Role of Zwitterions in Kindling Fluorescent Protein Photochemistry

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On the Role of Zwitterions in Kindling Fluorescent Protein Photochemistry Vladimir A. Mironov, Ksenia B. Bravaya, and Alexander V. Nemukhin J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp5075219 • Publication Date (Web): 03 Nov 2014 Downloaded from http://pubs.acs.org on November 5, 2014

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The Journal of Physical Chemistry

On the Role of Zwitterions in Kindling Fluorescent Protein Photochemistry Vladimir A. Mironov, † Ksenia B. Bravaya, ‡ Alexander V. Nemukhin *,†,§

†

Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3,

Moscow, 119991, Russian Federation ‡

§

Department of Chemistry, Boston University, Boston, MA 02215, USA N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4,

Moscow, 119334, Russian Federation

* Corresponding author: Prof. Alexander Nemukhin, Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow, 119991, Russian Federation Phone: +7-495-939-10-96 E-mail: [email protected]; [email protected]

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Abstract Kindling fluorescent protein (KFP) one of the chronologically first members of photoswitchable colored proteins from the GFP family, increasingly attracts efforts from experimental and theoretical sides. Ambiguous conclusions in solving puzzles of photochemistry of KFP and of its parent natural protein asFP595 are partially explained by lack of reliable theoretical data on chromophore properties in the electronically excited state. We report the results of state-of-the-art quantum chemistry calculations of the structure and energy of the KFP chromophore, 2-acetyl-,4-(phydroxybenzylidene)-1-methyl-5-imidazolone (AHBMI), both in the ground and excited states. Ground state equilibrium structures of anionic and zwitterionic protonation states of AHBMI were computed by the conventional MP2 method while excited state structures were characterized by the extended multireference perturbation theory method XMCQDPT2 including optimization of geometry parameters at this level. In particular, the computational results demonstrate that basicity of the N2 nitrogen atom of imidazolinone ring should noticeably increase upon electronic excitation thus affecting excited state proton transfer events in proteins. The results of these simulations as well as of QM/MM calculations for model protein systems evidence that KFP conformations with the zwitterionic chromophore are hardly expected to occur in the ground state, but may be populated upon excitation.

Keywords: fluorescent proteins; organic chromophores; GFP; excited states; proton transfer; zwitterions; quantum chemistry; QM/MM


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Introduction Chromoprotein asFP595 and its Ala143Gly mutated variant called kindling fluorescent protein (KFP)

1-7

belong to the species for which photoinduced cycling between conformations containing

trans and cis chromophore isomers has been proposed. This reversible photoswitching is highly attractive, in particular, for developments in optical microscopy much below the diffraction limit. The chromophore of asFP595 (or KFP) shown in Fig.1 is formed as a result of autocatalytical cyclization and oxidation of the Met63-Tyr64-Gly65 residues. Unlike many fluorescent proteins,8,9 chromophore maturation involves additional oxidation and hydrolysis steps, leading to the backbone cleavage between 62 and 63 residues. As a result the chromophore’s conjugated system is elongated including an extra π-type bond CA1=O1.

Figure 1. Molecular model of the KFP chromophore. Carbon atoms are colored in green, oxygen in red, nitrogen in blue. Few nearby molecular groups including a fraction of the Glu215 side chain (cycled by a dashed line) are also shown. The inset demonstrates the chemical formulae of the chromophore. 3 ACS Paragon Plus Environment


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Fig. 1 shows the chromophore in deprotonated anion trans form (with respect to mutual orientation of the C2-O2 and CB2-H groups). This particular species is believed to occur in the crystal structures

