Quantum Chemical Benchmark Studies of the ElectronicProperties of the Green Fluorescent Protein

Chromophore: 2. Cis-Trans Isomerization in Water

Igor Polyakov,† Evgeny Epifanovsky,*,‡ Bella Grigorenko,† Anna I. Krylov,*,‡ andAlexander Nemukhin†,§

Department of Chemistry, UniVersity of Southern California, Los Angeles, California90089, Department of Chemistry, M.V. LomonosoV Moscow State UniVersity, Moscow119991, Russia, and Institute of Biochemical Physics, Russian Academy of Sciences,

Moscow 119334, Russia

Received March 25, 2009

Abstract: We present quantum chemical calculations of the properties of the anionic form ofthe green fluorescent protein (GFP) chromophore that can be directly compared to the resultsof experimental measurements: the cis-trans isomerization energy profile in water. Calculationsof the cis-trans chromophore isomerization pathway in the gas phase and in water reveal aproblematic behavior of density functional theory and scaled opposite-spin-MP2 due to themulticonfigurational character of the wave function at twisted geometries. The solvent effectstreated with the continuum solvation models, as well as with the water cluster model, are foundto be important and can reduce the activation energy by more than 10 kcal/mol. Strong solventeffects are explained by the change in charge localization patterns along the isomerizationcoordinate. At the equilibrium, the negative charge is almost equally delocalized between thephenyl and imidazolin rings due to the interaction of two resonance structures, whereas at thetransition state the charge is localized on the imidazolin moiety. Our best estimate of the barrierobtained in cluster calculations employing the effective fragment potential-based quantummechanics/molecular mechanics method with the complete active space self-consistent fielddescription of the chromophore augmented by perturbation theory correction and the TIP3Pwater model is 14.8 kcal/mol, which is in excellent agreement with the experimental value of15.4 kcal/mol. This result helps to resolve previously reported disagreements betweenexperimental measurements and theoretical estimates.

1. Introduction

The properties of the green fluorescent protein (GFP), whichconverts blue light to green light, have inspired numerousexperimental and theoretical studies as well as manyimportant applications (see ref 1–3 and references therein).This paper is the second in a series4 that focuses on accuratecalculations of the properties of biological chromophores

with ab initio methods using the model GFP chromophore,4′-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI)anion, as a benchmark system (Figure 1).

The cis-trans isomerization (or Z/E diastereomerization)of the photoswitchable fluorescent chromophores plays anessential role in their photophysical properties. For example,their functionality is believed to be driven by photoinducedcis-trans isomerization inside a protein matrix.5 Kindlingand blinking phenomena, as well as loss of fluorescence yieldof bare chromophores in solution, are also related to thisprocess. In a broader context, the photophysics of GFP issimilar to that of other fluorogenic unsymmetric methine

* Corresponding authors. E-mail: epifanov@usc.edu (E.E.) andkrylov@usc.edu (A.I.K.).

† M.V. Lomonosov Moscow State University.‡ University of Southern California.§ Russian Academy of Sciences.

J. Chem. Theory Comput. 2009, 5, 1907–1914 1907

10.1021/ct9001448 CCC: $40.75 2009 American Chemical SocietyPublished on Web 06/25/2009

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

dyes6–11 and is of interest with connection to organicphotovoltaic materials.

The majority of the experimental and computationalstudies of this process focused on the excited-state dynamics.However, the ground-state electronic potential-energy surface(PES) also needs to be considered for two main reasons. First,after photoisomerization has completed, the photoswitchableprotein returns to its initial state to prepare for the next cycle.The recovery apparently takes place on the ground-state PES.Second, the details of the ground-state isomerization canelucidate the chromophore’s rearrangements along the morecomplicated excited-state route. Despite previous studies ofthe cis-trans photoisomerization of GFP-like chromopho-res12–17 using quantum chemistry modeling,18–24 manyquestions remain unanswered.

The process of cis-trans isomerization of the HBDI anionin aqueous solution was investigated experimentally by Heet al.12 who estimated the free-energy differences and theactivation energies using the NMR technique. The authorsstressed that the activation energy of 15.4 kcal/mol derivedfrom their measurements in aqueous solution is in distinctdisagreement with the results of calculations,18,19 whichestimated that the barrier is above 21 kcal/mol. The relativelylow value of the barrier (as compared to other isomerizationreactions involving exocyclic double bonds) has also beenemphasized in subsequent studies of the isomerization andseveral explanations have been suggested.13,17 For example,thermal isomerization studies of model GFP-like com-pounds13 suggested that different substituent groups mayhave a significant effect on the activation energy by changingthe interaction between two resonance structures of thechromophore. Tolbert and co-workers considered mecha-nisms involving changes in the chemical structure of thechromophore, e.g., addition/elimination pathway.17 No abinitio calculations have been reported so far to resolve thisdisagreement between experimental measurements and theo-retical estimates and to explain the low value of the barrier.

