Excited States of Fluorescent Proteins, mKO and DsRed

J. Phys. Chem. B 2010, 114, 2971–2979

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Excited States of Fluorescent Proteins, mKO and DsRed: Chromophore-Protein Electrostatic Interaction Behind the Color Variations Jun-ya Hasegawa,*,†,‡ Takehiko Ise,† Kazuhiro J. Fujimoto,† Akihiro Kikuchi,§ Eiko Fukumura,§,¶ Atsushi Miyawaki,| and Yoshitsugu Shiro§ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniVersity, Kyoto-Daigaku-Katsura, Nshikyo-ku, Kyoto 615-8510, Japan, Quantun Chemistry Research Institute, Kyodai Katsura Venture Plaza, 1-36 Goryo Oohara, Nishikyo-ku, Kyoto 615-8245, Japan, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Koto, Sayo, Hyogo 679-5148, Japan, Department of Biological Sciences, Graduate School of Science, Osaka UniVersity, 1-1, Machikaneyama-cho, Toyonaka, Osaka, and Brain Science Institute, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan ReceiVed: October 17, 2009; ReVised Manuscript ReceiVed: December 21, 2009

The emitting states of green fluorescent protein (GFP), monomeric Kusabira orange (mKO), and Discosoma red (DsRed) were studied using QM/MM and SAC-CI methods. By comparing the electronic structures among the green-, orange-, and red-emitting states as well as their electrostatic and quantum mechanical interactions within the protein cavity, the basic mechanisms for determining emission colors have been clarified. We found that the orange and red emissions of mKO and DsRed, respectively, result from cancellation between two effects, the π skeleton extension (red shift) and protein electrostatic potential (blue shift). The extension of the π skeleton enhances the intramolecular charge-transfer character of the transition, which makes the fluorescence energy more sensitive to the protein’s electrostatic potential. On the basis of this mechanism, we predicted amino acid mutations that could red shift the emission energy of DsRed. A novel single amino acid mutation, which was examined computationally, reduced the DsRed emission energy from 2.14 (579 nm) to 1.95 eV (636 nm), which is approaching near-infrared fluorescence. 1. Introduction The green fluorescent protein (GFP) has been extensively developed and has become an indispensable tool for molecular imaging in the field of molecular and cellular biology.1,2 The protein was discovered by Shimomura et al. in the jellyfish Aequorea,3 and the gene was cloned by Prasher et al.4 Its heterologous expression in other organisms was achieved by Chalfie et al.5 and Tsuji et al.6 Since then, GFP has been established as an excellent marker of gene expression and protein targeting in living cells.1 Color variations are one of the most important aspects in developing FPs to maximize specificity in gene expression. A wide variety of fluorescence colors have become available after extensive mutation studies1,2 and the discovery of the Anthozoa species.7,8 However, “clearer vision” is still necessary, especially for in vivo imaging.9 Because the absorption coefficients of hemes and water are smallest in the near-infrared region (around 650-900 nm), it is desirable to have a FP with its absorption/ emission peaks in this region. The fluorescence wavelength of mPlum, a red FP mutant, is 649 nm (1.91 eV), which is one of the lowest fluorescence energies among the FPs reported so far.10 More efforts are still necessary to move the fluorescence energy into the target region. In attempting molecular design of the fluorophore to modulate emission energy, the most direct approach is to change the * To whom correspondence should be addressed. E-mail: hasegawa@ sbchem.kyoto-u.ac.jp. † Kyoto University. ‡ Quantrum Chemistry Research Institute. § Harima Institute, RIKEN SPring-8 Center. ¶ Osaka University. | Brain Science Institute, RIKEN.

molecular structure and electronic structure of the fluorophore. Blue FP (BFP),11 cyan FP (CFP),12 and red FP (RFP)7 all belong to this class of FPs. To create BFP and CFP, the tyrosine residue in GFP was replaced by histidine and tryptophan, respectively, because the fluorophores in FPs originate from amino acid residues. In the case of yellow FP,13 the fluorophore has a π-stacking interaction with a tyrosine residue at the position of 203. Although the RFP fluorophore is composed of a tyrosine residue, as in GFP (see Figure 1), the π conjugation is extended toward a neighboring residue via a peptide bond. Recently, some of the authors created a novel Kusabira orange fluorescent protein with a three-ring fluorophore, monomeric Kusabira orange (mKO),14,15 which also has a π system extending to the peptide bond (see Figure 1). There are a number of theoretical studies on the excited states of FPs, especially on GFP.16–21 In our previous studies, we analyzed the protonation states of the fluorophore17 and the emission color-tuning mechanism of GFP, BFP, CFP, and Y66F18 using a symmetry-adapted cluster configuration interaction (SAC-CI22) method (see the next section for more description). Regarding DsRed, a first theoretical calculation23 was performed to analyze the red shift of the fluorescence. On the basis of time-dependent (TD) density functional theory (DFT) calculations, this study proposed that the π system in the DsRed chromophore extended along the main chain before the crystal structure revealed by an X-ray diffraction study24 confirmed this. The photoabsorption of the DsRed fluorophore in the gas phase was also studied with TD-DFT, where the intramolecular chargetransfer character of the emitting state was pointed out.25 However, without including the effect of the protein in the calculations, it is impossible to know how such a charge-transfer characteristic affects the emission color-tuning mechanism.