3-5

of the dark state of proteins. Possible photoinduced transformations leading to

an increase of fluorescence are attributed to the trans-cis conversion of the chromophore; however, details of the mechanism are still under debates.6,10-15 A model suggested in the computational works of Schäfer et al.12,13 puts forward the zwitterionic chromophore as a main player. As seen in Fig.1 the side chain of Glu215 is close to the chromophore, and a proton between OE2 oxygen of Glu and N2 nitrogen of the chromophore can migrate between these two sites. If proton is captured by the chromophore, the latter appears as a zwitterionic species. Quantum mechanical – molecular mechanical (QM/MM) calculations by Schäfer et al.12 based on modest theoretical approaches in QM (RHF/3-21G(d) ground state geometry optimization followed by ZINDO and TD-DFT estimates of vertical excitations) prompted the authors to attribute the observed absorption band of the dark state of asFP595 to excitation from the ground state conformation of the protein with the trans zwitterionic chromophore. The authors estimated pKa of the imidazolinone N2 center of the trans chromophore in aqueous solution as 9.1 favoring formation of zwitterion in the ground state. Correspondingly, a kindling mechanism in asFP595 has been formulated assuming, in particular, photoinduced trans-cis isomerization of the zwitterion inside the protein cavity followed by sequential changes in protonation states of the chromophore.12 However, this mechanism seems to be at variance with results of previous and present computational approaches. In QM/MM calculations described by Grigorenko et al.10,11 also based on the RHF equilibrium structures, a larger fraction of KFP was included to the model system than in Ref.(12). A stationary point on the ground state potential energy with the zwitterionic chromophore, i.e., corresponding to the system in which a proton was shifted from Glu215 to the trans anionic chromophore (Fig.1),


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was located; however, the energy of such conformation was 1.4 kcal/mol higher than that of the structure with the anionic chromophore. Calculations of pKa values for the asFP595 chromohore in aqueous solution were carried out by Nemukhin et al.16 using conventional quantum based computational protocols at the appropriate accuracy level. The reported pKa value for the imidazolinone N2 center (trans form) of 4.6 was consistent with the estimates for the green fluorescent protein (GFP) chromophore performed by Scharnagl and Raupp-Kossmann,17 but twofold smaller than that reported in Ref. (12). Such small pKa value contradicts with the formation of the zwitterion by proton transfer from Glu215 to the chromophore. In recent paper,

18

Topol et al. applied a computational strategy, in a sense, resembling that of

Ref.(12), to simulate structures and optical spectra of the chromophore containing pockets of asFP595. The authors constructed a large molecular cluster, optimized its geometry parameters, this time, applying a more appropriate level (DFT at the ground state and CIS at the excited state) and used ZINDO for calculations of transition energies. Excellent agreement with the experimental results provides support to an assignment of the observed absorption and emission bands to the anionic form of the asFP595 chromophore, but not to the zwitterionic form. Structures and spectra of the KFP (or asFP595) chromophore in vacuo and in solution were studied in several works.19-24 The most comprehensive description of the anionic, zwitterionic and neutral (in the latter case, the anionic chromophore is protonated over the O3 center, Fig.1) forms in both trans and cis conformations is given by Bravaya et al.24 Equilibrium geometry parameters were optimized in the density functional theory approach PBE0/(aug)-cc-pVDZ, and the vertical excitation energies were computed using a version of the multiconfigurational quasidegenerate perturbation theory (MCQDPT2)

25,26

based on the CASSCF(14/12) and CASSCF(16/14)



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wavefunctions. These calculations evidence that the most red-shifted absorption bands originate from the anionic form of the chromophore. The motivation of the present study was twofold. Firstly, we intended to apply an extended version of MCQDPT2 (XMCQDPT226 ) to characterize excited state structures of the chromophore. Development and implementation of the numerical gradient code of the XMCQDPT2 method the Firefly package

27

26

to

promise an accurate treatment of dynamic electron correlation when

considering excited state properties.28,29 Secondly, we intended to re-visit ground state configurations of the protein model systems with zwitterionic chromophores by using QM/MM methodology with an improved description of quantum subsystems. These results allow us to review the role of zwitterionic forms of the chromophore when considering photochemistry of KFP and asFP595. Models and Methods Ab initio quantum chemistry calculations were carried out for the model molecule, 2-acetyl-4(p-hydroxybenzylidene)-1-methyl-5-imidazolone (AHBMI), constructed from the parent species (see Fig.1) by cutting the CA1-C and N3-C bonds and adding the terminal -CH3 groups to CA1 and N3 atoms of the chromophore. We considered the anionic and zwitterionic forms in the trans conformations corresponding to the dark state of KFP. Ground state equilibrium structures of AHBMI in vacuo were optimized with the MP2 method. The obtained structures were used as starting points for excited state calculations. First, we optimized geometry parameters of the anionic and zwitterionic variants of AHBMI in the S1 state using the state-specific (SS) CASSCF approach. The active space included 11 orbitals populated by 12 electrons, i.e., the SS-S1-CASSCF(12/11) option was explored. The obtained structures were then re-optimized by using the XMCQDPT2 method26 based on the SS-S1-CASSCF(12/11) reference. Vertical and adiabatic S0→S1 and S1→S0 transition energies for the anionic and zwitterionic protonation states of AHBMI were computed 6 ACS Paragon Plus Environment