2. Computational Methods

The equilibrium geometries were optimized by densityfunctional theory (DFT) with the PBE0 variant25 of the

Perdew-Burke-Ernzerhof (PBE) hybrid functional26 andby complete active space self-consistent field (CASSCF)(14/12). The cc-pVDZ basis set27 was used in both calculations.The cis-trans isomerization pathways of the chromophorewere studied with the DFT and CASSCF methods. Weestimated the solvent effects by using continuum solvationmodels as well as by explicit treatment of water moleculesin a quantum mechanics/molecular mechanics (QM/MM)scheme. The Cartesian coordinates of optimized structuresalong the isomerization pathway are given in the SupportingInformation.

The methods are outlined below and in the first paper inthis series,4 and the computational details are given in theResults and Discussion section. Multireference second-orderperturbation theory (MRMP2) and dielectric polarizablecontinuum model (D-PCM)33 calculations were carried outwith the PC GAMESS version28 of the GAMESS(US)quantum chemistry package.29 Scaled opposite-spin-MP2(SOS-MP2) calculations were performed with Q-Chem.30

GAMESS(US)29 was employed for C-PCM and surface andvolume polarization for electrostatic (SVPE) approach35

computations. The QM/MM implementation is based on PCGAMESS28 and the Tinker molecular mechanics package.31

2.1. Continuum Solvation Models. To simulate solventeffects on the chromophore’s cis-trans isomerization energyprofile in an aqueous solution, we employ three versions ofthe continuum solvation model:32 D-PCM, C-PCM, andSVPE. In the simplest approach, the D-PCM,33 the watersolvent is treated as a continuous unstructured dielectric witha dielectric constant of 78.39. C-PCM34 is a version of PCMthat takes into account certain corrections in the boundaryconditions of the electrostatic problem in accord with theconductor-like screening model. The SVPE approach35

provides an improved description of the volume polarizationcontributions. This effect can be significant for reactionbarriers, especially for ionic solutes.35

2.2. Effective Fragment Potential-Based QM/MMMethod. Solvent effects can be described explicitly in acombined QM/MM approach based on the effective fragmentpotential (EFP) model.36,37 In this scheme, solvent molecules(water in our case) are represented by effective fragmentsin the MM-part. The fragments affect the Hamiltonian ofthe QM-part (HBDI anion) by their electrostatic potentialsexpanded up to octupole terms. The parameters of these one-electron electrostatic potentials, as well as contributions frominteractions between polarizable effective fragments and theQM-region, are computed in preliminary ab initio calcula-tions of the electronic densities of individual fragments. Theexchange-repulsion potentials, which are combined with theelectrostatic and polarizability terms, are obtained frompreliminary ab initio calculations as well.

The original EFP approach36 treats interactions betweensolvent molecules as EFP-EFP interactions. Studies ofchemical reactions in aqueous solution38,39 have shown thatreplacing the EFP-EFP terms by the empirically calibratedTIP3P potential yields a faster computational scheme forlarge water clusters around the solute.

Figure 1. Chemical structure and atomic labels of the anionicform of the model GFP chromophore HBDI in the cis (top)and trans (bottom) conformations.

1908 J. Chem. Theory Comput., Vol. 5, No. 7, 2009 Polyakov et al.

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

3. Results and Discussion

3.1. Cis-Trans Isomerization Pathway in the GasPhase. On the ground-state PES, conversion between thelower-energy cis-isomer and the higher-energy trans-isomertakes place when the two rings are rotated along the C1I-C1B

bond as shown in Figure 1. The dihedral angleN-C1I-C1B-H, which defines the reaction coordinate, isdenoted as τI.

The shape of the isomerization pathway can be character-ized by: (i) the activation energy Ea defined as the relativeenergy of the transition state (TS) with respect to the cis-isomer; and (ii) the energy difference Etc between the trans-and cis-forms. We began with gas-phase DFT calculationswith the PBE0 functional and the 6-31+G(d,p) basis set andlocated stationary points that correspond to the cis-, trans-and TS structures. The latter is characterized by a singleimaginary frequency of 725i cm-1. The intrinsic reactioncoordinate (IRC) profile was computed by starting steepestdescent pathways from the TS point in both directions alongthe Hessian eigenvector that corresponds to the imaginaryfrequency (shown in Supporting Information).

The results of this calculation are discouraging in twoaspects: the cusp-like shape of the profile (Figure 2), whichis consistent with the large value of the imaginary frequencyat the saddle point, and the value of the activation energyEa(DFT) ) 34.5 kcal/mol, which is more than twice as highas the experimental estimate of 15.4 kcal/mol in aqueoussolution.12 The computed energy difference between thetrans- and cis- structures Etc(DFT) ) 2.3 kcal/mol is inexcellent agreement with the experimental estimate insolution,12 most likely due to error cancellation.