10.1021/jp9099573  2010 American Chemical Society Published on Web 02/04/2010

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Hasegawa et al. 2. Computational Details

Figure 1. (a) Structures of RFP model chromophores. (b) QM regions, “Cro+Wat” models, of the QM/MM calculations. (b-1) GFP. (b-2) mKO. (b-3) DsRed. The “Cro” model has no water molecule in the vicinity of the phenolate O atom. L denotes linked-atom (hydrogen atom).

Extensive TDDFT calculations were also performed to investigate the variation of spectral properties of FP chromophores in solution.19 Protein effect was included using the fragment MO (FMO)26 method.20,21 Multilayer FMO-CIS(D) calculations for DsRed and mFruits were performed,20,21 and the calculated excitation and emission energies showed agreement with experimental data. It was observed that the protein effect blue shifts the excitation energy.21 However, the crucial relationship between the protein effect and the π skeleton extension effect was not studied. In the present study, we point out that these two factors are mutually indispensable and essential for controlling the emission color in mKO and RFP. In this study, we performed SAC-CI22,27 calculations and investigated the mechanism of emission color tuning among GFP, mKO, and DsRed. Because the fluorescence energies of the FPs are 2.46 (green), 2.22 (orange), and 2.14 eV (red), respectively, this set of FPs provides a good range of color variation suitable for investigation. Employing several computational models, the fluorophore structural and protein environmental origins behind the color variation were analyzed. We obtained a color-tuning mechanism common to retinal proteins and firefly luciferin, suggesting a universal feature in the color tuning of photofunctional proteins. The electrostatic effect of each amino acid was summarized in diagrams, which clearly show specific roles of amino acids. We found an interesting correlation between the structural and the electrostatic factors, indicating a strategy to control the fluorescence energy of DsRed. The idea was further tested by a computational mutagenesis simulation. The result showed a reasonable red shift, as expected in the analysis. This article is organized as follows. Computational Details are described in the next section. In section 3, the results of the calculations are compared with experimental data. The physical origin of the color tuning (fluorophore structural, protein electrostatic, and electronic polarization of the protein environment) is clarified. On the basis of the results in the models, the origins of the emission color variation are discussed. In subsection 3.5, we propose a mutation to red shift the emission energy, and the idea was supported by a computational mutation.

