Molecular-Level Insight into the Spectral Tuning Mechanism of the

Letter pubs.acs.org/JPCL

Molecular-Level Insight into the Spectral Tuning Mechanism of the DsRed Chromophore Nanna H. List,† Jógvan Magnus H. Olsen,† Hans Jørgen Aa. Jensen,† Arnfinn H. Steindal,‡ and Jacob Kongsted*,† †

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, DK-5230 Odense M, Denmark Centre of Theoretical and Computational Chemistry, Department of Chemistry, N-9037 Tromsø, Norway

‡

S Supporting Information *

ABSTRACT: We present a detailed study of the protein environmental effects on the one- and two-photon absorption (1PA and 2PA, respectively) properties of the S0-S1 transition in the DsRed protein using the polarizable embedding density functional theory formalism. We find that steric factors and chromophore− protein interactions act in concert to enhance the 2PA activity inside the protein while adversely blue-shifting the 1PA maximum. A twostate model reveals that the 2PA intensity gain is primarily governed by the increased change in the permanent dipole moment between the ground and the excited states acquired inside the protein. Our results indicate that this mainly is attributable to counter-directional contributions stemming from Lys163 and the conserved Arg95 with the former additionally identified as a key residue in the color tuning mechanism. The results provide new insight into the tuning mechanism of DsRed and suggest a possible strategy for simultaneous improvement of its 1PA and 2PA properties. SECTION: Spectroscopy, Photochemistry, and Excited States from local variations in the internal electric field of the protein.9 The sensitivity to the local electrostatic environment of the one-photon absorption (1PA) maximum of the red chromophore has been supported by several computational studies.10−14 Moreover, comparison of the 2PA spectra along the same series of RFPs also reveals that unlike the 1PA intensity of the S0-S1 transition, which is not appreciably changed by the amino acid substitutions, its 2PA counterpart exhibits a strong dependency on the protein surroundings.15,16 A molecular-level description of the field-induced color and intensity tuning of the red chromophore is not only of fundamental interest but also necessary to facilitate rational design of mutants tailored for specific applications. Unfortunately, experimental efforts into scrutinizing the spectral tuning mechanism governed by the protein barrel are complicated by the high electrophilicity of the crucial acylimine group, which prevents measurements from being conducted in the absence of the protein.17 Progress in this area can be assisted by computational studies, and theoretical rationalization of the optical properties of FPs has indeed attracted considerable attention; for more recent studies, see, for example, refs 10−14 and 18−24. So far, most studies on the red chromophore have been focusing on 1PA,

ince the pioneering work on the green fluorescent protein (GFP),1 fluorescent proteins have, because of their application as genetically encoded fluorescent biomarkers, become indispensable tools within life sciences.2 Besides their use in conventional one-photon induced fluorescence imaging, these proteins have been recognized to possess fairly large twophoton absorption (2PA) cross sections and are now increasingly being utilized as labels in two-photon laser scanning microscopy (2PLSM).3−5 The ability to reach an excited state through the simultaneous absorption of two photons offers considerable advantages over linear microscopy. The use of a longer wavelength enables deeper tissue penetration and reduces cellular autofluorescence, and along with the spatial confinement, due to the quadratic dependence on the intensity, two-photon techniques are well-suited for noninvasive studies of biological systems.5−7 To further minimize phototoxicity and optimize thick tissue imaging, application of red fluorescent proteins (RFPs) is particularly attractive. The chromophore shared by the RFPs is characterized by an extension of the π-conjugated phydroxybenzylideneimidazolinone core with an acylimine group, thereby yielding a structural motif with an intrinsically red-shifted absorption compared with GFP. Since the discovery of the first of its kind (DsRed8), prolific mutation studies have produced a range of different hues in the red part of the spectrum. Recently, it has been shown that the color palette available by the members of the mFruit series of RFPs arises

S

© 2012 American Chemical Society

Received: September 21, 2012 Accepted: November 13, 2012 Published: November 13, 2012 3513

dx.doi.org/10.1021/jz3014858 | J. Phys. Chem. Lett. 2012, 3, 3513−3521

The Journal of Physical Chemistry Letters

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decomposition into indirect and direct effects of the protein, that is geometrical changes and electrostatics interactions, respectively, we consider three systems: (1) the bare chromophore at the vacuum optimized geometry (QM-vac), (2) the bare chromophore at the geometry in the protein (QMprotein), and (3) the chromophore embedded in the protein (PE-QM); that is, the chromophore in the two latter cases adopts the same geometry, corresponding to the one acquired from the QM/MM optimization outlined above. Thus, comparison of (1) and (2) gives the indirect effects while the direct effects can be examined by comparing (2) and (3). The 1PA results for the electronic S0-S1 transition of the three systems are presented in the upper part of Table 1

