Chromophore Structure of Photochromic Fluorescent Protein Dronpa

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Chromophore Structure of Photochromic Fluorescent Protein Dronpa: Acid-Base Equilibrium of Two cis Configurations Asuka Higashino, Misao Mizuno, and Yasuhisa Mizutani J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b01752 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 21, 2016

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The Journal of Physical Chemistry

Chromophore Structure of Photochromic Fluorescent Protein Dronpa: Acid-base Equilibrium of Two cis Configurations Asuka Higashino, Misao Mizuno, and Yasuhisa Mizutani* Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

*The author to whom correspondence should be addressed. Phone: +81-6-6850-5776, Fax: +816-6850-5776, E-mail: [email protected]

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ABSTRACT Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid-base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pKa of 5.2±0.3 and maintains the cis configuration. The observed resonance Raman bands showed that the other photochromic state of the chromophore is in a trans configuration. The results demonstrate that Raman bands selectively enhanced for the chromophore yield valuable information on the molecular structure of the chromophore in photochromic fluorescent proteins after careful elimination of the fluorescence background.


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INTRODUCTION Photochromic florescent proteins have emerged as an important subset of the green fluorescent protein (GFP) family.1 Dronpa is a prototypical example and was engineered from a coral protein.2 Dronpa exists in two interconvertible forms; one is fluorescent with a high fluorescence quantum yield (0.85)2 and the other is non-fluorescent. Irradiation with ~500 nm light can convert the fluorescent form to a non-fluorescent form that absorbs at 390 nm. The nonfluorescent protein can then be switched back to the fluorescent form by irradiation at around 400 nm.3-4 The ability of photochromic florescent proteins to undergo repeated cycles of forward and reverse photoconversion has resulted in these proteins finding unique utility in superresolution fluorescence microscopy in living cells.5-7 The photophysical scheme showing the photoswitching of Dronpa conformers provided in Figure 1A shows that a pH-induced non-fluorescent form (A1) also exists. The photoswitching can be described with a three-state model that includes the fluorescent (B), the photoswitched non-fluorescent (A2), and the pH-induced non-fluorescent (A1) forms.8 The bright and dark conditions indicated in Figure 1A refer to the states saturated by ~400 and ~500 nm light, respectively. The B and A1 forms are in an acid–base equilibrium in the bright condition. Furthermore, whereas the B form is interconvertible with the A2 form upon irradiation, the A1 form is not. Dronpa has been the subject of intense structural study to understand how alternative chromophore states exist and interconvert within a protein. The chromophore can adopt either the cis or trans configuration, as shown in Figure 1B, and X-ray crystallographic studies showed that the B and A2 forms are in the former and latter configurations, respectively.9-11 However, the conformation of a chromophore within a protein may be affected by crystal packing effects in a


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protein crystal. Consequently, the small conformational changes induced by irradiation should be studied in solution. Resonance Raman spectroscopy is a versatile technique for studying the structures of proteins, and resonance enhancements of Raman scattering make it possible to selectively obtain vibrational spectra of a chromophore embedded in a protein matrix, thereby providing detailed information on the chromophore structure and its interactions with the environment.12 For example, preresonance Raman spectra were reported to help elucidate the chromophore structure of wild-type GFP.13 Furthermore, preresonance Raman spectra of the two metastable states of a red photochromic protein were compared to provide insights into the possible involvement of cis-trans isomerization in the photoswitching process.14 Raman measurements on synthetic analogues of the GFP chromophore were conducted to allow mode assignments15-17 and to reveal the dependence of the vibrational spectra on the protonation states18 and the configurations19 of the chromophores. Therefore, systematic measurement of the Raman spectra of Dronpa in the A1, A2, and B forms and comparison of the chromophore structures would further our understanding of how these alternative chromophore states exist and interconvert. In this article, we report the resonance and preresonance Raman spectra of Dronpa in the A1, A2, and B forms for the first time. Resonance Raman bands were observed in the spectra recorded upon excitation at 400 nm both for the bright and dark conditions. The pH-dependence of the band intensities observed for the bright condition were very similar to that of the absorption of the A1 form and of the reduction in fluorescence intensity of the B form, indicating that the resonance Raman bands are due to the A1 form. The spectral features showed that the A1 form is a protonated cis chromophore. The Raman spectrum of the A2 form observed for the dark condition confirmed that this form of the chromophore is protonated and adopts the trans


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configuration. The B state provided a preresonance Raman spectrum showing that the chromophore is deprotonated and adopts the cis configuration. Based on the obtained spectra, we discuss the structural marker bands of the chromophore and the role of acid-base equilibrium in the cis configuration of Dronpa.