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with the XMCQDPT2 method based on the state averaged (SA) reference SA2-CASSCF(12/11). The cc-pVDZ basis set was used in all these calculations. To estimate the difference in the pKa values attributed to imidasolinone nitrogen N2 of the chromophore in the ground S0 and excited S1 states we considered the corresponding deprotonation energies as illustrated in Fig.2 (see, e.g., Ref.(30)). A simplified formulae used in this work ignores contributions from the vibrational zero point energies: pK (S ) − pK (S ) ≈ (ΔE (AN) − ΔE (ZW)) ∕ (2.3 RT)

S1 '()*

+,- (./)

(./)

# # (./)

# # ()

+,- ()

$%& ()

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'()*

$%#

S0

ZW

−

!

"

!

AN

Figure 2. The energy diagram illustrating estimates of changes in the pKa values of imidasolinone nitrogen N2 upon excitation of the chromophore.



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QM/MM geometry optimizations and transition energies were performed using the mechanical embedding

31

and electronic embedding variants, respectively, of the QM/MM approach.

32

In the

latter case a balanced QM electron density polarization (in both ground and excited state) by the electrostatic field of the rest of the proteins was taken into account. The QM subsystem included the chromophore and the nearby aminoacid residues and water molecules forming the hydrogen bond network around the chromophore (Fig.3). More specifically, the QM part consisted of the chromophore, the side chains of His197, Glu215, Ser158, Cys62, Arg92 and three water molecules. Initial coordinates of heavy atoms were taken from the crystal structure (PDB ID: 2A504) corresponding to the dark state of asFP595. Geometry parameters of the QM part were optimized in the density functional PBE0/(aug)-cc-pVDZ approximation. Diffuse functions were added to the oxygen atoms only. The MM part was described by the force field parameters from the CHARMM library. Artificial charge at the QM-MM interface residue arising due to the truncation was eliminated by redistributing the removed charges over the all atoms of the residue. Upon geometry optimization molecular groups located farther than 5 Ǻ from the QM part were kept fixed as in the crystal structure. To compute positions of optical spectral band maxima, the XMCQDPT2 approach26 based on CASSCF(16/14) reference was applied in the QM subsystem. To avoid artificially large polarization effects at the QM/MM boundary, the partial charges on few atoms located close to the QM part were set to zero and their contributions to the one-electron part of the Hamiltonian were excluded. All calculations were carried out with the Firefly quantum chemistry package.27


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Figure 3. Molecular groups included to the QM subsystem in QM/MM calculations.

Results 1. Equilibrium structures of AHBMI: importance of electron correlation All computed equilibrium structures (except that of the zwitterion excited state) correspond to perfectly planar chromophore conformations. The electronic structure of GFP-like chromophores may be represented by several resonance patterns,33 the most important of which for the KFP (or asFP595) chromophore are illustrated in Fig.4. Computational results obtained for equilibrium bond lengths and corresponding bond orders (shown in Tables S1, S2 in Supporting Information) are consistent with this resonant π- electron density delocalization



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Figure 4. Chemical resonance structures for anionic AHBMI. Geometry parameters of the anionic and zwitterionic forms of AHBMI were optimized in the MP2/cc-pVDZ approach in the ground state and the XMCQDPT2/cc-pVDZ approach in the excited state. Correspondingly, we have an opportunity to compare ground state and excited state geometry parameters obtained at a balanced theoretical level: namely, within the perturbation theory to the second order and the same basis set. We present the most important interatomic distances optimized with the MP2 or XMCQDPT2 approaches for anion and zwitterion in Fig.5. Corresponding changes in equilibrium geometry parameters noticed upon the S0→S1 excitation (the top and bottom rows in Fig.5) as well upon protonation of the anionic chromophore (left column in Fig.5) are also specified. The ground state anionic and zwitterionic structures (left column in Fig.5) are better consistent with a quinoid-type π-bond conjugation pattern with a shorter CG-CB2 bond compared to CB2-CA2 (the right-side structure in Fig.4). This result agrees with previous considerations of AHBMI.23,24 Upon excitation (right column in Fig.5), the balance is shifted toward another structure (the left-side structure in Fig.4) with alternating CG-CB2 and CB2-CA2 bond lengths. Changes in bond lengths upon protonation account up to 0.09 Å (the CB2-N2 bond in zwitterion). Geometry changes upon protonation of the anionic chromophore are small enough and do not exceed 0.03 Å.