and only recently, theoretical investigations of its 2PA properties have been pursued.23,24 However, the microscopic origin of the environmental implications on the 2PA properties of the red chromophore remains to be explored. Exactly with this quest in mind, the goal of the present letter is to improve the understanding of the physics underlying the protein environmental effects on the 1PA and 2PA properties of the S0-S1 transition of the red chromophore, focusing on the progenitor DsRed protein. To account for the protein environment in the 1PA and 2PA calculations, we employ the recently developed polarizable embedding density functional theory (PE-DFT) scheme,25 which comprises a detailed electrostatic and polarizable force-field representation of the protein consisting of higher-order localized multipoles and anisotropic dipole−dipole polarizabilities as implemented in a development version of DALTON2011.26 This method not only incorporates the mutual polarization between the protein and the chromophore in its electronic ground state but also includes, within a linear response formulation, the instantaneous relaxation of the electronic degrees of freedom of the environment to the changing electron density of the chromophore upon excitation. Because it describes a change in the induced dipole moments of the environment to the firstorder change in the electronic density of the chromophore caused by the oscillating electromagnetic field, we refer to this as a first-order relaxation of the environment. The calculations are performed in a nonequilibrium formulation; that is, only the electronic part of the environment is allowed to respond to the excitation as appropriate for vertical electronic transitions. All 1PA and 2PA properties were computed with TD-DFT, and to address the charge-transfer like nature of the S0-S1 transition of DsRed, we employed the range-separated CAMB3LYP27 functional in combination with the 6-31+G*28−30 basis set. As expected,31 the CAM-B3LYP functional significantly overshoots the absolute transition energies; however, this functional has recently been demonstrated to provide spectral shifts for chromophore-residue systems of the photoactive yellow protein in excellent agreement with RI-CC2/aug-ccpVTZ,32 which supports its use in the presented analysis of the shifts caused by the surrounding protein environment. A detailed description of the computational protocol is supplied in the Supporting Information (SI). Compared with previous quantum mechanics/molecular mechanics (QM/MM) studies on the related DsRed.M1 protein,12,14 we use in the present PE-DFT calculations an enlarged QM region (63 atoms) comprising the chromophore residue CRQ66, the entire Phe65 residue, in addition to the N and HN atoms of Gln64 and hydrogen link atoms. This choice is based on our subsequent analysis of residue-specific contributions, as described in the SI. The structural model used in the subsequent PE-DFT calculations was constructed by a point-charge electrostatic embedding QM/MM optimization of the PDB entry 1ZGO33 using the Qsite34 program. The discrepancies between the crystallographic structure and the QM/MM-optimized geometry mainly concern the acylimine group of the chromophore. These differences are likely due to the limitations in the X-ray crystallographic refinement, which relies on an MM-based guiding strategy, as demonstrated by the fact that similar substantial rearrangements have been reported for the related DsRed.M1 protein by applying a QMrather than an MM-based guiding algorithm in the refinement of the X-ray structure.35 A comparison of the QM/MMoptimized and 1ZGO structures is given in the SI. To enable

Table 1. One- and Two-Photon Absorption Properties for the S0-S1 Transition of the DsRed Chromophore Obtained at the PE-CAM-B3LYP/6-31+G* Level of Theorya model

ω1

f

σ2PA

|Δμ|

QM-vac QM-protein PE-QM exp

2.392 2.528 2.826 (2.746) 2.22b

1.093 1.163 1.308 (1.072)

16.6 46.5 105.9 (74.6) 96c

2.356 3.534 4.845 3.5d

2.891 3.000

1.196 1.155

51.0 59.1

3.663 4.006

NPE FPE

Vertical excitation energies ω1 (eV), corresponding oscillator strengths f (length gauge), and 2PA cross sections (σ2PA), the latter as calculated from quadratic response theory and eq 3 of the SI. Results from the PE-cluster calculations are given in parentheses. The change in permanent dipole moment (Δμ) is defined relative to the coordinate system in Figure 3, and its length is given in Debye. bRef 8. c Value for DsRed2, ref 9. dEstimate based on ref 46 using local field factor from ref 9. a