MATERIALS AND METHODS Sample preparation. The expression plasmid for His×6-tagged Dronpa was obtained by inserting the coding sequence of Dronpa-Green (pDG1-S1) (Medical & Biological Laboratories) into the pET-28a(+) expression vector (Novagen) between the Nde I and Hind III restriction sites. The expression plasmid was transformed into BL21(DE3) (Nippon Gene) cells. Single colonies were isolated and inoculated in LB medium supplemented with kanamycin (30 mg/L). Cells were grown at 37 °C with shaking to an OD600 of 0.8 before induction with 0.4 mM isopropyl βD-thiogalactopyranoside. Incubation was continued at 25 °C overnight, then the cells were harvested to obtain colored cell pellets. The cells were disrupted by sonication and the protein was purified using a HisTrap HP column (GE Healthcare). The buffer in the protein sample was exchanged with phosphate buffered saline (PBS) by dialysis. The expression plasmid for His×6tagged GFP was obtained by inserting the coding sequence of GFP (Clontech) into the pET-15b expression vector (Novagen) between the Xho I and BamH I restriction sites. The same procedure as performed for Dronpa was followed to express and purify GFP, except for that the cells were grown in LB medium supplemented with ampicillin (50 mg/L). The pH of the protein solutions was adjusted by the addition of HCl or NaOH aqueous solutions prior to pH titration measurements. For spectroscopic measurements of samples in the bright condition, we used the purified protein solution as obtained. To prepare samples in the dark condition, we irradiated 15


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ml solution in the bright condition with 514.5 nm line of an Ar+ ion laser (BeamLok 2060, Spectra Physics) for 100 minutes before spectroscopic measurements. Spectroscopic measurements. The probe light for the resonance Raman measurements with 400 nm excitation was the second harmonic of the output of a Ti:sapphire laser pumped by a Qswitched LD-pumped Nd:YLF laser (DM laser system, Photonics Industries). The probe light source for the preresonance Raman measurements with 561 nm excitation was a cw DPSS laser (Jive, Cobolt). The energies of the 400 and 561 nm probe lights were set to 0.1-1.0 and 14-75 mW at the sample point, respectively. The sample solution was circulated in a rectangularshaped (1×4 mm2) quartz cell to prevent sample degradation. The probe light was linearly focused onto the sample solution by using planoconvex and cylindrical lenses. The 45º back scattering light was collected and focused onto the entrance slit of a Czerny-Turner configured Littrow prism prefilter (PF-200MP, Bunkoukeiki) coupled to a single spectrograph with a focal length of 550 mm (iHR550, HORIBA Jobin Ybon) by using two achromatic doublet lenses. The dispersed light was detected with a liquid nitrogen cooled CCD camera (SPEC-10:400B/LN-SNU, Roper Scientific). The Raman shifts were calibrated using the Raman bands of cyclohexane, acetone, ethanol and 2-propanol. Strong fluorescence background from the protein samples was observed in the resonance Raman spectra. Sensitivity variations at each pixel in the detector would severely affect the spectral shape, and thus sensitivity calibration of the CCD camera was carried out using the fluorescence spectrum of standard samples as described previously20 but with modifications. First, a fluorescent standard sample was excited using the 400 nm wavelength of the Raman probe light and the fluorescence spectrum was measured using a fluorometer (F-2700, Hitachi). The fluorescence intensity in the spectral region of the Raman measurements of the intensity


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standard was obtained by polynomial fitting of the fluorescence spectrum measured by the fluorometer. Then, the fluorescence spectrum of the standard sample was measured using exactly the same setup and settings, thus allowing calibration of the pixel-to-pixel sensitivity variations of the CCD camera. The fluorescence standard sample for the 400 nm excitation resonance Raman spectra was quinine sulfate, and the standard for the 561 nm excitation preresonance Raman spectra was rhodamine 6G.

RESULTS Figure 2 shows the absorption and fluorescence spectra of Dronpa in the bright (A) and dark conditions (B). The sample in the bright condition shows absorption and emission bands at 503 and 519 nm, respectively, and these bands are attributed to the B form.4 The sample in the dark condition showed weak fluorescence, and a strong broad absorption band centered at 390 nm arising from the A2 form.4 We measured the resonance Raman spectrum of the sample in the dark condition using an excitation wavelength of 400 nm because the sample has an absorption band at 390 nm and is weakly emissive; this spectrum is shown as trace a in Figure 3. The fluorescence of the sample was much weaker compared to in the bright condition but the spectrum still contained strong fluorescence background. This fluorescence background was removed by curve fitting using polynomials (see Figure S1 in Supporting Information). For comparison, we also measured the resonance Raman spectrum of the sample in the bright condition using an excitation wavelength of 400 nm; the result is shown as trace b in Figure 3. Noticeable resonance Raman bands were unexpectedly observed on the fluorescence background, despite the very weak absorbance of the sample at around 400 nm in the bright condition. As previously described, Dronpa in the bright condition is predominantly in the B form but is in acid-base equilibrium with the A1 form.4, 8 The