The structures of the proteins were optimized with a combined quantum mechanics/molecular mechanics (QM/MM)28 method. Our QM/MM code was successfully applied in our previous studies on retinal proteins29 and GFP.18 The QM(B3LYP30)/ MM(AMBER9631) and the QM(CI-Singles)/MM(AMBER96) calculations were performed for ground and excited states, respectively. For the optimization, 6-31g*32 basis sets were used. The emitting states of the FPs studied here are dominated by one-electron transitions from the highest occupied molecular orbital (π, HOMO) to the lowest unoccupied MO (π*, LUMO) of the fluorophore (see Figure 2 for the shapes of the orbitals). In these cases, the optimized structures with the CI-Singles (CIS) are qualitatively correct. In our previous study on firefly emission, the structure of the first excited state calculated using the CIS method qualitatively agreed with that obtained with SAC-CI method.33 The fluorescence energies calculated with these two structures were similar to each other.33 This is because the weight of the HOMO-LUMO excitations reaches 91-93% in the wave functions. Another group calculated fluorescence energies of various heteroaromatic compounds and also obtained similar results.34 We adopted several computational models to investigate the mechanism of the emission energy tuning. In the “All” model, the fluorophore unit and a water molecule were included in the QM region, as shown in Figure 1b. The other part of the system was described by the MM method. The symbol “L” denotes a hydrogen atom used for the linked atom, which was introduced instead of the sp3 carbon atoms. Some amino acids contain a QM/MM border, and their atomic charges become noninteger values. In these cases, the atomic charges of the amino acid were redefined so as to have an integer charge.35 The charge of the atom replaced by the linked atoms was set to 0. In the “Cro+Wat” model, the protein electrostatic potential (ESP), which is the MM part of the All model, was not considered. In the “Cro” model, the water molecule in the Cro+Wat model was removed. The atomic coordinates used in the Cro and Cro+Wat models were the same as those in the All model. The structures used for the initial guesses in the optimization were taken from X-ray crystallographic structures 1GFL,36 2ZMU,15 and 1GGX24 for GFP, mKO, and DsRed, respectively; their resolutions are 1.9, 1.65, and 1.9 Å, respectively. Excitation and fluorescence energies were calculated with the SAC37 and SAC-CI22 methods.27,38 The SAC method is a coupled cluster method with symmetry-adapted excitation operators and describes the electron correlations in the ground state. SAC-CI is a theory for the excited state and is based on the correlated ground-state SAC wave function. The SAC/SAC-CI method was proposed by one of the authors and has been applied to a number of systems and established as a reliable method for calculating the excited state.27,38 The computational code was continuously developed by Nakatsuji’s group and has been available in the Gaussian program package. In the present study, we adopted single and double excitation operators for the SAC and SACCI wave functions, that is, the SAC-SD and the SAC-CISD methods, respectively. We also evaluated the performance of the TD-DFT method. As we describe in subsection 3.2, the calculated excitation energies overestimated the experimental data by about 0.46-0.52 eV, although the relative shifts in the excitation energies were reproduced. In the SAC-CI calculations, the 1s orbitals of the C, N, and O atoms and the 1s, 2s, and 2p orbitals of the S atom were treated as frozen core orbitals. Corresponding virtual orbitals having very high orbital energies were also treated as frozen

Excited States of Fluorescent Proteins, mKO and DsRed

J. Phys. Chem. B, Vol. 114, No. 8, 2010 2973

Figure 2. Molecular orbitals (HOMO and LUMO), difference electron density (∆F ) Fex - Fg), and atomic charge difference (∆Q ) Qex - Qg) of (a) GFP, (b) mKO, and (c) DsRed. The symbols Fex and Fg represent the electron densities of the excited and the ground states, respectively. Blue and red colors show that the density increases and decreases in the excited state. Each chromophore is divided into two fragments by a dotted line. The numbers shown are the sum of the atomic charge differences in each fragment.

virtual orbitals. All single excitation operators and selected double excitation operators were included in the SAC/SAC-CI wave functions. The perturbation selection procedure39 with the “LevelThree” set of energy thresholds (1.0 × 10-6 and 1.0 × 10-7 au for the SAC and the SAC-CI wave functions, respectively) was used for selecting the doubles. The QM/MM and SAC-CI calculations were performed using a development version of Gaussian03.40 3. Results and Discussion 3.1. Excitation Energy in the Gas Phase. First, we performed several SAC-CI calculations on the DsRed model fluorophores, RFP(1) and RFP(2),41 in the gas phase. Experimental excitation energies of these compounds were measured in the gas phase using the electrostatic ion storage ring in Aarhus (ELISA).41 The structures of RFP(1) and RFP(2) have extra ethylenic units extending π conjugations, as shown in Figure 1 (a-1 and a-2). We used this result to check the dependence on the basis sets and the numerical accuracy of the present computational procedure. The atomic coordinates of these compounds were optimized by B3LYP/6-31G* calculations. The basis sets used were D95(d), D95(d,p), D95+(d,p), and 6-311G(d). Starting from the D95(d) sets, we augmented polarization on the H atoms in the D95(d,p) sets, further added diffuse functions on the C, N, O, and S atoms in the D95+(d,p) sets, and improved the valence space contractions to the triple-ζ level in the 6-311G(d) sets. As shown in Table 1, excitation energies calculated with the SAC-CI method show reasonable agreement with the experimental data, 2.38 eV for RFP(1) and 2.26 eV for RFP(2). The calculated excitation energies for RFP(1) and RFP(2) were 2.52-2.61 and 2.20-2.24 eV, respectively. Deviations from the experimental data were 0.14-0.23 eV for RFP(1) and 0.06-0.02 eV for RFP(2). In a previous study,21 CIS(D)42 and CIS(Ds)43 and their partially renormalized versions43 gave very similar results. This is reasonable because the CIS(D) method can be derived as a perturbative approximation to the SAC-CI

TABLE 1: Excitation Energies of Model Chromophores of Red Fluorescent Proteina chromophore

basis setsb

Eexc (eV)

exptl.d (eV)

