation of the structure of the chromophore [9,10]. The large steric hindrance due to the protein cavity interferes with the non-radiative transition of the chromophore, resulting in the increase in fluorescence quantum yield [9,10]. The protein environment surrounding the chromophore is also responsible for other fluorescence properties of GFP such as fluorescence wavelength, relative contributions between the neutral and anionic chromophores [3,4]. The strong GFP fluorescence has been used for in vivo imaging of cells and organisms; however, fluorescence intensity in itself depends on a variety of biophysical and experimental factors such as excitation intensity fluctuations and photobleaching, and thus is * Corresponding author. E-mail address: [email protected] (N. Ohta). 0009-2614/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2008.04.018
difficult to be analyzed quantitatively. To overcome these problems, imaging of fluorescence lifetime has been proposed [11–13] because fluorescence lifetime is independent of photobleaching, excitation power, and other factors that limit intensity measurements. The fluorescence lifetime images of GFP could also be used for probing cellular environments [11] and analyzing fluorescence resonance energy transfer [12]. However, environmental factors that affect the fluorescence decay profile of GFP have not been fully understood [11]. In the present study, we have focused on the external electric field effects on the photoexcitation dynamics of the GFP chromophore; how the excitation dynamics of the fluorescent state of the GFP chromophore is affected by an electric field. As mentioned above, the GFP chromophore is rigidly embedded in the protein cavity and is surrounded by both apolar and polar residues of a polypeptide chain [6–8]. These charged residues produce a strong electric field inside the protein cavity, which may affect the photoexcitation dynamics of the GFP chromophore [14,15]. Charged and polar groups within protein structures have been reported to produce an electric field of 1–80 MV cm1 for embedded molecules [16–18]. Thus it is conceivable that the electric field produced by the protein cavity is one of the vital factors that control the rate of the non-radiative process of the GFP chromophore. To investigate the electric field effects on the photoexcitation dynamics of the GFP chromophore, we prepared a poly(vinyl alcohol) (PVA) film in which GFP is isotropically distributed and examined the external electric field effects on fluorescence spectrum and fluorescence decay profile of the GFP chromophore [19–21]. Since the molecular structure of the GFP chromophore remains unchanged in PVA, there is fundamental importance in investigating external electric field effects on the fluorescence of GFP-doped films to understand effects of the surrounding environment on the photoexcitation dynamics of the GFP chromophore. We also evaluated the difference in electric dipole moment between the
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Fig. 1. Schematic structure of the p-hydroxybenzylidene-imidazolidinone group of the chromophore of GFP.
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cipitation of GFPuv5. Then the maximum of the absorbance of GFPuv5 in PVA at 490 nm was in the order of 103 at the thickness of 9000 Å. The mixture was then cast on an indium-tinoxide (ITO)-coated quartz substrate by a spin coating method. A semitransparent aluminum (Al) film was deposited on the polymer film by vacuum vapor deposition. The ITO and Al films were used as electrodes. 3. Results and discussion
ground state and the fluorescent state of the GFP chromophore from the analysis of the field-induced change in fluorescence spectrum. 2. Experimental All the measurements were performed under vacuum conditions at room temperature. Plots of the electric field-induced change in fluorescence intensity as a function of wavelength, i.e., the so-called electrofluorescence spectrum, was obtained using electric field modulation spectroscopy with the same apparatus as reported previously [19,21]. A sinusoidal ac voltage was applied to a sample with a modulation frequency of 40 Hz. Field-induced change in fluorescence intensity was detected with a lock-in amplifier at the second harmonic of the modulation frequency. A field-free component of the fluorescence intensity was simultaneously observed. Applied field strength was evaluated from the applied voltage divided by the thickness of a film. Hereafter, electrofluorescence spectrum is abbreviated