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A1 form has an absorption band around 390 nm and generates resonance Raman bands upon excitation at 400 nm. The population of the A1 form increases as the pH decreases. The assignment of the Raman bands in trace b of Figure 3 to the A1 form was confirmed by performing a spectroscopic pH titration to shift the equilibrium between the A1 and B forms. Figure 4 shows the resonance Raman spectra of Dronpa in the bright condition at pH 8.0, 6.3, 5.4, 4.5 and 3.0. The intensities of the observed resonance Raman bands increased as the pH decreased and the intensities were low at neutral and weakly alkaline conditions. Figure 5A shows the intensity of the 1558-cm−1 band plotted against pH, together with the change in absorbance at 390 nm (Figure 5B) and in fluorescence intensity at 550 nm (Figure 5C). It was previously reported that Dronpa in the bright condition shows pH-dependent absorption spectral changes,4 compatible with the present observed changes in the absorption spectra (see Figure S2 in Supporting Information). The changes in Figures 5A, 5B and 5C are consistent with an acidbase equilibrium pKa value of 5.2±0.3 and this pKa value is consistent with the reported pKa based on absorption changes.2 Accordingly, the resonance Raman bands in trace b of Figure 3 were assigned to the A1 form. In addition, these data showed that the change in the chromophore structure occurs in a single step at around pH 5. Figure 6 shows the resonance Raman spectra of the A1 and A2 forms of Dronpa excited at 400 nm compared to that of GFP measured under the same conditions and after background subtraction. Vibrational analysis based on isotopic labelling and DFT calculation was performed for a model GFP chromophore.15 The bands around 1640 cm−1 are assigned to ν(C=C) for the exocyclic C=C double bond, the bands around 1560 cm−1 are assigned to a normal mode delocalized over the imidazolinone ring and exocyclic double bond, the bands around 1600 cm−1


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are assigned to ν(C=C) for the phenol ring; and the bands around 1170 and 1090 cm−1 are assigned to vibrational modes of angle deformation and C−H rocking for the phenol ring.17 The most striking difference in the three traces in Figure 6 is that a Raman band at 1145 cm−1 was observed only for the A2 form of Dronpa. A study using a model compound of a typical chromophore in photochromic fluorescent proteins revealed that the trans form provided a Raman band in the frequency region whereas the cis form did not.19 X-ray crystallographic analyses of the A2 form of Dronpa9 and GFP21-22 showed that the former is in the trans configuration and the latter is in the cis configuration, consistent with the assignment suggested by the Raman marker band elucidated by the model compound study. This implies that the 1145cm−1 band can be utilized as a marker band to determine the configuration of the Dronpa chromophore. Moreover, the present Raman spectral data showed that the A1 form, for which there are no crystallographic data, has a cis configuration. The A1 and A2 forms gave a Raman band around 1560 cm−1 (traces a and b, respectively, in Figure 6). It was previously reported that the frequency of the mode delocalized over the imidazolinone ring and the exocyclic double bond is sensitive to the protonation state of the phenol ring in the chromophore.17-18 The chromophore of wild-type GFP is predominantly in the protonated form and the major band was observed at 1565 cm−1 (trace c, Figure 6); in contrast, S65T GFP has a predominantly deprotonated chromophore and the major band is observed at 1537 cm−1.18 The relationship between the frequency of the major band and the protonation state has been explained based on the resonance structure of the chromophore.17 The frequency of the major band at around 1560 cm−1 in the spectra of the A1 and A2 forms (Figure 6) suggests that their chromophores are protonated. Figure 7 shows close-up views of the resonance Raman spectra of the A1 (a-c) and A2 forms (d-f) in the 1510-1670 cm−1 region: traces (a) and (d) show


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the spectra measured in H2O buffer, traces (b) and (e) show the spectra measured in D2O buffer, and traces (c) and (f) are difference spectra ([(b)-(a)] and [(e)-(d)], respectively). The 1556-cm−1 band in (a) was downshifted in (b). This downshift upon deuteration was confirmed by a derivative-like feature in the 1550-1560 cm−1 region in the difference spectrum (c). A similar downshift was observed for the 1553-cm−1 band of the A2 form (difference spectrum (f)). These downshifts provide further experimental evidence that the chromophores of the A1 and A2 forms are protonated. We measured the preresonance Raman spectra of the B form excited at 561 nm to avoid strong fluorescence in the wavelength region during observation of Raman scattering. Figure 8 shows the preresonance Raman spectrum of the B form (trace a) together with that of GFP (trace b). There was no Raman band around 1145 cm−1 in the spectrum of the B form, suggesting that the chromophore in the B form is in a cis configuration, consistent with the crystallographic data.10, 23-24