together with the corresponding experimental absorption maximum. As evident, the computed absolute excitation energy inside the protein (PE-QM) is significantly overestimated with respect to its experimental counterpart; however, as discussed above, we expect the spectral shifts caused by the geometrical and electrostatic effects of the protein to be reasonably described. In all three systems, the first excited and intense state is due to a ππ*-type transition mainly governed by a promotion of an electron from HOMO to LUMO. It is evident that the structural changes imposed by the protein matrix lead to a blue shift of the excitation energy, as previously reported.13,14,36 Although a clear distinction between strain and electrostatic effects of the protein is not possible due to the use of the enlarged QM part, this blue shift can be rationalized on the basis of the qualitative particle-in-a-box model14 by the near-perpendicular twist of the terminal carbonyl group of the chromophore induced by the protein. Indeed, a shortening of the conjugation pathway is apparent from a comparison of the respective orbitals (Figure S6 of the SI). From the orbital pictures it is also worth noting that the electron density is shifted from the phenolate ring toward the acylimine part of the chromophore upon excitation, as discussed in previous work.13 This charge redistribution is quantified by a change in the permanent dipole moment (Δμ = μ11 − μ00) going from the ground to the excited state, predominantly in the direction of the π-conjugated chain of the chromophore (Table 1). As detailed below, the smaller dipole moment is found to be associated with the excited state, consistent with the 3514



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Figure 1. Total electrostatic potential, Vtot, of the ground-state environment of the DsRed protein as well as the constituent static and induced contributions, Vstatic and Vind, respectively, at the atomic sites along the π-conjugation pathway of the chromophore.

Figure 2. Electrostatic potential, Vtot, (in a.u.) of the ground-state environment projected onto the van der Waals surface of the chromophore and colored according to the color scale bar. To highlight smaller fluctuations in the potential along the chromophore, the potential is locally higher than the maximum values on the scale, as illustrated in Figure 1. Selected close-lying residues are shown in wire representation.

hypsochromic behavior observed for DsRed chromophore models in aqueous solution,37,38 and the obtained |Δμ| in the protein is in the same range as the experimental value (cf. Table 1). In passing, we note that the dipole moment obtained for QM-vac (2.356 D) is smaller than the value reported by Nifosı ̀ et al. (4.69 D). This discrepancy is likely related to (i) the differences in the applied chromophore models (an enlarged chromophore in the present work) and (ii) the different methods employed. The |Δμ| value reported in ref 39 is based on an estimate of the excited-state wave function by transferring one electron from HOMO to LUMO, whereas the present study applies quadratic response theory, in which all virtual orbitals are included in the description of the excited state.

Inspection of Table 1 also reveals that the excitation energy is shifted further to the blue when the chromophore is embedded in the protein. Compared with the solvent phase, the chromophore is buried within the heterogeneous, spatially organized protein matrix, which makes it less straightforward to rationalize spectral shifts without knowledge of the strength and direction of the internal electric field set up by the protein. Hence, to shed light on the origin of the preferred stabilization of the ground state by the electrostatic effects of the protein, we have computed the electrostatic potential of the ground-state environment, Vtot, around the chromophore. Figure 1 gives the electrostatic potential at the atomic positions along the conjugation pathway of chromophore and, for a more complete 3515



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Figure 3. Coordinate system that defines the vector and matrix quantities given in Table 2 as well as the atom nomenclature used for the DsRed chromophore. The origin is located at the C1 atom, and the xy plane corresponds approximately to the plane of the two rings. For clarity, the chromophore structure has been truncated to include only the methyl-terminated π-conjugated chain.

first-order relaxation of the environment turned off as well as those obtained by disregarding environmental responses altogether, hereafter referred to as the frozen polarizable environment (FPE) and nonpolarizable environment (NPE) approximations, respectively. More specifically, the NPE approximation corresponds to the use of electrostatic embedding so that the environment is represented only by the permanent multipole moments obtained for the groundstate geometry, whereas the FPE approximation also includes the relaxation of the electronic degrees of freedom of the environment to the chromophore in its ground state; however, it retains the resulting ground-state electrostatic potential upon excitation. As shown in the lower part of Table 1, employing the NPE approximation (corresponding to Vstatic in Figure 1) yields a 0.06 eV overestimation of the spectral shift compared with the full model (PE-QM). As expected, the larger variations in the potential across the chromophore present in the FPE approximation (corresponding to Vtot in Figure 1) are reflected in a more prominent effect that amounts to a 0.17 eV overestimation of the shift. In agreement with physical intuition, this shows that the environmental adaptation to the electronic transition itself serves to modulate the electrostatic potential according to the excited state, thereby reducing the spectral shift. We now consider the 2PA properties obtained from quadratic response theory and compiled in Table 1. Because of the complexity in acquiring accurate experimental absolute 2PA cross sections and the consequent variation in the reported values,16 we provide the recent experimental 2PA cross section for the DsRed2 mutant42 (six mutations peripheral to the chromophore) conducted by Drobizhev et al.15 instead of the first reported 2PA measurement on the wildtype DsRed protein.43 From our predictions, one can clearly see that the 2PA cross section is strongly intensified by the geometrical changes caused by the spatial restrictions imposed by the protein surroundings. More interesting is the finding that the direct influence of the protein barrel gives rise to an additional increase in the 2PA cross section. To decipher the microscopic