as E–F spectrum, and applied electric field is denoted by F. Measurements of the field-induced change in fluorescence decay were carried out using a single-photon counting system combined with a pulse generator supplying a bipolar square wave [20]. The second harmonic of the output from a mode-locked Ti:sapphire laser (Spectra Physics, Tsunami) was used for the excitation, and the repetition rate of the laser pulse was selected to be 5.8 MHz with a pulse picker (Conoptics, 350-160). Fluorescence from the sample was dispersed with a monochromator (Nikon, G-250) and detected with a microchannel-plate photomultiplier (Hamamatsu, R3809U-52). Color glass filters were also used to eliminate scattered excitation light. Fluorescence decays were obtained with a multichannel pulse height analyzer (SEIKO EG&G, 7700). The instrumental response function had a pulse width of 60 ps (full-width at a half-maximum). Applied voltage was a repetition of rectangular waves of positive, zero, negative, and zero bias. The time duration of each bias was 30 ms, but the first 2 ms was a deadtime to exclude an overshooting effect of applied field just after the change in applied voltage. Four different decays were collected, corresponding to positive, zero, negative, and zero sample bias, respectively. In the present study, GFPuv5, which is a UV-excited GFP variant, was used for the preparation of the polymer film because it exhibits fluorescence brighter than those of wild-type GFP (wtGFP) and enhanced GFP (EGFP) [22–25]. The GFPuv5 has the GFPuv (F99S/ M153T/V163A), the EGFP (F64L/S65T), the S208L, and the I167T mutations relative to wtGFP [23,24] that do not affect the molecular structure of the chromophore. The crystal structure of GFPuv indicates that all the three mutations are present on the surface of the cylinder of b-sheets [26]. PVA (molecular mass 124 000– 186 000) was obtained from Sigma–Aldrich and purified several times by precipitation with water and methanol. A 0.4-ml portion of the aqueous solution (5 ml total) containing PVA (88 mg) was mixed with 50 ll of ca. 100 lM GFP buffer solution and stirred for several minutes at room temperature. Organic reagents are generally used for precipitation of proteins, so that the concentration of GFPuv5 was adjusted to be as low as possible to avoid pre-
3.1. Optical properties in a PVA film The optical properties of GFP in a PVA polymer film are firstly examined by measuring fluorescence spectra, fluorescence excitation spectra, and fluorescence decay profiles. As mentioned in Section 1, the GFP chromophore in the ground state exists as equilibrium between the neutral and anionic forms (see Fig. 1), the magnitude of which depends on mutation and physiological parameters. Thus the absorption spectra of wtGFP and its mutants are generally characterized by two bands around 400 and 480 nm, which arise from the neutral and anionic forms of the chromophore, respectively [3,22,27–31]. Fluorescence emission mostly comes from the anionic chromophore. The absorption of GFPuv5 is dominated by the one of the anionic chromophore with a peak at 490 nm, and the fluorescence intensity is stronger than those of other GFP mutants [22–24]. Fig. 2a shows the fluorescence spectra of GFPuv5 in a PVA film and in phosphate-buffered saline (PBS) solution at pH 7.3 excited at 440 nm. It is clearly seen that the fluorescence spectra are identical between PVA and PBS, although the fluorescence intensity seems to be lower in PVA. The fluorescence spectra of GFPuv5 exhibit a strong peak at 513 nm and a shoulder around 540 nm [22,23], which roughly mirrors the absorption spectrum of the anionic chromophore. Such a fluorescence spectrum is also observed for wtGFP, EGFP and other GFP mutants [3,22,27–31]. Fig. 2b shows the fluorescence excitation spectra of GFPuv5 in a PVA film and in PBS solution with 540-nm detection. The excitation spectrum of GFPuv5 is very similar in shape to those of EGFP and S65T, indicating that the absorption spectrum of GFPuv5 is also very similar in shape to those of EGFP and S65T [3,22,28,30,31].
Fig. 2. (a) Fluorescence spectra of GFPuv5 in a PVA film (solid line) and in PBS solution (dotted line) excited at 440 nm. (b) Excitation spectra of GFPuv5 in a PVA film (solid line) and in PBS solution (dotted line) with 540-nm detection. (c) Fluorescence decay profiles of GFPuv5 in a PVA film (solid line) and in PBS solution (dotted line). Excitation and fluorescence wavelengths were 440 and 515 nm, respectively. Fluorescence is normalized to unity in every case.