Traces (a) and (b) are distinctly different in the 1500-1600 cm−1 region: the B form exhibited

a strong band at 1537 cm−1 while GFP showed a strong and a weak band at 1564 and 1538 cm−1, respectively. The Raman band at 1537 cm−1 of the B form suggests that this form of the chromophore is deprotonated. GFP in neutral pH conditions is a mixture composed mostly of the protonated form together with some deprotonated form. The relative intensity of the 1564 and 1538 cm−1 bands of GFP is consistent with the relative population between the protonated and deprotonated forms. The 400-nm excited Raman spectrum (Figure 6c) showed only one band at 1565 cm−1 due to the Raman bands of the protonated form being resonantly enhanced by the 400-nm excitation. The ν(C=C) bands of the exocyclic C=C double bond were observed at 1623 and 1641 cm−1 for B form Dronpa and GFP, respectively. The frequency of this mode is also known to be sensitive to the protonation state of the phenol ring in the chromophore.17-18 Taken


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together, the resonance and preresonance Raman spectra obtained in the present study allowed determination of the configuration and protonation state of the Dronpa chromophore in the A1, A2, and B forms.

DISCUSSION Acid-base equilibrium in the bright condition. The present study revealed the configuration and protonation state of the chromophore in Dronpa in the A2, B, and most importantly, in the A1 form. Prior to this study, the chromophore structure of the A1 form was poorly characterized, although it was known that the A1 and B forms are in acid-base equilibrium. The importance of acid-base equilibrium was previously revealed for other photoswitchable fluorescent proteins such as Padron25 and EYQ1.26 The present study clearly showed that protonation occurs at the phenolate oxygen of the chromophore and that the cis configuration is maintained upon lowering the pH below 6. GFP also exists as an equilibrium mixture of the protonated and deprotonated forms, and photoexcitation of the protonated form provides a fluorescence spectrum which is identical to that of the deprotonated form, indicating that excited state proton transfer (ESPT) occurs. In contrast, in Dronpa photoexcitation of the A1 form resulted in very weak fluorescence, indicating that ESPT is hindered in the A1 form. pH dependence of the chromophore structure in the bright condition. The absorbance and fluorescence properties of wild-type GFP are relatively insensitive to changes in pH,27 whereas interestingly the properties of the GFP S65T mutant are sensitive to solvent pH,28 with the chromophore exhibiting a pKa of 6.029 due to stabilization of the phenolate form. This pKa value is very close to that of the chromophore in Dronpa. The replacement of Ser65 by Cys has roughly similar effects30 to that observed with the GFP S65T mutant, although the replacement of Ser65 by Thr is the most commonly used mutation to ionize the phenol of the chromophore in


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GFP. At low pH, the S65T mutant is non-fluorescent,28-29 similar to Dronpa in the bright condition. The chromophore of Dronpa differs from that of GFP at the site corresponding to Ser65 of GFP, which in Dronpa is Cys62. Thus, the S65T GFP mutant provides insights into explaining the Raman data for the deprotonated form of the B form chromophore in Dronpa. The crystal structures of S65T GFP at both high and low pH have been solved,21, 29 and the changes in the arrangement of side chains close to the phenolic hydroxyl group have been interpreted in terms of titration of this group. The distinct pH-dependence of the phenolic hydroxyl group can be explained based on the crystal structures as follows. Glu222 is a key residue for controlling the ionization state of the chromophore in GFP, and mutation of Glu222 to Gly provides the same spectral shape and wavelengths as Ser65 mutations.31 The neutral form is maintained by the carboxylate of Glu222 because electrostatic repulsion between the carboxylate and the chromophore prevents the chromophore from becoming an anion. In addition, a hydrogen bond via a bound water molecule and Ser205 stabilizes the neutral form of the chromophore. Replacement of Ser65 of GFP with Thr or Cys promotes the neutral state of Glu222 because only Ser65 can donate a hydrogen bond to the buried side chain of Glu222 to allow ionization of the carboxylate. The Thr and Cys side chains are too bulky for the crowded interior of the protein and prevent adoption of the same protein conformation as that of wild-type GFP; consequently, these mutations force the carboxyl of Glu222 to remain neutral. A hydrogenbonding network around the chromophore similar to that found in the S65T GFP mutant is likely adopted by the Dronpa B form, where Glu211 and His193 play the same roles as Glu222 and Ser205 in GFP. Protonation/deprotonation equilibrium and the pH-dependent properties of fluorescent proteins are important. The present study revealed that the chromophore in the A1 form is protonated and is in protonation/deprotonation equilibrium with the B form, which is also


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in the cis configuration in the bright condition. These insights will be useful in various applications of pH-sensitive fluorescent proteins by providing a better general understanding of the mechanism of pH-dependent change of the fluorescence properties of fluorescent proteins. Configuration marker band of the chromophore in fluorescent proteins.