picture, we also provide the projection of the electrostatic potential onto the van der Waals surface of the chromophore buried inside the DsRed protein (see Figure 2, which has been prepared using VMD40). The atom labeling is shown in Figure 3. As clearly seen, the highest potential is found at the phenolic oxygen, and with the exception of the peak at the carbonyl O2 atom, it gradually decreases toward the acylimine moiety, as also reported in ref 13. With the chromophore dipole moment roughly aligned with the electric field in the two rings produced by the protein, the more charge-separated ground state becomes favored, thereby explaining the observed blue shift in the excitation energy (electric field components and norm along the conjugated chain are supplied in Figures S7 and S8 in the SI). Although, similar sizable effects on the excitation energy have been predicted when applying a uniform external electric field along the conjugation path of the bare chromophore,41 we find that the intraprotein electric field exerted on the DsRed chromophore is far from being uniform (Figures S7 and S8 in the SI). To move toward a more detailed understanding of the tuning mechanism of the protein environment, we dissected the total electrostatic potential of the ground-state environment into static and induced contributions, respectively. Here Vstatic designates the contribution to the electrostatic potential due to the permanent multipole moments of the ground-state environment, while Vind is the contribution arising from relaxation of the electronic degrees of freedom of the environment to the ground-state density of the chromophore. As seen in Figure 1, the profile of the potential is determined by the static charge distribution while the induced potential acts as a tuning factor by enhancing the electrostatic potential difference between the phenolate ring and the acylimine group. So far we have not discussed the effects of the environmental response to the change in the electronic density of the chromophore induced by the absorption of light. With the considerable redistribution of charge that occurs in the excitation process, it is pertinent to investigate the influence of the nonequilibrium response of the surrounding protein. Therefore, we re-evaluated the absorption properties with the 3516



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Table 2. Two-Photon Absorption Probability for the S0-S1 Transition of the DsRed Chromophore As Calculated from the TwoState-Model (δ2SM) and Parameters Constituting Equation 1b 01

model

ω1

δ2PA

δ2SM

|μx01|

|μy01|

μx00

μy00

μx11

μy11

|μ01|

|Δμ|

θμΔμa

QM-vac QM-protein PE-QM

2.392 2.528 2.826

3.96 9.94 18.10

3.85 11.24 19.66

1.561 1.305 1.776

4.026 4.131 3.962

−3.104 1.990 2.477

−2.254 −7.558 −10.937

−3.585 1.697 2.858

−1.461 −6.200 −9.070

4.318 4.333 4.342

0.927 1.389 1.906

52.46 29.71 12.61

NPE FPE

2.891 3.000

9.20 8.98

9.40 9.82

1.565 1.504

3.796 3.962

3.319 2.477

−10.466 −10.937

3.471 2.671

−9.033 −9.373

4.106 3.964

1.441 1.576

16.38 15.23

b Vertical excitation energies ω1 (eV) and x,y-components (a.u.) of the transition dipole moment (μ01 α ) and permanent dipole moments of the 11 01 ground and first excited states (μ00 α and μα , respectively), as defined by the coordinate system in Figure 3 as well as the norms, |μ | and |Δμ|, of the 01

transition dipole moment and change in permanent dipole moment, respectively, and the angle θμΔμ (degree) between μ01 and Δμ projected onto the 01

01

xy plane. For comparison, we also include the quadratic-response-derived δ2PA values. aAngle between Δμ and μ01 can either be θμΔμ or 180° − θμΔμ.