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It is found that the excitation spectrum in PVA is almost the same in shape as that in PBS, indicating that the dominance of the anionic form as well as the molecular structure of the chromophore remains unchanged in a PVA film. From the measurements of the fluorescence and fluorescence excitation spectra, no clear difference was observed between PVA polymer film and buffer solution. However, a significant difference was observed in the fluorescence decay. Fig. 2c shows the fluorescence decay profiles of GFPuv5 observed at 515 nm in a PVA film and in PBS solution. The fluorescence of GFPuv5 in PBS shows a bi-exponential decay with the average fluorescence lifetime of 2.62 ns, which is within the range reported for the fluorescence lifetimes of other GFP families [11,25,29–32]. However, the average fluorescence lifetime of GFPuv5 becomes shorter in PVA to 432 ps (the detailed analysis is given later). It is known that the fluorescence of GFP is completely quenched by denaturation [33,34], indicating that the fluorescence lifetime of GFP becomes drastically shorter during denaturation. If the denaturation completely occurs, therefore, no fluorescence is detected. The short lifetime of fluorescence in PVA, which is equivalent to the low fluorescence quantum yield, may indicate that small conformational changes in protein structure occur in PVA, which weakens the hindrance to the structural relaxation of the excited chromophore. It is also conceivable that the chromophore is not shielded completely from PVA by the change in protein structure, and the interaction between the chromophore and PVA should be very small because both the fluorescence and excitation spectra remain unchanged in a PVA film. To summarize, the fluorescence lifetime of the chromophore of GFPuv5 is shorter in PVA than in solution, which is probably due to the protein folding not in the native conformation [33,34], but the molecular structure of the chromophore of GFPuv5 with the anionic form remains unchanged in a PVA film. Then, it is possible to examine the electric field effects on photoexcitation dynamics of the GFP chromophore at the fluorescent state by using GFPuv5doped polymer films. It is conceivable that the results are very useful to discuss the local fields produced by the surroundings of the GFP chromophore. 3.2. Electric field effect on fluorescence spectrum Fig. 3a shows the E–F spectrum of GFPuv5 in a PVA film with a field strength of 0.5 MV cm1, together with the fluorescence spectrum simultaneously observed. We could not obtain the reliable electroabsorption spectrum of GFPuv5 because of the low concen-
Fig. 3. (a) Electrofluorescence spectrum of GFPuv5 in a PVA film (thick solid line) and the fluorescence spectrum (thin solid line) simultaneously observed. The simulated spectrum is shown by a dotted line. Excitation wavelength was 440 nm and applied field strength was 0.5 MV cm1. (b) The first (dotted line) and second (solid line) derivatives of the fluorescence spectrum.
trations of GFPuv5 in PVA, but the absorption spectrum was essentially the same in shape as the excitation spectrum shown in Fig. 2b. In the present study, therefore, excitation wavelength was chosen to be 440 nm because the field-induced change in absorption intensity is negligible at 440 nm for S65T [35]. Note that the absorption spectra of GFPuv5 and S65T are nearly the same [3,35]. The first and second derivatives of the fluorescence spectrum are shown in Fig. 3b. A field-induced quenching of the fluorescence is clearly observed in the E–F spectrum. The dominance of the field-induced fluorescence quenching was also confirmed at the excitation wavelength of 478 nm. The E–F spectrum is well reproduced by a linear combination of the zeroth and second derivatives of the fluorescence spectrum (see Fig. 3a). In the presence of F, each energy level of GFPuv5 is shifted, depending on electric dipole moment (l) and molecular polarizability (a) of the state concerned. When the magnitude of l or a in the excited state is different from the one in the ground state, the fluorescence spectrum is shifted since the magnitudes of the level shift in both states are different from each other. For an isotropic distribution of chromophores in rigid matrices such as PVA, the E–F spectrum (DIF(m)) is given by the following equation [15,36]: " # 2 3 3 2 3 dfI F ðmÞ=m g 3 d fI F ðmÞ=m g þ Cm DIF ðmÞ ¼ ðf FÞ AIF ðmÞ þ Bm ð1Þ dm dm2 where f is the internal field factor. The coefficient A corresponds to the field-induced change in fluorescence quantum yield. B and C correspond to the spectral shift and spectral broadening resulting from the differences in molecular polarizability (Da) and in electric dipole moment (Dl), respectively, between the ground state and the excited state, which are given as follows: D a=2 þ ðDam D aÞð3 cos2 v 1Þ=10 hc 2 2 2 ½5 þ ð3 cos n 1Þð3 cos v 1Þ C ¼ jDlj 2 30h c2
B¼
ð2Þ ð3Þ