Raman

spectroscopy is particularly suited for determining the configurations of chromophores in proteins. In retinal proteins, for example, the configuration of the chromophore was determined by resonance Raman spectroscopy. The C-C single bond stretches and vinyl hydrogen in-plane rocks of the retinal chromophore appear in the distinctive 1100-1400 cm−1 region.32-34 The intensities and frequencies of these modes are sensitive to the cis-trans configuration of the C=C double bonds and the conformation of the C-C single bonds of retinal. Luin et al. investigated the preresonance Raman spectra of a model compound representative of the chromophores in fluorescent proteins and found that the intensity of the band observed around 1130 cm−1 was sensitive to the configuration of the compound. The atomic displacements of the 1130 cm−1 mode were calculated and showed that the mode results from deformation of the imidazolinone ring. This deformation mode of the ring involves C-C single bond stretches and vinyl hydrogen in-plane rocks and is therefore sensitive to the cis-trans configuration of the C=C double bond. Resonance Raman intensities are a function of the displacement of the electronic excited state geometry relative to the ground state along the various normal coordinates, which is described by Albrecht’s A-term in vibronic theory.35 Thus, the intensity difference between the trans and cis configurations resulting from the displacement of the ring geometry in the excited states relative to the ground state differs depending on the configuration. Luin et al. measured the preresonance Raman spectra of GFP mutants19 and found that the intensity difference of the ~1140 cm−1 band between the cis and trans samples of the protein was


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less pronounced than that of the model compound of the chromophore. Our resonance Raman spectra of the A1 and A2 forms of Dronpa and of GFP show significant intensity differences in this band between the cis and trans forms. This inconsistency is probably due to differences in the excitation wavelength for the Raman measurements. Luin et al. chose a long excitation wavelength (647 nm) to minimize fluorescence and to prevent photoconversion during data accumulation, whereas we used an excitation wavelength of 400 nm to obtain large Raman intensity enhancement in resonance with the electronic transition around 390 nm of the A1 and A2 forms. The present study clearly demonstrates the feasibility of using the marker band around 1140 cm−1 to determine the chromophore configuration in a protein.

CONCLUSIONS We determined the chromophore structure of the A1, A2 and B forms of Dronpa by using resonance and preresonance Raman spectroscopy and investigated the acid-base equilibrium between the B and A1 forms by spectroscopic pH titration. In the bright condition, the chromophore shows a protonation/deprotonation transition with a pKa of 5.2±0.3 and maintains a cis configuration. The present study reports the first resonance Raman spectra of a photochromic fluorescent protein. We demonstrated that Raman bands selectively enhanced for the chromophore yield valuable information on its molecular structure after careful elimination of the fluorescence background. Systematic application of resonance Raman spectroscopy would provide novel insights into understanding the photoswitching mechanism of photochromic fluorescent proteins.


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Figure Captions Figure 1. (Upper) Model of reversible photoswitching in Dronpa, including the fluorescent (B), the photoswitched non-fluorescent (A2), and the pH-induced non-fluorescent (A1) forms. The bright and dark conditions refer to the states saturated by ~400 and ~500 nm light, respectively. (Lower) The two chromophore configurations of Dronpa. The chromophore is formed from Cys62, Tyr63, and Glu64. Figure 2. Absorption (red) and fluorescence (blue) spectra of the bright (A) and dark conditions (B) of Dronpa. A solution of 50 µM Dropa in PBS at pH 7.4 was used for the measurements. The fluorescence spectra were obtained by excitation with 400-nm light. Figure 3. Resonance Raman spectra of the samples in the dark (a) and bright (b) conditions excited at 400 nm. A solution of 50 µM Dropa in PBS at pH 7.4 was used for the measurements. The accumulation times for obtaining traces (a) and (b) were 10 and 40 minutes, respectively. Figure 4. pH dependence of resonance Raman spectra of the bright condition excited at 400 nm. The pH values of the samples were 3.0 (a), 4.5 (b), 5.4 (c), 6.3 (d), and 8.0 (e). The accumulation time for obtaining each spectrum was 40 minutes. Figure 5. Raman intensity at 1560 cm−1 (A), absorbance at 390 nm (B), and fluorescence intensity at 550 nm (C) as a function of pH. The fluorescence intensity at 550 nm was adopted to determine the population because the longer wavelength side of the fluorescence band is not affected by self-absorption. The fluorescence spectra were obtained by excitation at 400 nm. Figure 6. Resonance Raman spectra of the A1 (a) and (b) A2 forms of Dronpa and of GFP (c) upon excitation at 400 nm. The fluorescence background was removed by curve fitting using