S4 in the SI. Hence, we find a positive correlation between the variation in bond distance and difference in permanent dipole moment, irrespective of whether we consider the CZ−OH bond alone or the average over the three bonds CZ−OH, CB2−CG2, and N2−CA2, as can be expected from the larger charge separation present in the phenolate form. Therefore, the combined action of the decreased Δμ and the reduced degree of alignment between μ01 and Δμ explains the decreased 2PA cross section characterizing the gas-phase optimized chromophore (QM-vac) compared with the bare chromophore adopting the structure inside the protein (QM-protein). To validate the plausibility of a 2PA intensity gain effectuated by the electrostatic effects of the protein, we performed PEDFT calculations using an enlarged QM region (242 atoms) that in addition to the chromophore encompasses the nearby amino acids and biological water molecules, as described in the SI. Although we are well aware of the limits of such a comparison (inherent discrepancies due to, e.g., basis set incompleteness and neglect of local field factors), the similar although less accentuated effect found for this PE-QM cluster system (cf. Table 1) supports a field-induced 2PA enhancement. On the basis of this combined PE-QM cluster calculation, the magnitude of the 2PA cross section intensification is ca. 60%, and the resulting absolute value is in reasonable agreement with the experimental counterpart. (See Table 1.) Having established that the electrostatic effects of the DsRed protein enhance the 2PA intensity, it is enlightening to consider the corresponding results provided by the lower-level polarization models (FPE and NPE) introduced above. The results are compiled in Table 1. As clearly seen, the 2PA cross section is sensitive to the applied force-field representation of the environment, and the two lower-level approximations completely fail to account for the enhancement of the 2PA by the electric field produced by the protein. As elucidated by the 2SM, the associated underestimation of Δμ accounts for the approximate halving of the 2PA cross section. (See Table 2.) Stepwise comparisons of the ground and the first excited-state dipole moments predicted by the FPE and NPE models to those obtained from the full PE description allow us to identify a tuning mechanism: the ground-state polarization environment (Vtot in Figure 1) leads to an increased |Δμ| via a preferential increase in the ground-state dipole moment. By incorporating the environmental response to the electronic transition itself, the aforementioned increase in the excited-state dipole moment is almost canceled, thereby yielding an even larger |Δμ|. Cumulatively, these oppositely directed effects on the ground-

origin of the enhanced 2PA activity mediated by the protein, we examined the qualitative two-level approximation for the twophoton transition elements. This analysis is motivated by the fact that the higher excited states are either strongly dipole forbidden or energetically well-separated from the ground and first excited states. For ease of interpretation, we exploit the additional simplification offered by the pseudo two-dimensionality of the chromophore, and thus we concentrate only on the components in the plane of the π-conjugation (xy plane, see Figure 3). This assumption is supported by negligible out-ofplane components of the transition dipole moments and change in the permanent dipole moment. Further assuming linearly polarized light and orientational averaging, the planeprojected two-state model (2SM) for the 2PA transition probability (in a.u.) is given by44,45 δ 2SM =

01 16 |μ01 |2 |Δμ|2 (1 + 2 cos2 θΔμμ ) 2 15 ω1

(1)

where ω1 is the excitation energy of the first excited state while 01 θμΔμ denotes the angle between the transition dipole vector, μ01, and the change in the permanent dipole moments between the ground and the first excited state, Δμ, as previously defined. These quantities are listed in Table 2 and are defined relative to the molecular axes and origin depicted in Figure 3. It is clear from the similarity between the 2PA transition probability predicted by the 2SM and the quadratic response counterpart that the simple model indeed captures the dominant part of the 2PA transition probability and thus is a reliable platform for physical interpretation. By comparing the magnitude of the quantities appearing in eq 1, cf. Table 2, we find that the intensified 2PA cross section is linked to an increased change in the permanent dipole moment acquired inside the protein and a concomitant closer alignment of the transition dipole moment and permanent dipole moment difference. Within the protein, the transition dipole and change in permanent dipole vectors are nearly aligned, as demonstrated by the fairly small angle of 12.7°, which is perfectly in line with the value (13°) derived from Stark effect measurements.46 The corresponding angles are markedly larger for the bare chromophore structures (QMprotein: 29.7° and QM-vac: 52.4°), which diminishes the 2PA intensity, as evident from eq 1. In line with previous findings,12,14,35 we observe a transition from the quinoid-like resonance form prevalent in the gas phase to a more phenolate dominated form in the protein, as can be seen from the differences in the bond length alternation patterns of the gasphase and QM/MM-optimized structures, illustrated in Figure 3517



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Figure 4. Contributions to the electrostatic potential of the ground-state environment at the atomic sites along the π-conjugation pathway of the chromophore originating from key residues of the DsRed protein compared with the total electrostatic potential Vtot (black line). PDB nomenclature has been employed. (See Figure 3.)

hydrogen bonding to the phenolate while the peak at the carbonyl O2 can be traced to the nearby positively charged Arg95. Hence, Lys163 and Arg95 seem to hold the greatest tuning potential. However, bearing in mind the impact of the environmental response to the electronic structure of the chromophore, one needs to consider the transition itself to quantify the tuning capability of a given residue. Accordingly, we conducted a set of PE-DFT calculations in which each of the six amino acids considered above was separately replaced by glycine. The consequent shifts of the excitation energy, the oscillator strengths, and the 2PA cross sections with respect to wildtype DsRed are presented in Figures 5 and 6.