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Figure 5. Equilibrium interatomic distances (Å) and their changes upon excitation and protonation. The ground state structures were optimized in the MP2/cc-pVDZ approximation; excited state structures – in the XMCQDPT2/cc-pvDZ approximation. The top row illustrates geometry changes upon excitation of the anionic chromophore. The left column shows changes upon protonation of the anionic chromophore. The bottom row illustrates geometry changes upon excitation of the zwitterionic chromophore. Differences smaller than 0.01 Å are not shown in the diagrams. Since geometry optimization of organic dyes in the excited state within the CASSCF approach is a very popular strategy (for the GFP-like chromophores see, e.g. Ref.(34)), it is important to estimate contributions from dynamical correlation missing in the CASSCF scheme. To this goal we compare the results of SS-CASSCF and XMCQDPT2 approaches for the excited state optimization. Fig.6 shows such comparison for the anionic form, Fig.7 – for the zwitterionic form. In both figures the top values refer to the XMCQDPT2 results, bottom values to the CASSCF data. 11 ACS Paragon Plus Environment


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Figure 6. Comparison of equilibrium geometry parameters of the excited state structure of the anionic form of AHBMI. Distances between heavy atoms of the skeleton are given in Å: top values XMCQDPT, bottom – CASSCF.

Figure 7. Comparison of equilibrium geometry parameters of the excited state structure of the zwitterionic form of AHBMI. Distances between heavy atoms of the skeleton are given in Å: top values XMCQDPT, bottom – CASSCF.


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We notice that differences in bond lengths between MP2/XMCQDPT2 and CASSCF equilibrium structures are quite notable and reach 0.05 Å. Account of dynamical correlation provides systematical improvement over CASSCF results. For example, the six-membered ring in the anionic chromophore in the excited state shows the quinoid character in the CASSCF approach, while the quinoid motive is not so pronounced in the XMCQDPT2 approximation. Another result refers to the differences in the lengths of the anionic chromophore bridge bonds in the excited state. In the CASSCF approach the CG2-CB2 and CA2-CB2 bond lengths are almost equal, while in the XMCQDPT2 approach the latter is noticeably shorter. It is known that chromophore de-excitation proceeds through the twisted chromophore structures.35 Lengths of the chromophore bridge bonds are important parameters that determine the relative barriers of these processes. Therefore, the lack of dynamical correlation may lead to incorrect predictions in the studies of twisted chromophore structures.

2. Absorption and emission spectral band maxima of AHBMI in vacuo The vertical excitation and emission energies referred to the respective equilibrium geometry configurations of AHBMI and corresponding wavelengths computed in the perturbation theory approaches both at the stage of geometry optimization and of energy calculations are shown in Fig.8.



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Figure 8. Absorption and emission energies and corresponding wavelengths computed for the zwitterionic and anionic forms of AHBMI in the XMCQDPT2/cc-pVDZ approximation at the geometry configurations optimized in the MP2/cc-pVDZ (S0) and XMCQDPT2/cc-pVDZ (S1) approaches.

The S0 → S1 excitation energies of trans anionic and zwitterionic AHBMI computed with different methods are listed in Table 1. The method of calculation of energy differences and the method of geometry optimization in the ground state S0 are cited in the second column. First of all, we note that the approaches to compute excitation energies which are based on different variants of perturbation theory (rows № 1-5) result in consistent data for the most studied anionic species: the corresponding values are between 2.17 and 2.46 eV. Comparison of rows № 5 and 6 illustrates a known observation that neglect of dynamical correlation leads to serious errors of about 1 eV. Differences due to variations in the ground state geometries (DFT, MP2, CASSCF, cf. rows № 3, 4, 5) are noticeable but not critical. 14 ACS Paragon Plus Environment

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Table 1. Excitation energy, eV, and corresponding wavelengths (nm) for the trans AHBMI chromophore in the anionic and zwitterionic forms. № Method 1

Anion

N-electron valence state PT(8/7)//DFT(B3LYP)

Zwitterion

Ref.