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polynomials (see Figure S1 in Supporting Information). Protein solutions (50 µM) in PBS at pH 7.4 were used for the measurements. The accumulation times for obtaining traces (a), (b) and (c) were 40, 10 and 10 minutes, respectively. Figure 7. Close-up views of the resonance Raman spectra of the A1 form of Dronpa and GFP in H2O and D2O buffers in the 1510-1670 cm−1 region. The excitation wavelength was 400 nm. Traces (a), (b), (d) and (e) show the spectra of the A1 form in H2O buffer, the A1 form in D2O buffer, GFP in H2O buffer, and GFP in D2O buffer, respectively. Traces (c) and (f) are difference spectra ([(b)-(a)] and [(e)-(d)], respectively). The difference spectra are multiplied by a factor of 3. Protein solutions (50 µM) in PBS at pH 7.4 and deuterated PBS at pD7.4 were used for the measurements. The accumulation time for obtaining traces (a) and (b) was 40 minutes, while that for obtaining traces (d) and (e) was 10 minutes. Figure 8. Preresonance Raman spectra of the B form of Dronpa (a) and GFP (b) excited at 561 nm. The asterisk denotes the Raman band of sulfate ion added as an internal standard of Raman intensity. Protein solution (50 µM) in PBS at pH 7.4 was used for the measurements. The accumulation time for obtaining each spectrum was 10 minutes.


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Higashino et al., Figure 1


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ASSOCIATED CONTENT Supporting Information. An example of subtraction of fluorescence background from a raw spectrum to yield a resonance Raman spectrum, pH dependence of absorption and fluorescence spectra of Dronpa. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Yasuhisa Mizutani, Tel.: +81-6-6850-5776. Fax: +81-6-6850-5776. E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Soft Molecular Systems” (No. 25104006) to Y.M. from The Ministry of Education, Culture, Sports, Science and Technology of Japan.


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REFERENCES 1. Zhou, X. X.; Lin, M. Z., Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications. Curr. Opin. Chem. Biol. 2013, 17, 682-690. 2. Ando, R.; Mizuno, H.; Miyawaki, A., Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting. Science 2004, 306, 1370-1373. 3. Dedecker, P.; Hotta, J.-i.; Ando, R.; Miyawaki, A.; Engelborghs, Y.; Hofkens, J., Fast and Reversible Photoswitching of the Fluorescent Protein Dronpa as Evidenced by Fluorescence Correlation Spectroscopy. Biophys. J. 2006, 91, L45-L47. 4. Habuchi, S.; Ando, R.; Dedecker, P.; Verheijen, W.; Mizuno, H.; Miyawaki, A.; Hofkens, J., Reversible Single-Molecule Photoswitching in the GFP-Like Fluorescent Protein Dronpa. Proc. Natl. Acad. Sci. USA 2005, 102, 9511-9516. 5. Dedecker, P.; Hotta, J.-i.; Flors, C.; Sliwa, M.; Uji-i, H.; Roeffaers, M. B. J.; Ando, R.; Mizuno, H.; Miyawaki, A.; Hofkens, J., Subdiffraction Imaging through the Selective DonutMode Depletion of Thermally Stable Photoswitchable Fluorophores: Numerical Analysis and Application to the Fluorescent Protein Dronpa. J. Am. Chem. Soc. 2007, 129, 16132-16141. 6. Dedecker, P.; Mo, G. C. H.; Dertinger, T.; Zhang, J., Widely Accessible Method for Superresolution Fluorescence Imaging of Living Systems. Proc. Natl. Acad. Sci. USA 2012, 109, 10909-10914. 7. Shroff, H.; Galbraith, C. G.; Galbraith, J. A.; White, H.; Gillette, J.; Olenych, S.; Davidson, M. W.; Betzig, E., Dual-Color Superresolution Imaging of Genetically Expressed Probes within Individual Adhesion Complexes. Proc. Natl. Acad. Sci. USA 2007, 104, 2030820313. 8. Habuchi, S.; Dedecker, P.; Hotta, J.-i.; Flors, C.; Ando, R.; Mizuno, H.; Miyawaki, A.; Hofkens, J., Photo-Induced Protonation/Deprotonation in the GFP-Like Fluorescent Protein Dronpa: Mechanism Responsible for the Reversible Photoswitching. Photochem. Photobiol. Sci. 2006, 5, 567-576. 9. Andresen, M.; Stiel, A. C.; Trowitzsch, S.; Weber, G.; Eggeling, C.; Wahl, M. C.; Hell, S. W.; Jakobs, S., Structural Basis for Reversible Photoswitching in Dronpa. Proc. Natl. Acad. Sci. USA 2007, 104, 13005-13009. 10. Wilmann, P. G.; Turcic, K.; Battad, J. M.; Wilce, M. C. J.; Devenish, R. J.; Prescott, M.; Rossjohn, J., The 1.7 Å Crystal Structure of Dronpa: A Photoswitchable Green Fluorescent Protein. J. Mol. Biol. 2006, 364, 213-224. 11. Yadav, D.; Lacombat, F.; Dozova, N.; Rappaport, F.; Plaza, P.; Espagne, A., Real-Time Monitoring of Chromophore Isomerization and Deprotonation During the Photoactivation of the Fluorescent Protein Dronpa. J. Phys. Chem. B 2015, 119, 2404-2414. 12. Mathies, R. A.; Smith, S. O.; Palings, I., Determination of Retinal Chromophore Structure in Rhodopsins. In Biological Applications of Raman Spectroscopy, Spiro, T. G., Ed. John Wiley & Sons: New York, 1987; Vol. 2, pp 59-108. 13. Bell, A. F.; Stoner-Ma, D.; Wachter, R. M.; Tonge, P. J., Light-Driven Decarboxylation of Wild-Type Green Fluorescent Protein. J. Am. Chem. Soc. 2003, 125, 6919-6926. 14. Loos, D. C.; Habuchi, S.; Flors, C.; Hotta, J.-i.; Wiedenmann, J.; Nienhaus, G. U.; Hofkens, J., Photoconversion in the Red Fluorescent Protein from the Sea Anemone Entacmaea Quadricolor: Is Cis−Trans Isomerization Involved? J. Am. Chem. Soc. 2006, 128, 6270-6271.