and excited-state dipole moments controlled by the responsive environment as well as the better alignment of μ01 and Δμ bring about the observed 2PA intensity gain exhibited by the chromophore inside the protein. We should note that the conclusions are based on a single configuration obtained from a QM/MM optimization and thus do not incorporate the effects of conformational sampling as well as bulk solvation. However, as suggested by recent studies on DsRed.M1,14 mCherry, and mStrawberry,47 we do not expect either substantial structural fluctuations of the chromophore or large variations in the internal electric field exerted on the chromophore. Together, this indicates that the trends observed for the single configuration are likely to hold also in the time-averaged picture. Furthermore, the neglect of bulk solvation is justified by the fact that the chromophore is situated in the center of the protein and thus not exposed to the bulk solvent. We note that Drobizhev et al.9 reached the opposite conclusion regarding the direction of the protein-mediated contribution to Δμ. Our comparative analysis strongly suggests that an assumption of a frozen protein environment, such as that employed in ref 9, does not apply in this case due to the intramolecular chargetransfer nature of the S0-S1 transition of the DsRed protein. Parenthetically, we also note that the observed lower sensitivity of the excited state to its environment compared with the ground state hints that the excited state features a smaller polarizability, as supported by previous calculations on a DsRed chromophore model.38 Finally, to understand the principal determinants of the spectral tuning of the red chromophore inside DsRed, we decomposed the total electrostatic potential of the ground-state environment into contributions from the individual amino acids. The results are displayed in Figure 4 for the polar amino acids in the immediate environment of the chromophore. (See Figure 2.) We clearly see that the positively charged Lys163 is a major contributor to the higher electrostatic potential at the phenolic ring in line with its location close to the phenolate oxygen in line with a previous finding.13 The potential difference along the conjugated system is further increased by local contributions generated by the neutral Ser146 and biological water molecule (W238), directly engaged in

Figure 5. Shift of the excitation energy of the S0-S1 transition with − ωWT respect to wildtype DsRed (Δω1 = ωmutant 1 1 ) induced by substitutions of amino acids close to the chromophore, cf. Figure 2.

Several important conclusions can be extracted from Figures 5 and 6. First, it is obvious that the K163G replacement induces a dramatic red shift (0.2 eV) of the excitation energy, as anticipated from the charge-localizing effect of Lys163 illustrated by the potential decomposition analysis discussed above. Because none of the other substitutions leads to 3518



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Figure 6. Shifts of the oscillator strength Δf (length gauge) and 2PA cross section (Δσ2PA) of the S0-S1 transition with respect to wildtype DsRed (ΔX = Xmutant − XWT, X = f,σ2PA) caused by mutations of polar residues located in the vicinity of the chromophore, confer Figure 2.

comparatively large shifts and the set of considered residues covers all charged residues with a formal charge within 8.4 Å (a complete residue-based decomposition of the potential at the atomic positions along the conjugated chain of the chromophore is supplied in Figure S9 in the SI), it can be concluded that Lys163 is the amino acid mainly responsible for the blue-shifting effect of the DsRed protein barrel. The important role played by this residue in the tuning of the 1PA maximum of these RFPs has previously been recognized48,49 and also confirmed by theoretical studies.10,13 Second, the Lys163 residue is also identified as the single residue among the considered to provide by far the strongest 2PA enhancement, as can be explained by its charge state and location in continuation of the conjugation chain of the chromophore, which collectively 01 lead to an increased Δμ and a smaller θμΔμ. In other words, mutation of this residue will presumably cause the desired red shift of the absorption spectrum, however, it will inevitably be accompanied by a reduction of the 2PA activity. This result is supported by the experimentally observed trend of a lower 2PA activity15,16,50 and a simultaneous red-shifted absorption pertinent for the S0-S1 transition of mCherry, mStrawberry,49,51 and their precursor mRFP152 compared with the parental DsRed and corroborates its direct linkage to the mutations K163Q and K163 M (in the two latter), which both lead to the loss of an attractive electrostatic interaction with the phenolic oxygen of the chromophore. Third, the effect of the substitution of Arg95 with glycine reveals a critical feature possibly operating also in other DsRed variants with the same chromophore. The replacement of this conserved residue shows that Arg95 quenches the 2PA intensity. Within the 2SM, this effect can be traced to a decrease in the permanent dipole moment primarily of the excited state. Mutation of this residue is, however, likely to be incompatible with a functional protein, as it has been proposed to be directly implicated in the catalysis of the chromophore formation.53,54 Fourth, we find that the oscillator strength varies only slightly relative to the variations of the 2PA cross section when modifying the protein surroundings. The difference in sensitivity of the extinction coefficient and 2PA cross section to the local electrostatic environment has previously been pointed out on the basis of measurements on the mFruits family of fluorescent proteins.15,16