RFP(1)

D95(d) D95(d,p) D95+(d,p) 6-311G(d) D95(d) D95(d,p) D95+(d,p) 6-311G(d)

2.57 2.55 2.61 2.52 2.23 2.20 2.24 2.24

2.38

RFP(2)

2.26

a

See Figure 1 for the structures. b See text in the Computational Details section. c Excitation energy. d Experimental data.41,44

method.42 The results shown in Table 1 indicate that the basis set extensions over the D95(d) set give no significant improvement in the excitation energies. The deviations from the D95(d) sets are -0.05 to +0.04 eV in RFP(1) and -0.03 and +0.01 eV in RFP(2). Therefore, D95(d) sets would be a requisite minimum and are used as default basis sets in the rest of the study, unless otherwise noted. 3.2. Excited States in the Protein Environment. Table 2 shows the calculated fluorescence energies of FPs. The SACCI results for the All model are 2.55, 2.24, and 2.14 eV for GFP, mKO, and DsRed, respectively. We also performed the SAC-CI calculations with basis sets that are partially extended in the valence region, the 6-311G(d) sets for the π skeleton and the 6-31G(d) sets for the others. The calculated fluorescence energies were 2.57, 2.29, and 2.25 eV for GFP, mKO and DsRed, respectively. These are in good agreement with the experimental data, 2.46 eV for GFP, 2.22 eV for mKO, and 2.14 eV for DsRed. The observed red shift was reproduced with the present model. The TD-B3LYP results reproduced the relative values, although they overestimated the fluorescence energy of each FP by about 0.5 eV. To figure out the red-shift mechanism of the emission energy, we also performed several calculations with two different computational models. The atomic coordinates of these models

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TABLE 2: Photoabsorption and Fluorescence Energies of Fluorescent Proteins fluorescence energy (eV) B3LYPa CISc b

proteins

All

GFP mKO DsRed Phe14Lys

2.95 2.70 2.66 2.57

All

b

3.71 3.46 3.42 3.27

SAC-CId Cro

b

2.45 1.98 1.91 1.85

Cro+Watb

Allb,h

exptl.

2.45 1.99 1.90 1.87

2.55 (2.57) 2.24 (2.29) 2.21 (2.24) 1.98 (2.08)

2.46e 2.22f 2.14g

a TD-B3LYP/6-31g+(d) result. b The “Cro” model is composed of only fluorophores in the QM region. In the “Cro+Wat” model, a neighboring water molecule was also included. See Figure 1. In the “All” model, the protein electrostatic effect was also included. c D95(d) basis sets for excitation energy. Structure optimization at the QM(B3LYP/6-31G*)/MM(AMBER96) level. d D95(d) basis sets for excitation energy. Structure optimization at the QM(CIS/ 6-31G*)/MM(AMBER96) level. e Reference 1. f References 14 and 15. g Reference 7. h The number in the parentheses was calculated with the 6-311G(d) sets for the π and the 6-31G(d) sets for the others.

were the same as those of the All model. The “Cro” model was used for evaluating structural effects, in which all of the MM region and a water molecule in the QM region were removed. The “Cro+Wat” model, which has an additional water molecule in the QM region, is to estimate the solvation effect of a water molecule on the fluorophore because the protonation state at the phenolate group significantly affects the excitation energy of GFP.45 Finally, with the All model, the protein electrostatic effect was estimated by adding a MM environment. As shown in Table 2, the results of the “Cro” model indicate that the extension of the π skeleton causes a red shift very effectively. The emission energies of mKO and DsRed in their Cro model are 1.98 and 1.91 eV. Compared with that of GFP, the emission red shifts by 0.47 and 0.54 eV, respectively. The effect of the hydrogen bonding with the water molecule is negligibly small. The results of the All models show that the protein electrostatic effect significantly increases fluorescence energies, particularly in mKO and DsRed. The amount of blue shift becomes larger in the order of DsRed (0.31 eV) > mKO (0.25 eV) > GFP (0.10 eV). It is very interesting that the blue shift caused by the protein ESP effect cancels out the red-shift effect that arose from the π skeleton extension. To understand the reason, we analyzed the electronic structures of the FPs. The wave functions of the emitting states were dominated by a one-electron transition from the HOMO to LUMO. Figure 2 shows the orbital distributions of the HOMO and LUMO for GFP, mKO, and DsRed. These MOs have π character, as reported elsewhere.19,25,43 Because the π conjugations in mKO and DsRed extend to the peptide bond, the amplitudes of the HOMOs and LUMOs spread to the extended π skeletons. In mKO and DsRed, the amplitude of the LUMO in the extended part is larger than that of the HOMO, which suggests intermolecular charge-transfer character in the emitting state. The reason why the LUMOs can delocalize more than the HOMOs is as follows. As we can see in Figure 2, both HOMOs and LUMOs have bonding interactions with the π* orbital of the extended skeleton. The orbital energy of the π* orbital is close to the LUMO level of the original chromophore’s skeleton. On the other hand, the HOMO of the extended π skeleton is a lone pair orbital of the N and O atoms. The HOMO level is by 0.3 hartree lower than the HOMO level of the GFP chromophore skeleton. Because of the symmetry, the lone pair orbital is not allowed to mix with the HOMO and LUMO of the chromophore skeleton.