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15. He, X.; Bell, A. F.; Tonge, P. J., Isotopic Labeling and Normal-Mode Analysis of a Model Green Fluorescent Protein Chromophore. J. Phys. Chem. B 2002, 106, 6056-6066. 16. Esposito, A. P.; Schellenberg, P.; Parson, W. W.; Reid, P. J., Vibrational Spectroscopy and Mode Assignments for an Analog of the Green Fluorescent Protein Chromophore. J. Mol. Struct. 2001, 569, 25-41. 17. Tozzini, V.; Nifosì, R., Ab Initio Molecular Dynamics of the Green Fluorescent Protein (GFP) Chromophore: An Insight into the Photoinduced Dynamics of Green Fluorescent Proteins. J. Phys. Chem. B 2001, 105, 5797-5803. 18. Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J., Probing the Ground State Structure of the Green Fluorescent Protein Chromophore Using Raman Spectroscopy. Biochemistry 2000, 39, 4423-4431. 19. Luin, S.; Voliani, V.; Lanza, G.; Bizzarri, R.; Nifosì, R.; Amat, P.; Tozzini, V.; Serresi, M.; Beltram, F., Raman Study of Chromophore States in Photochromic Fluorescent Proteins. J. Am. Chem. Soc. 2008, 131, 96-103. 20. Iwata, K.; Hamaguchi, H.; Tasumi, M., Sensitivity Calibration of Multichannel Raman Spectrometers Using the Least-Squares-Fitted Fluorescence Spectrum of Quinine. Appl. Spectrosc. 1988, 42, 12-14. 21. Ormö, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J., Crystal Structure of the Aequorea Victoria Green Fluorescent Protein. Science 1996, 273, 1392-1395. 22. Yang, F.; Moss, L. G.; Phillips, G. N., The Molecular Structure of Green Fluorescent Protein. Nat. Biotech. 1996, 14, 1246-1251. 23. Nam, K.-H.; Kwon, O. Y.; Sugiyama, K.; Lee, W.-H.; Kim, Y. K.; Song, H. K.; Kim, E. E.; Park, S.-Y.; Jeon, H.; Hwang, K. Y., Structural Characterization of the Photoswitchable Fluorescent Protein Dronpa-C62S. Biochem. Biophys. Res. Commun. 2007, 354, 962-967. 24. Stiel, A. C.; Trowitzsch, S.; Weber, G.; Andresen, M.; Eggeling, C.; Hell, S. W.; Jakobs, S.; Wahl, M. C., 1.8 Å Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants. Biochem. J. 2007, 402, 35-42. 25. Fron, E.; Van der Auweraer, M.; Hofkens, J.; Dedecker, P., Excited State Dynamics of Photoswitchable Fluorescent Protein Padron. J. Phys. Chem. B 2013, 117, 16422-16427. 26. Bizzarri, R.; Serresi, M.; Cardarelli, F.; Abbruzzetti, S.; Campanini, B.; Viappiani, C.; Beltram, F., Single Amino Acid Replacement Makes Aequorea Victoria Fluorescent Proteins Reversibly Photoswitchable. J. Am. Chem. Soc. 2010, 132, 85-95. 27. Ward, W. W.; Prentice, H. J.; Roth, A. F.; Cody, C. W.; Reeves, S. C., Spectral Perturbations of the Aequorea Green-Fluorescent Protein. Photochem. Photobiol. 1982, 35, 803808. 28. Kneen, M.; Farinas, J.; Li, Y.; Verkman, A. S., Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator. Biophys. J. 1998, 74, 1591-1599. 29. Elsliger, M.-A.; Wachter, R. M.; Hanson, G. T.; Kallio, K.; Remington, S. J., Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH. Biochemistry 1999, 38, 5296-5301. 30. Heim, R.; Cubitt, A. B.; Tsien, R. Y., Improved Green Fluorescence. Nature 1995, 373, 663-664. 31. Ehrig, T.; O'Kane, D. J.; Prendergast, F. G., Green-Fluorescent Protein Mutants with Altered Fluorescence Excitation Spectra. FEBS Lett. 1995, 367, 163-166.