One can now ponder over which modifications one can make for enhancing the 2PA cross section of the S0-S1 transition without blue-shifting the position of its absorption maximum. Our analysis suggests that an increase in the ground-state permanent dipole moment and thus a potentially associated 2PA enhancement is intimately related to a blue shift of the excitation energy. An alternative route to achieve the desired optical properties consists of modulating the first excited state. One would need to create a mutant that stabilizes the S1 state and simultaneously decreases its permanent dipole moment while affecting the ground state minimally. The F14K mutation proposed in ref 13 to red-shift the absorption and emission spectrum generates a positive contribution to the field at the acylimine group of the chromophore corresponding to its close proximity (cf. Figure 2). As the charge is redistributed toward this region upon excitation, it seems reasonable to expect that the F14K replacement could result in a further displacement of charge, preferably in the excited state, with a resultant lowering of the excited-state dipole moment. Thus, it appears that the F14K variant could also be a candidate for improving the 2PA intensity. In summary, our results show that the protein matrix plays a decisive role in the tuning of the optical properties of the DsRed protein and thereby supports previous appreciations of the significance of the intraprotein field based on experiments9,15,16,48,49 as well as calculations.10,11,13,14 The sequential in silico screening of the electrostatic effects of the amino acids elucidates that the predicted blue shift of the 1PA maximum of the DsRed protein with respect to the bare chromophore is primarily dictated by interactions with the positively charged Lys163. More importantly, this residue is found to provide the largest contribution, among the residues near the chromophore, to the predicted 2PA enhancement inside the protein, although its effect is counteracted by quenching contributions, particularly stemming from the conserved Arg95 residue. On the basis of the well-motivated 2SM, this 2PA gain can be understood by an increased Δμ and a better alignment of μ01 and Δμ mediated by the electric-field contribution along the conjugation chain set up by the Lys163 residue. Accordingly, our results suggest that mutation of this residue will lead to the requested red shift of the absorption spectrum, however, at the expense of a decrease in the 2PA activity. An apparently more 3519