D a denotes the trace of Da; Dam is the diagonal component of Da with respect to the direction of the transition dipole moment; v is the angle between the direction of F and the electric vector of the light; and n is the angle between the direction of Dl and the transition dipole moment. It is worth mentioning that the first-order term in F, i.e., any odd term, becomes zero when this term is integrated over the full space in a randomly distributed system; however, the second-order term in F, i.e., any even term, can give the nonzero value even when this term is integrated over the full space. The magnitude of the field-induced change in fluorescence intensity is proportional to the square of the applied field strength, as expected from Eq. (1). The zeroth derivative component in the E–F spectrum indicates that the fluorescence of GFPuv5 is quenched in the presence of F. The magnitude of the quenching is 0.1% with a field strength of 0.5 MV cm1. The second derivative component in the E–F spectrum arises from |Dl| following the transition from the fluorescent state to the ground state (see Eqs. (1) and (3)). The |Dl| value between the fluorescent state and the ground state of the anionic chromophore is evaluated to be 3D by assuming that the angle-dependent term in Eq. (3) is negligible and f = 1. The angle-dependent term induces the uncertainty of ±1D for the evaluated |Dl| value. It is difficult to determine the f value precisely, thus |Dl| is evaluated using f = 1 or the unit of D/ f in a variety of cases [17,27,37]. The magnitude of Dl for the fluorescence is smaller than those for the absorption of wtGFP and the S65T mutant (7D) [27,35]. This may arise from the geometrical relaxation from the Franck-Condon state to the fluorescent state [10]. The effect of the medium on |Dl| may also have to be considered. It is computed that the anionic form of the p-hydroxybenzylidene-imidazolidinone structure (see Fig. 1) has the |Dl| value of
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1–2 D for the Franck-Condon transition [10,38]. The first derivative component is negligibly small in the E–F spectrum, indicating that the magnitude of Da is very small following the transition. 3.3. Electric field effect on fluorescence decay profile From the steady state measurements, it is difficult to confirm whether the field-induced change in fluorescence intensity is ascribed to a change in fluorescence lifetime or a change in initial population of the fluorescent component. The field-induced change in fluorescence intensity resulting from the Stark shift of the absorption spectrum can be examined by measuring the field-induced change in fluorescence decay. This is because the field-induced change in absorption intensity only affects the initial population of the fluorescent component. In the present study, therefore, we have measured the external electric field effects on fluorescence decay profile of GFPuv5 in a PVA film. Fig. 4a shows the fluorescence decay of GFPuv5 in a PVA film in the absence of F. Excitation and monitoring wavelengths were 440 and 515 nm, respectively. The difference between the decays observed at zero field (I0(t)) and at 0.7 MV cm1 (IF(t)), i.e., IF(t) I0(t), referred to as DIf(t), is shown in Fig. 4b. The DIf(t) value is negative during the full decay, showing that the fluorescence is quenched by F in the whole time region. This result agrees with the one obtained from the E–F spectrum. The time dependence of DIf(t) is different from that of I0(t), indicating that the fluorescence lifetime of GFPuv5 is influenced by F. If the fluorescence lifetime is independent of F, the intensity ratio between the two decays observed in the presence and absence of F should remain constant over the whole time region. As shown in Fig. 4c, however, the IF(t)/I0(t) value is not constant but decreases with time. These results clearly indicate that the fluorescence lifetime of GFPuv5 is reduced in the presence of F. It should also be noted that the IF(t)/I0(t) value is almost unity just after photoexcitation, indicating that the field-induced change in the initial population of the fluorescent component is very small. Thus, the field-induced change in fluorescence intensity originating from the Stark shift of the absorption spectrum is confirmed to be negligible at 440 nm excitation. GFPuv5 in PVA exhibits a multi-exponential fluorescence decay, which probably arises from inhomogeneous environments in a polymer film [20,21,39,40]. The lifetime change could be evaluated from the profiles of I0(t), DIf(t), and IF(t)/I0(t) by assuming a tri-exponential P decay, i.e., i Ai expðt=si Þ, where Ai and si denote the pre-exponen-