2.17 (571)

(22)

2.41 (514)

(23)

2.28 (543) 2.63 (470)

(24)

2.33 (533) 2.74 (452)

this

basis set: 6-31G(d) 2

multi-reference-multi-state PT2 //SA3-CASSCF(4/3) basis set: DZP

3

aug-MCQDPT2/SA2-CASSCF(16/14)//DFT(PBE0) basis set: (aug)-cc-pVDZ

4

XMCQDPT2/SA2-CASSCF(12/11)//MP2 basis set: cc-pVDZ

5

work

XMCQDPT2/SA2-CASSCF(12/11)//SS-CASSCF(12/11)

2.46 (504) 2.63 (471)

basis set: cc-pVDZ 6

this work

SA2-CASSCF(12/11)//SS-CASSCF(12/11)

3.35 (370) 3.50 (354)

basis set: cc-pVDZ

this work

7

ZINDO//DFT

2.23 (556)

(18)

8

ZINDO//MP2

2.20 (564) 2.02 (614)

this work

ZINDO technique (rows № 7, 8) results in estimates of the anionic chromophore excitation energies that are close to the values obtained with more advanced approaches. Yet, the approach fails to reproduce the higher level data for the zwitterionic species (row № 8, values distinguished in bold). Analysis of compositions of multiconfigurational wavefunctions shows that the excitation S0 → S1 in the case of anion can be predominantly characterized as a simple HOMO-LUMO transition, while other contributions (including two-electron terms) are essential for zwitterion leading to a strong mixing of S1 and S2 states at the ground state geometry configuration of a molecule. Such 15 ACS Paragon Plus Environment


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difficulties in description of electronic structure changes upon excitation of specific protonation forms of GFP-like chromophores are documented.36-41 The zwitterionic form of the KFP chromophore belongs to the same category, and application of ZINDO in this case leads to a wrong ordering of computationally predicted absorption band maxima for the anion and zwitterion species. The experimental absorption band maximum 520 nm for the KFP chromophore in aqueous solution at neutral pH reported by Yampolsky et al.19 seems to be better consistent with the anionic species (see Table 1). In Table 2 we collect computational results for the S1 → S0 emission. The theoretical level, at which both geometry optimization and energy estimates are carried out in the perturbation theory approaches, is represented by data in the upper row (also, in Fig. 8). We may conclude that the contributions to the transition energies due to effects of dynamical correlation upon excited state geometry optimization are about 0.1 eV (cf. rows 1,2), while the contributions from dynamical correlation upon calculations of energy differences are large enough: 0.4 – 0.7 eV (cf. rows 2,3). Interestingly, the ZINDO method results in fair estimates for both anionic and zwitterionic species. In the latter case, a reasonable performance is easily explained. Upon geometry relaxation on the excited state potential surface towards the S1 minimum energy point, the mixing of S1 and S2 states occurring near the S0 minimum is removed, and the HOMO-LUMO character of the vertical transition well described by ZINDO is restored.


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Table 2. Emission energy, eV, and corresponding wavelengths (nm) for the trans AHBMI chromophore in the anionic and zwitterionic forms. Method

Anion

XMCQDPT2/SA2-CASSCF(12/11)//XMCQDPT2/SS-CASSCF(12/11)

Zwitterion

2.14 (580) 1.67 (744)

basis set: cc-pVDZ XMCQDPT2/SA2-CASSCF(12/11)//SS-CASSCF(12/11)

2.26 (550) 1.74 (712)

basis set: cc-pVDZ SA2-CASSCF(12/11)//SS-CASSCF(12/11)

2.99 (415) 2.13 (582)

basis set: cc-pVDZ ZINDO//CIS/6-31+G(d,p) 18

2.19 (565)

ZINDO//XMCQDPT2/SS-CASSCF(12/11)/cc-pVDZ

2.10 (588) 1.61 (770)