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32. Braiman, M.; Mathies, R., Resonance Raman Evidence for an All-trans to 13-cis Isomerization in the Proton-Pumping Cycle of Bacteriorhodopsin. Biochemistry 1980, 19, 54215428. 33. Curry, B.; Broek, A.; Lugtenburg, J.; Mathies, R., Vibrational Analysis of All-transRetinal. J. Am. Chem. Soc. 1982, 104, 5274-5286. 34. Curry, B.; Palings, I.; Broek, A.; Pardoen, J. A.; Mulder, P. P. J.; Lugtenburg, J.; Mathies, R., Vibrational Analysis of 13-cis-Retinal. J. Phys. Chem. 1984, 88, 688-702. 35. Tang, J.; Albrecht, A. C., Developments in the Theories of Vibrational Raman Intensities. In Raman Spectroscopy-Theory and Practice, Szymanski, H., Ed. Plenum Press: New York, 1970; Vol. 2, pp 33-67.


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Table of Contents Graphic


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Figure 1. (Upper) Model of reversible photoswitching in Dronpa, including the fluorescent (B), the photoswitched non-fluorescent (A2), and the pH-induced non-fluorescent (A1) forms. The bright and dark conditions refer to the states saturated by ~400 and ~500 nm light, respectively. (Lower) The two chromophore configurations of Dronpa. The chromophore is formed from Cys62, Tyr63, and Glu64. 80x109mm (300 x 300 DPI)


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Figure 2. Absorption (red) and fluorescence (blue) spectra of the bright (A) and dark conditions (B) of Dronpa. A solution of 50 µM Dropa in PBS at pH 7.4 was used for the measurements. The fluorescence spectra were obtained by excitation with 400-nm light. 86x69mm (300 x 300 DPI)



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Figure 3. Resonance Raman spectra of the samples in the dark (a) and bright (b) conditions excited at 400 nm. A solution of 50 µM Dropa in PBS at pH 7.4 was used for the measurements. The accumulation times for obtaining traces (a) and (b) were 10 and 40 minutes, respectively. 60x39mm (300 x 300 DPI)


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Figure 4. pH dependence of resonance Raman spectra of the bright condition excited at 400 nm. The pH values of the samples were 3.0 (a), 4.5 (b), 5.4 (c), 6.3 (d), and 8.0 (e). The accumulation time for obtaining each spectrum was 40 minutes. 108x123mm (300 x 300 DPI)



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Figure 5. Raman intensity at 1560 cm-1 (A), absorbance at 390 nm (B), and fluorescence intensity at 550 nm (C) as a function of pH. The fluorescence intensity at 550 nm was adopted to determine the population because the longer wavelength side of the fluorescence band is not affected by self-absorption. The fluorescence spectra were obtained by excitation at 400 nm. 177x310mm (300 x 300 DPI)


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Figure 6. Resonance Raman spectra of the A1 (a) and (b) A2 forms of Dronpa and of GFP (c) upon excitation at 400 nm. The fluorescence background was removed by curve fitting using polynomials (see Figure S1 in Supporting Information). Protein solutions (50 µM) in PBS at pH 7.4 were used for the measurements. The accumulation times for obtaining traces (a), (b) and (c) were 40, 10 and 10 minutes, respectively. 108x123mm (300 x 300 DPI)



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Figure 7. Close-up views of the resonance Raman spectra of the A1 form of Dronpa and GFP in H2O and D2O buffers in the 1510-1670 cm-1 region. The excitation wavelength was 400 nm. Traces (a), (b), (d) and (e) show the spectra of the A1 form in H2O buffer, the A1 form in D2O buffer, GFP in H2O buffer, and GFP in D2O buffer, respectively. Traces (c) and (f) are difference spectra ([(b)-(a)] and [(e)-(d)], respectively). The difference spectra are multiplied by a factor of 3. Protein solutions (50 µM) in PBS at pH 7.4 and deuterated PBS at pD7.4 were used for the measurements. The accumulation time for obtaining traces (a) and (b) was 40 minutes, while that for obtaining traces (d) and (e) was 10 minutes. 119x145mm (300 x 300 DPI)


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Figure 8. reresonance Raman spectra of the B form of Dronpa (a) and GFP (b) excited at 561 nm. The asterisk denotes the Raman band of sulfate ion added as an internal standard of Raman intensity. Protein solution (50 µM) in PBS at pH 7.4 was used for the measurements. The accumulation time for obtaining each spectrum was 10 minutes. 134x176mm (300 x 300 DPI)



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TOC Graphic 84x47mm (300 x 300 DPI)


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Chromophore Structure of Photochromic Fluorescent Protein Dronpa

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