Figure 3. Protein electrostatic potential at the nuclei of the π skeleton of the fluorophore. Numbers in the inset show the atom indices.

The difference electron density upon the transition (∆F ) Fex - Fg) is also shown in Figure 2. In the excited states of mKO and DsRed, the electron density around the extended part is larger than that in the ground state. We also calculated atomic charges using Mulliken’s population analysis. Figure 2 shows a partial sum of the atomic charges. The calculated charges clearly show that the electron density shifts into the extended π skeleton upon the transition. The amount of the shift in DsRed (0.13) is the largest, that of mKO (0.08) is the second largest, and that of GFP (0.01) is smallest of the three. Therefore, the MO character, difference electron density, and atomic charge show that the electron distribution shifts to the extended part upon the transition. The shift in the emission energy caused by the protein ESP is written as

∆VES )

∫ ∆F(r)VESP(r)dr

(1)

where ∆F ) Fex - Fg is the difference electron density of the QM region, and VESP(r) is the protein ESP at a position r. Therefore, the shift occurs when both ∆F and the ESP have meaningful magnitude. As shown in Figure 3, we calculated ESP at the nuclei of the π system. The results for GFP, mKO, and DsRed show that the ESPs are small around atoms 15-18 in the extended π skeletons and high in the two rings. Because the negative charge of the extended π skeleton increases in the excited states of mKO and DsRed, the excited state becomes relatively unstable compared with the ground state. This is the reason the protein ESP effect causes the blue shift in mKO and DsRed. In the case of GFP, because the charge shift to the extended part is negligibly small, as shown in Figure 2, the amount of the spectral shift becomes much smaller than that in mKO and DsRed. 3.3. Quantum Mechanical Effect of the Protein Environment in the Vicinity of the Chromophore Investigated with the ONIOM Method. The above-mentioned QM/MM calculations adopted a fixed point charge set to describe the electrostatic effect of the protein environment. The protein effects missing are, therefore, exchange repulsion, polarization, charge transfer, and van der Waals interactions46 between the fluorophore and its environment. A QM/QM/MM study showed that, in the case of retinal proteins, the polarization effect of protein media reduced the excitation energy by 0.1 eV.47 The electron density of the fluorophore changes upon transition, especially in mKO and RFP, which could induce the reorganization of the electron density of the protein environment. To take into account the



TABLE 3: ONIOM Calculations to Include the Quantum Mechanical Effect from the Protein Environment within a 3 Å Distance from the Chromophorea SAC-CI

CIS

FPs

All modelb

All model

3 Å modelc

∆d

SAC-CI:CIS

exptl. (eV: nm)

GFP mKO DsRed F14K

2.55 2.24 2.21 1.98

3.71 3.46 3.42 3.27

3.60 3.42 3.31 3.21

-0.11 -0.05 -0.11 -0.07

2.44 2.19 2.10 1.91

2.46: 504e 2.22: 558f 2.14: 579g 1.95: 636h

a HF and CIS methods were used to calculate the excitation energy of the lower-level region. The units are eV. b The “All” model denotes that the structure shown in Figure 1 was included in the QM region. In addition, the electrostatic effect of the rest of the environment was treated by a point charge model. c Amino acids and waters within 3 Å of the chromophore were included in the QM region. The other atoms were approximated by a point charge model. d Correction to the excitation energy due to the quantum mechanical treatment of the protein environment with the 3 Å model. e Reference 1. f References 14 and 15. g Reference 7. h Estimated value. The fluorescence energy was shifted by +0.04 eV because the SAC-CI:CIS value for DsRed underestimates the experimental value by 0.04 eV.