3. Estimates of pKa*(N2) Using the strategy illustrated in Fig.2 we calculated the adiabatic energy differences ∆E0-0 (AN) and ∆E0-0 (ZW) and converted these values to pKa units. At the highest theoretical level applied in this work we obtain (∆E0-0 (AN) - ∆E0-0 (ZW)) = 4.8 kcal/mol, and correspondingly, the nitrogen atom N2 of imidazolinone ring becomes more basic by 3.5 pKa units upon excitation. Using ground state pKa of 4.6 18 and computed here ∆pKa= pKa*-pKa of 3.5 we arrive to pKa*(N2) value of 8.1. This value is consistent with the possible formation of the zwitterion in the electronically excited state. We checked how our estimates depend on computational methodology: ∆pKa = +4.2 for the CASSCF optimized structures and ∆pKa =4.0 for the mixed approach, XMQCDPT2/MP2 for S0 state and XMCQDPT2/CASSCF for S1 state. Use of the CASSCF method to calculate ∆E0-0 energies results in too high values of ∆pKa: +7.0 for SA2-CASSCF/CASSCF and SA217 ACS Paragon Plus Environment


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CASSCF/PT2 approaches. Therefore, accuracy of the pKa change depends mainly on the method of energy calculation. Scharnagl and Raupp-Kossmann calculated ∆pKa for the N2 atom in the model GFP chromophore (4-(p-hydroxybenzylidene)imidazolidin, or HBI) using only vertical absorption energies in the Förster thermodynamic cycle and obtained ∆pKa (N2) = +5.3.17 The equilibrium structures were obtained using RHF-SCF+CISD method with a small active space, which is similar to the CASSCF approach applied in our work. The energies of electronic transitions were estimated with the INDO/S method. It should be noted that, at least for the case of KFP chromophore, the S1 state of zwitterion at the S0 equilibrium structure carries a significant contribution of double excitations and thus the results of CIS-based ZINDO/S method may lead to a significant error. Also, in addition to large numerical deviations, the sign ∆pKa estimate based on absorption energies only is incorrect. The main reason of that is a large Stokes shift obtained in our work for the zwitterionic KFP chromophore. 4. Results of QM/MM simulations QM/MM calculations for the model systems specified in Models and Methods allowed us to locate several equilibrium geometry configurations on the S0 ground state potential energy surface and to estimate vertical excitation energies S0 → S1. First of all, this is the structure with the trans anionic form of the chromophore shown in Fig.1. The computed absorption band maximum corresponding to 561 nm (2.21 eV) perfectly matches the experimental values 568 nm in asFP595 or 575 nm in KFP.2 The previously reported values obtained with the ZINDO//DFT method for a model cluster described in Ref.(18) are also close (572 nm or 2.16 eV) to our results. We could not locate a stationary point on potential energy surface describing the minimum energy configuration in the case of the zwitterionic trans isomer. In this structure, the nearby Glu215 side chain is preferably protonated excluding location of the proton on the chromophore. 18 ACS Paragon Plus Environment

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We manually prepared a structure in which the anionic chromophore was in the cis form and reoptimized geometry parameters in QM/MM minimization. The computed excitation energy for the resulting structure is 576 nm (2.15 eV). We also located the minimum corresponding to the cis zwitterionic form (see Fig.9). Unlike the system with the trans chromophore, here the side chain of Glu215 is arranged in such a way that protonation of chromophore’s nitrogen is possible due to a longer distance between carboxyl group of Glu215 and the N2 atom of the chromophore. However, the energy of the optimized model system with the zwitterionic cis isomer is 7.3 kcal/mol higher than that with the cis anionic chromophore. Computed zwitterion vertical excitation energy corresponds to the band maximum at 528 nm (2.34 eV), considerably blue-shifted with respect to the band assigned to the anionic chromophores.