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tial factor and the fluorescence lifetime of component i, respectively. The profiles of DIf(t) and IF(t)/I0(t) are very sensitive to the parameters of a tri-exponential decay [20,21,39]. By simulating all the three time profile of I0(t), DIf(t) and IF(t)/I0(t), the field-induced change in lifetime and pre-exponential factor could be evaluated with an error as small as 0.05% [20,21,39]. The lifetime and the pre-exponential factor of each time component in the absence of F are evaluated to be as follows: s1 = 239.3 ps, s2 = 861.9 ps, s3 = 2498.7 ps, F1 = 0.6995, F2 = 0.2971, F3 = 0.0034. The corresponding values in the presence of 0.7 MV cm1 are determined to be as follows: s1 = 238.4 ps, s2 = 859.8 ps, s3 = 2498.2 ps, F1 = 0.7000, F2 = 0.2966, F3 P = 0.0034. The average fluorescence lifetime, defined by i Ai si = P i Ai , is determined to be 432 ps at zero field and 430 ps at 0.7 MV cm1. This result indicates that the field-induced quenching of the fluorescence in Fig. 3 results from the decrease in the fluorescence lifetime. The observed field-induced decrease in the fluorescence lifetime can be attributed to the field-induced acceleration of the rate of the non-radiative process from the fluorescent state. It has been shown that the presence of F induces significant changes in charge transfer (CT) dynamics of intramolecular as well as intermolecular donor–acceptor systems, while internal conversion including no CT process such as radiationless decay of pyrene and N-ethylcarbazole is not affected by F [15]. It is therefore concluded that the non-radiative process of the GFP chromophore includes significant CT process. The synthetic GFP chromophore derivative does not exhibit fluorescence in liquid ethanol but fluoresces when it is confined in an ethanol matrix at 77 K, indicating that the conformational relaxation is the main non-radiative pathway of the GFP chromophore [9]. Ab initio theoretical calculations suggest that the critical non-radiative pathway of the GFP chromophore is the twisting between the two rings of the chromophore, resulting in the formation of a non-radiative twisted intermediate state with CT character [10]. We recently showed that the trans–cis photoisomerization processes of diphenylpolyenes are accelerated by F, which arises from CT character of the twisted intermediate of the conformational relaxation [21]. It is thus concluded that the field-induced enhancement of the non-radiative process of the GFP chromophore comes from the CT character of the intermediate of the non-radiative process. It should be noted that the field-induced acceleration of the non-radiative rate of the chromophore should occur in normal GFP because the molecular structure of the chromophore itself remains constant in buffer solution and in PVA (see Fig. 2). Since the magnitude of the change in fluorescence yield in a randomly distributed system is proportional to the square of applied field strength (see Eq. (1)), the field-induced change in fluorescence lifetime (DsF) is written as follows: DsF/sF = A(fF)2, where sF is the fluorescence lifetime at zero field and A is the coefficient of Eq. (1). The magnitude of the field-induced decrease in fluorescence lifetime at 0.7 MV cm1 is 0.5%. Then, DsF of the GFP chromophore can be qualitatively estimated using the following equation: DsF = 0.01sF|F|2, where F is in MV cm1. By measuring the change in fluorescence lifetime, therefore, we can evaluate the change in magnitude of the electric field applied to the embedded chromophore. The obtained result is also applicable to the native condition because no specific interaction or relaxation is assumed in the present study.
4. Conclusions
Fig. 4. (a) Fluorescence decay (solid line) of GFPuv5 in a PVA film at zero field. (b) The difference between the decays at 0.7 MV cm1 and at zero field (solid line). (c) The ratio of the decay at 0.7 MV cm1 relative to that at zero field (solid line). The simulated curve is shown in each panel by a dotted line. Excitation and monitoring wavelengths were 440 and 515 nm, respectively.
External electric field effects on fluorescence spectrum and fluorescence decay profile of GFPuv5 in a PVA film have been measured. The E–F spectrum of GFPuv5 is reproduced by a linear combination between the fluorescence spectrum and its second derivative spectrum, indicating both the field-induced quenching
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of the fluorescence and the change of |Dl| following the transition. The measurements of the field-induced change in fluorescence decay profile indicate that the field-induced decrease in fluorescence intensity results from the field-induced increase in rate of the nonradiative process of the GFP chromophore. The enhancement of the non-radiative process in the presence of F may arise from the CT character of the twisted intermediate on the way of the non-radiative process. The present study indicates that the local electric field applied to the GFP chromophore is one of the significant factors that determine the fluorescence lifetime of GFPs. Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research in Priority Area ‘Molecular Nano Dynamics’ from the Ministry of Education, Culture, Sports, Science, and Technology in Japan. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
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