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(5) Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nonlinear Magic: Multiphoton Microscopy in the Biosciences. Nat. Biotechnol. 2003, 21, 1369−1377. (6) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W. Multiphoton Fluorescence Excitation: New Spectral Windows for Biological Nonlinear Microscopy. Proc. Natl. Acad. Sci. U.S.A 1996, 93, 10763−10768. (7) Williams, R. M.; Zipfel, W. R.; Webb, W. W. Multiphoton Microscopy in Biological Research. Curr. Opin. Chem. Biol. 2001, 5, 603−608. (8) Matz, M. V.; Fradkov, A. F.; Labas, Y. A.; Savitsky, A. P.; Zaraisky, A. G.; Markelov, M. L.; Lukyanov, S. A. Fluorescent Proteins from Nonbioluminescent Anthozoa Species. Nat. Biotechnol. 1999, 17, 969− 973. (9) Drobizhev, M.; Tillo, S.; Makarov, N. S.; Hughes, T. E.; Rebane, A. Color Hues in Red Fluorescent Proteins Are Due to Internal Quadratic Stark Effect. J. Phys. Chem. B 2009, 113, 12860−12864. (10) Mochizuki, Y.; Nakano, T.; Amari, S.; Ishikawa, T.; Tanaka, K.; Sakurai, M.; Tanaka, S. Fragment Molecular Orbital Calculations on Red Fluorescent Protein (DsRed). Chem. Phys. Lett. 2007, 433, 360− 367. (11) Taguchi, N.; Mochizuki, Y.; Nakano, T.; Amari, S.; Fukuzawa, K.; Ishikawa, T.; Sakurai, M.; Tanaka, S. Fragment Molecular Orbital Calculations on Red Fluorescent Proteins (DsRed and mFruits). J. Phys. Chem. B 2009, 113, 1153−1161. (12) Sanchez-Garcia, E.; Doerr, M.; Hsiao, Y.-W.; Thiel, W. QM/ MM Study of the Monomeric Red Fluorescent Protein DsRed.M1. J. Phys. Chem. B 2009, 113, 16622−16631. (13) Hasegawa, J.; Ise, T.; Fujimoto, K. J.; Kikuchi, A.; Fukumura, E.; Miyawaki, A.; Shiro, Y. Excited States of Fluorescent Proteins, mKO and DsRed: Chromophore-Protein Electrostatic Interaction Behind the Color Variations. J. Phys. Chem. B 2010, 114, 2971−2979. (14) Sanchez-Garcia, E.; Doerr, M.; Thiel, W. QM/MM study of the Absorption Spectra of DsRed.M1 Chromophores. J. Comput. Chem. 2009, 31, 1603−1612. (15) Drobizhev, M.; Tillo, S.; Makarov, N. S.; Hughes, T. E.; Rebane, A. Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins. J. Phys. Chem. B 2009, 113, 855−859. (16) Drobizhev, M.; Makarov, N. S.; Tillo, S. E.; Hughes, T. E.; Rebane, A. Two-Photon Absorption Properties of Fluorescent Proteins. Nat. Methods 2011, 8, 393−399. (17) Gross, L. A.; Baird, G. S.; Hoffman, R. C.; Baldridge, K. K.; Tsien, R. Y. The Structure of the Chromophore within DsRed, a Red Fluorescent Protein from Coral. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11990−11995. (18) Epifanovsky, E.; Polyakov, I.; Grigorenko, B.; Nemukhin, A.; Krylov, A. I. Quantum Chemical Benchmark Studies of the Electronic Properties of the Green Fluorescent Protein Chromophore. 1. Electronically Excited and Ionized States of the Anionic Chromophore in the Gas Phase. J. Chem. Theory. Comput. 2009, 5, 1895−1906. (19) Bravaya, K. B.; Grigorenko, B. L.; Nemukhin, A. V.; Krylov, A. I. Quantum Chemistry Behind Bioimaging: Insights from Ab Initio Studies of Fluorescent Proteins and their Chromophores. Acc. Chem. Res. 2011, 45, 265−275. (20) Kamarchik, E.; Krylov, A. I. Non-Condon Effects in the Oneand Two-Photon Absorption Spectra of the Green Fluorescent Protein. J. Phys. Chem. Lett 2011, 2, 488−492. (21) Bravaya, K. B.; Khrenova, M. G.; Grigorenko, B. L.; Nemukhin, A. V.; Krylov, A. I. Effect of Protein Environment on Electronically Excited and Ionized States of the Green Fluorescent Protein Chromophore. J. Phys. Chem. B 2011, 115, 8296−8303. (22) Steindal, A. H.; Olsen, J. M. H.; Ruud, K.; Frediani, L.; Kongsted, J. A Combined Quantum Mechanics/Molecular Mechanics Study of the One- and Two-Photon Absorption in the Green Fluorescent Protein. Phys. Chem. Chem. Phys. 2012, 14, 5440−5451. (23) Nifosı ̀, R.; Luo, Y. Predictions of Novel Two-Photon Absorption Bands in Fluorescent Proteins. J. Phys. Chem. B 2007, 111, 14043−14050.

promising, although complex, strategy to optimize the optical properties of DsRed mutants is centered on modulating the electronic structure of the first excited state. Because of the substantial charge redistribution toward the acylimine moiety that occurs upon excitation, a preferential stabilization of the excited state seems to be coupled to a reduction in its permanent dipole moment. The observed shifts upon mutation of the conserved Arg95 give credence to a rational design strategy for concurrent improvement of the 1PA and 2PA properties of the DsRed protein. The theoretical analysis presented here constitutes an important step toward a computational protocol for the rationalization of the color and intensity tuning in fluorescent proteins and other photoactive proteins as well. In particular, it will be interesting to see if a similar protein-environmental tuning mechanism explains the variations of the 2PA intensity among the mFruits mutants of DsRed.

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ASSOCIATED CONTENT

S Supporting Information *

(1) Description of the protein preparation, force-field derivation and details about the PE-DFT calculations. (2) Comparison of the QM/MM-optimized chromophore structure with the crystallographic structure (PDB entry 1ZGO). (3) xyz coordinates of the chromophore structures (QM-vac and QM-protein). (4) Molecular orbital pictures associated with the S0-S1 transition of the bare chromophore structures (QM-vac and QM-protein) and when embedded inside the protein (PEDFT). (5) Components and norm of the total electric field at the positions of the atoms along the conjugated chain of the chromophore. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the DCSC (Danish Center for Scientific Computing). N.H.L. and J.K. thank the Lundbeck Foundation for financial support. J.K. thanks The Danish Councils for Independent Research (STENO and Sapere Aude programmes) and the Villum Foundation for financial support. A.H.S. was supported by the Research Council of Norway through a Centre of Excellence Grant (grant no. 179568/V30).

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REFERENCES

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Molecular-Level Insight into the Spectral Tuning Mechanism of the

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