quantum mechanical effect of the protein environment, we adopted an ONIOM scheme48 using the SAC-CI and the CIS methods for the higher and lower levels, respectively. An enlarged QM region, a 3 Å model, was introduced; amino acids including atoms within a 3 Å distance from any QM atoms were taken into the model. With this model, all of the atoms in the first solvation layer were included. The atoms outside of the QM regions were treated by a MM model, as in the All model. With this treatment, all of the missing effects except van der Waals interactions were included in the excitation energy. We note, however, that polarization outside of the 3 Å model was not considered. The 6-31G* basis sets were used for the CIS calculations. The structures of the 3 Å models and computational details are given in section S1 in Supporting Information. The results of the ONIOM calculations are summarized in Table 3. The calculated emission energies with the 3 Å models were smaller by 0.05-0.11 eV than those from the All models. The excitation energies of GFP, mKO, and DsRed became 2.44, 2.19, and 2.10 eV, respectively, after the correction. These values are in reasonable agreement with the experimental data (2.46, 2.22, and 2.14 eV for GFP, mKO, and DsRed, respectively). Relative differences between the excitation energies were slightly improved after the correction, although the amount of shift is similar among FPs. The quantum mechanical effects of the environment considered within the present procedure would be necessary for a more accurate prediction of the emission energy, although this effect is not a primary factor of the emission color tuning among the FPs. 3.4. Role of the Protein ESP in the Emission Color-Tuning Mechanism. In subsection 3.2, using the SAC-CI results of the All and the Cro+Wat models, we evaluated the shift in emission energy (∆Eex) after the protein ESP was included in the Hamiltonian operator. The calculated shift includes two components, (i) direct electrostatic interaction (∆VES) and (ii) electronic structure relaxation of the QM region (∆RLX) after the ESP was introduced in the All model.

∆Eex ) ∆VES + ∆RLX

(2)

The direct interaction term is the Coulomb interaction and is expressed as MM

∆VES )

∑∫ A

∆F(r) · qA dr |r - rA |

(3)

where ∆F ) Fex - Fg is the difference electron density obtained by the SAC/SAC-CI calculations using the protein ESP, and

TABLE 4: Result of Decomposition Analysis for the Emission Energy Shift Due to the Protein ESP (in eV) FPs

direct ES interactiona ∆VES

WF relaxationb ∆RLX

total shift ∆Eex

+0.04 +0.19 +0.40 +0.29

+0.06 +0.06 -0.09 -0.17

+0.10 +0.25 +0.31 +0.11

GFP mKO DsRed F14K

a Defined in eq 3. b Wave function relaxation effect defined in eq 4. See SI for the definitions of these terms.

qA is the atomic charge of atom A in the MM region. The relaxation term is the rest of the shift and is written as ˆ

ˆ 0, Rˆex(I)]eS |HF〉 ∆RLX ) ∆Eex - ∆VES ) 〈Lex(I) |[H Sˆ0 ˆ 0, Rˆex(I) |[H 〈Lex(I) 0 0 ]e |HF0〉

(4)

Both terms in eq 4 express the excitation energy calculated using ˆ 0 is the Hamiltonian the SAC and the SAC-CI wave functions. H operator only for the QM region, which is common to the two terms. None of the QM/MM and MM/MM interactions such as ˆ 0 operator. A subscript 0 protein ESP are included in the H denotes quantities and operators without the ESP. Sˆ and Rˆ are excitation operators in the wave functions for the ground and excited states, respectively. Those without subscript 0 indicate that their variables are determined including the ESP operator. Therefore, the ∆RLX term denotes the amount of the excitation energy shift due to the change of the wave functions in the QM region. In the Supporting Information (SI), the derivation of the eqs 2-4 is described in more detail. The result of the decomposition analysis is shown in Table 4. In the cases of mKO and DsRed, it is obvious that the total shift is dominated by the direct ES term. The amount of the contribution reaches 76 (0.19 eV) and 129% (0.40 eV) of the total shift in mKO and DsRed, respectively. In DsRed, the calculated RLX term became negative (-0.09 eV). Because DsRed has more charged residues in the vicinity of the fluorophore, the stronger ES interaction probably introduces a larger reorganization in the wave function. Because the direct ES term (eq 3) is the main source of the spectral shift, contributions from each amino acid are the next target of the analysis. amino acids

∆VES )

∑ M

amino acids atoms ES ∆VM

∑ ∑ M

A∈M

∫

∆F(r) · qA dr |r - rA |

(5)

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Figure 4. Contribution to ∆VES from each amino acid and water (in hartree; see eq 3 for the definition). (a) GFP, (b) mKO, (c) DsRed, and (d) Phe14Lys mutant.