Figure 9. Fragment of QM/MM optimized structure with zwitterion in the cis form. Distances between heavy atoms and hydrogen atoms are given in Å. Values in parentheses refer to the anionic form of the chromophore. 19 ACS Paragon Plus Environment


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Discussion Possible role of the zwitterionic forms of the chromophore in photochemistry of GFP-like proteins has been discussed since the very first studies of these species.42-44 Although Raman spectroscopy investigations of the GFP chromophore by Bell et al.45 showed no evidence of zwitterions, their formation was not been ruled out. Wild type asFP595 and its variant Ala143Gly (KFP) were the two members of the GFP family for which the formation of zwitterion was strongly advocated for. The results of computational studies of asFP595 photoswitching mechanism by Schäfer et al.12 were interpreted by the authors in favor of possible formation of zwitterions both in the ground and excited states. The authors constructed a molecular model starting from the crystal structure (PDBid: 2A50),4 relaxed the model by MD simulation followed by brief QM/MM (HF/321G(d), ONIOM) minimizations with the chromophore only in the QM subsystem. The ZINDO method was used to compute the vertical excitation energies for the subsystem composed of the chromophore and the side chains of six nearby residues (including Glu215, see Fig.1).12 These calculations predicted that the most red-shifted absorption in the dark state of the protein at 568 nm 2 (2.18 eV) should be assigned to the chromophore in the trans-zwitterionic form (the corresponding computational data were 538 nm or 2.30 eV), while absorption of the trans-anionic chromophore’s form corresponded to 507 nm (2.44 eV).12 Based on these estimates a mechanism of photoswitching in asFP595 was suggested,12,13 in which a starting ground state conformation of the protein assumed the zwitterionic form of the chromophore. Our most accurate estimates of vertical excitation and emission energies are close to the experimental band maxima for proteins (asFP595 and KFP)2,6 and also to that of AHBMI in solution.19 Excitation energy of the zwitterionic chromophore is significantly blue-shifted in comparison to the anionic form, and the experimental absorption spectra of Ref.(19)

has no

matching peaks. According to our QM/MM simulations, the zwitterionic forms may correspond to 20 ACS Paragon Plus Environment

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shallow local minima on the ground state potential surface lying higher in energy than the anionic states. One of the reasons of ambiguous conclusions in previous papers may be attributed to ZINDO problems in describing excitations of zwitterionic species. According to the first ZINDO estimates,42 the excitation energy of the GFP chromophore molecule (HBI) in vacuo should be the smallest one for the zwitterionic form. The same conclusion refers to the calculation results for the KFP chromophore described here and in the previous papers.12,20 In Results, we provided a plausible explanation for a poor performance of ZINDO in this particular case. Unlike the ground state case, formation of zwitterions in the excited electronic state can be facilitated by noticeable increase of basicity of the imidasolinone nitrogen N2 upon excitation, as demonstrated in the present work. It is also shown here that emission from the zwitterionic species should be considerably red-shifted compared to absorption; separation of the corresponding wavelengths amounts up to almost 300 nm (from 452 to 744 nm, Tables 1,2). In this respect, we cite another computational paper,46 in which QM/MM simulations of the KFP properties have predicted emission at 680 nm from the system with the zwitterionic chromophore. Finally, we mention the role of zwitterions in the process of chromophore’s photoswitching in KFP. We showed recently 15 that the chromophore trans-cis isomerization in the ground state can be explained by considering anionic forms only; the corresponding free energy profiles are consistent with the kinetic experimental data. Analysis of the photo-induced trans-cis isomerization in KFP is a topic of our current project, but preliminary data do not assume possible involvement of zwitterions.



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Conclusion The results of the present calculations as well as the analysis of previous studies allow us to conclude that the zwitterionic forms of the chromophore do not play a role in the ground state properties of the chromoprotein asFP595 and its variant KFP. On the other hand, formation of a zwitterion due to excited state proton transfer from the neighboring Glu residue can be a likely event because of increasing basicity of the imidasolinone nitrogen N2 upon excitation.

Acknowledgments. We thank Prof. A. Savitsky, Dr. B. Grigorenko and Dr. A. Bochenkova for discussion and advice. This work was supported in part by the Program on Molecular and Cell Biology from the Russian Academy of Sciences and by the Russian Foundation for Basic Research (project 13-03-00207). We acknowledge the use of supercomputer resources within the XSEDE supported project TG-CHE140041.

Supporting Information Available: Supporting Information to the paper includes tables with all computed bond lengths and bond orders (Table S1, S2) as well as with atomic charges (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org.


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Role of Zwitterions in Kindling Fluorescent Protein Photochemistry

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