Figure 5. Amino acid residues contributing to the emission color tuning via electrostatic interaction. Labels of the residues are ordered as (GFP)/ (mKO)/(DsRed) in (a) and (b) and (GFP)/(mKO)/(DsRed)/(Phe14Lys) in (c). For example, in (a), the amino acid residue above the chromophore is Ile167, Met162, and Lys163 in GFP, mKO, and DsRed, respectively. Atomic coordinates were optimized using QM/MM calculations for the emitting states.

Figure 4 clearly shows the contributions from amino acids, ∆VES M. The magnitudes of the contributions become larger in the order of DsRed > mKO > GFP, which indicates that the emission energy is sensitive to the protein ESP in this order. As described above, if the difference electron density ∆F is larger, the amount of the shift ∆VES M increases. The amount of the charge displacement upon the transition is largest in DsRed and smallest in GFP. In the case of mKO, Arg94 and His197 give positive contributions to the electrostatic interaction energy. In the case of DsRed, the positive electrostatic interaction energy originates from the contributions of Lys163, Lys70, Phe65, and Arg65. Most of these amino acids are positively charged, except for Phe65 in DsRed. All of these amino acids are in the first solvation layers, as shown in Figure 5. These results indicate

that the emission colors of the FPs are regulated by the positively charged amino acids in the vicinity of the chromophore. Next, we investigated how these charged amino acids provide their positive contributions. As shown in Figure 5c, Arg94 in mKO and Arg95 in DsRed form a hydrogen bond with the CdO group of the imidazolinone ring. As shown in Figure 3, the positive ESPs produced by these residues are particularly large at the oxygen atom, which looks like a “cusp” at the 11th atom. Figure 2 shows the difference electron density upon the transition (∆F ) Fex - Fg). The red surface around the 11th oxygen atom clearly shows that the electron density decreases in the excited state, which means that the oxygen atom becomes less negative in the excited state. Because the arginines generate positive ESP, their contributions to the emission energy become



Figure 6. ESP at the nuclei of the π skeleton produced by amino acids that give dominant contributions to total ESP. The numbers in the inset in (a) show the atom indices. (a)GFP, (b)mKO, (c)DsRed, and (d)Phe14Lys.

positive, resulting in a blue shift. In the case of GFP, there is little contribution from Arg96. One of the reasons is that the cusp shape in the total ESP is less sharp than those in mKO and DsRed. The other reason is that the red surface around the oxygen atom is smaller, as seen in Figure 2a, indicating that the amount of density change in GFP is smaller than that in mKO and DsRed. In addition, because of the difference density of the carbon atom in the CdO group, the electrostatic contributions at the C and O sites cancel each other. Lys163 of DsRed and His197 of mKO lie close to the phenolate group of the chromophore. They work as counterions to the chromophore, as shown in Figure 5a. On the basis of inspecting the molecular interactions observed in the X-ray structures 2ZMU and 2ZMW,15 His197 is expected to be cationic.15 These positively charged residues produce a gradient of the ESP as shown in Figure 6b and c. The ESP around the phenolate group (the 1st to 7th atoms) is relatively higher, and that around the peptide bond (the 15th to 18th atoms) is relatively lower. As described above, the electronic transitions in mKO and DsRed are accompanied by changes in the charge distribution, as shown in Figure 2b and c. Because transfer of negative charge under the ESP is energetically unfavorable, the ESP contributions of Lys163 of DsRed and His197 of mKO become blue-shifted. We point out the similarity of the color-tuning mechanisms among retinal proteins,29,49 firefly luciferins,33 and DsRed. In the case of retinal Schiff base, the counteranion creates negative ESP along the π skeleton of the retinal chromophore. Because the character of the excited state is an intermolecular CT toward the Schiff base, the protein ESP increases the excitation energy. Therefore, the mechanism is just opposite to that of DsRed, where the countercation contributes to the blue shift. In the case of firefly luciferin, charges of the carbonyl O atoms change. A positively charged arginine and a negatively charged phosphate group play crucial roles in the emission color tuning. A common feature in these mechanisms is an intermolecular charge-transfer

character and a charge-polarized electrostatic environment of the protein close to the chromophore. Because the natural mutation of amino acids would be the easiest way to change the electrostatic environment, a chromophore with an intramolecular CT character in its excited state could serve as an efficient chromophore in the history of the molecular evolution. 3.5. Molecular Design of the Electrostatic Interaction in DsRed: Phe14Lys Mutation. Before closing the paper, we will show the result of a theoretical mutation that should red shift the emission energy of DsRed. This is to verify the current conclusion on the emission color-tuning mechanism and to suggest a possible new mutation experiment. As clearly seen in Figure 2, atomic charges, difference electron densities, and distributions of the HOMO and LUMO show that the electron population of the oxidized peptide bond (OdC-NdC-) in the first excited state becomes larger than that in the ground state. If a positively charged residue is in the vicinity of the peptide bond, the energy of the excited state should decrease due to the electrostatic interaction. The 14th amino acid in DsRed, which is ideally close to the carbonyl group, is occupied by phenylalanine (see Figure 5c). We replaced this Phe14 with a positively charged lysine residue, as shown in Figure 5c. A possible alternative to lysine would be arginine. The structure of this mutant, Phe14Lys (F14K), in the excited state was optimized by the QM/MM calculations in the same way as described above. The optimized structure does not significantly deviate from that of DsRed (see Figure 5c). The electrostatic potential in Figure 3 clearly shows the effect of the mutation; atoms close to the lysine (15th-18th atoms) are exposed to a positive electrostatic potential from the lysine. Figure 6d shows that this increasing component originates from Lys14. As shown in Table 2, the calculated emission energy of the Phe14Lys mutant was 1.98 eV in the All model, which is smaller by 0.23 eV than that of DsRed. Basis sets of the 6-311G(d) quality (described in Table 2) gave 2.08 eV for the emission

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energy, and the amount of the red shift was 0.13 eV. After the ONIOM correction with the 3 Å model (SAC-CI:CIS result in Table 3), the emission energy became 1.91 eV, which is 0.19 eV less than that of DsRed. Because the calculated emission energy of DsRed underestimated the experimental value by 0.04 eV, we expect the Phe14Lys mutant to show an emission peak at around 1.95 eV (636 nm). Compared with the result of mPlum (649 nm, 1.91 eV),10 the Phe14Lys mutant has the potential to red shift the emission to very close to that of mPlum using only a single mutation. We hope that this result indicates a new direction toward a new generation of near-infrared fluorescent proteins. 4. Conclusion In this study, the emitting states of green fluorescent protein (GFP), monomeric Kusabira orange (mKO), and Discosoma red (DsRed) were studied using QM/MM and SAC-CI methods. The emission energies of these FPs gradually decrease in the order of GFP (2.46 eV) > mKO (2.22 eV) > DsRed (2.14), depending on the π conjugation, as shown in Figure 1. We analyzed the emission color-tuning mechanism in terms of structural differences, solvation of a water molecule, and electrostatic and quantum mechanical interactions with the protein environment. The structures of the emitting states were calculated with the QM(CIS/D95(d))/MM(Amber96) method. The fluorescence energies calculated with the QM(SAC-CI/D95(d))/MM(Amber96) method are in reasonable agreement with the experimental data. The excitation energies of the DsRed model compounds RFP(1) and RFP(2) were also calculated, and the results are in good accord with the experimental data. Within the basis sets examined in this study, D95(d,p), D95+(d,p), and 6-311G(d), no significant deviation from the D95(d) sets was observed in the calculated excitation energies. Using several model calculations, we found that the protein electrostatic effect increases the emission energies in mKO and DsRed, although the contribution of the π skeleton extension effect is to reduce the emission energies. Because of the structure of the protein ESP, the charge displacement in the excited state is energetically unfavorable. Because the amount of charge transfer in DsRed is the largest of the three FPs, the emission energy of DsRed is sensitive to the protein ESP and to the modulation of ESP. The specific roles of amino acids that control the emission energy were also clarified in detail. We further proposed and examined a novel mutation for DsRed intended to red shift the emission energy. A single mutant Phe14Lys reduces the calculated emission energy to 1.95 eV (636 nm), which is close to that of mPlum (1.91 eV, 649 nm) and is approaching a near-infrared fluorescent protein. Acknowledgment. This study was supported by a Grant-inAid for Young Scientists (A) and Scientific Research on Priority Areas “Molecular theory for real systems,” from the Japan Society for the Promotion of Science (JSPS) and by JST-CREST and a Grant-in-Aid for Young Scientists from ACCMS and IIMC, Kyoto University. A portion of the computations was carried out at RCCS (Okazaki, Japan). Supporting Information Available: Description of the computational models. Derivation of the equations in subsection 3.3. Full author list of the Gaussian 03 package. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Tsien, R. Y. Annu. ReV. Biochem. 1998, 67, 509–544. (2) Tsien, R. Y. FEBS Lett. 2005, 579, 927–932.

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Excited States of Fluorescent Proteins, mKO and DsRed

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