In the CASSCF energy profile (Figure 2), the stationarypoints were fully optimized with CASSCF(12/11)/cc-pVDZ.The points in between representing the minimum-energy path(MEP) were computed by varying the value of the dihedralangle τI and minimizing the energy by relaxing all otherdegrees of freedom. Although this curve does not representthe true IRC path, it is expected to be a good approximationto it. Along with a smoother curvature in the vicinity of the

saddle point (imaginary frequency 77i cm-1, see SupportingInformation), this profile yields a reasonable value of thetrans-cis energy difference Etc(CASSCF) ) 3.5 kcal/mol anda much lower (and closer to the experimental estimate) valueof the activation energy Ea(CASSCF) ) 22.5 kcal/mol.

When calculating the energy profile in the CASSCF(12/11) approximation with a fairly large active space, weperformed careful selection of the orbitals in order to avoiddubious solutions of the variational problem. The finallyoptimized orbitals and the corresponding occupation numbersat selected points along the energy graph are presented inthe Supporting Information. To better understand changesin the electronic structure along the isomerization pathway,we also computed the energy profile with the smallest activespace CASSCF(2/2). Selection of active orbitals in thisapproach was performed on the base of previously optimizedorbitals at the TS point. Then the descent in both directionstoward minimum-energy structures was easy to accomplish.As expected, the corresponding value of the activationenergy, 25 kcal/mol, was slightly larger than that computedwith CASSCF(12/11).

As discussed in more detail below (and illustrated by thepopulation analysis presented in Supporting Information),there is an increase in charge localization at the TS relativeto that the minimum-energy points.

The transition state located with SOS-MP2/cc-pVDZ ischaracterized by an imaginary frequency of 188i cm-1.Starting from that point, the MEP was taken to the cis- andtrans-configurations (Figure 2). Both DFT and SOS-MP2exhibit a cusp at the transition state, which is a manifestationof the multireference character of the ground-state wavefunction. The composition of the CASSCF wave functionin the region discussed below confirms that.

Table 1 presents computed equilibrium geometry param-eters in the bridging region: the C1P-C1B and C1I-C1B

bond lengths and the τP (C2P-C1P-C1B-H) and τI (N-C1I-C1B-H) dihedral angles (Figure 1). The parametersoptimized at the PBE0/6-31+G(d,p), CASSCF(12/11)/cc-pVDZ, and SOS-MP2/cc-pVDZ levels are in agreement withthose obtained by Olsen and Smith23 with SA3-CAS(4/3)/DZP, which stands for CASSCF with the (4/3) active spaceaveraged over three states computed with the DZP basis set.Overall, the geometry parameters at the stationary points arerather insensitive to the level of theory: DFT, large activespace state-specific CASSCF, small active space state-averaged CASSCF, and SOS-MP2 yield bond lengths thatagree within 0.02 Å and angles that agree within 2°.

At both cis- and trans-equilibrium points, the molecule isessentially planar except that the methyl groups naturallyhave out-of-plane atoms. Analysis of the structures revealsthat the difference between C1P-C1B and C1B-C1I is less thanexpected for the structure shown in Figure 1. This can beexplained by considering two resonance structures of theHBDI anion, see figure 2 from ref 4. The interaction betweenthese two structures results in charge delocalization betweentwo oxygen atoms and in scrambling CC bond orders asdiscussed, for example, in ref 23. The C1P-C1B bond acquiresa double-bond character, whereas the order of C1B-C1I bondis reduced. Natural bond orbital (NBO)40,41 charges and bond

Figure 2. Computed energy profiles of cis-trans ground-state isomerization of the HBDI anion in the gas phase: (1)IRC calculated with DFT(PBE0)/6-31+G(d,p); (2) MEP cal-culated with CASSCF(12/11)/cc-pVDZ; and (3) MEP calcu-lated with SOS-MP2/cc-pVDZ.

Protein Chromophore: Cis-Trans Isomerization J. Chem. Theory Comput., Vol. 5, No. 7, 2009 1909

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

orders (see ref 4) are consistent with computed bond lengths.Overall, CASSCF slightly exaggerates bond alternationrelative to DFT (or MP2, see ref 4) in favor of the canonicalstructure (Figure 1).

At the TS, the two rings are nearly perpendicular to eachother (τI ) 87-88°), which disturbs the π system and breaksthe resonance interaction. The bond alternation pattern isreversed such that C1P-C1B becomes shorter than C1B-C1I,suggesting that one of the two resonance structures becomesdominant. All the methods agree on the magnitude of thechange relative to the equilibrium structures: C1P-C1B, whichis longer at the cis- and trans-geometries, becomes shorterby about 0.04 Å at the TS, whereas the C1B-C1I bond is

about 0.07 Å longer at the saddle point. NBO charges (Table2) and the molecular orbital picture (Figure 3) reveal almostcomplete charge localization on the imidazolin ring.

Despite the similarity of the computed geometry param-eters and charge distributions at the minima and the TScomputed with DFT and CASSCF, respective activationenergies differ by more than 10 kcal/mol (34.5 versus 22.5kcal/mol). Note that the corresponding cis-trans energydifferences are very close: 2.3 and 3.5 kcal/mol, respectively.A close inspection of the charges from Table 1 revealsslightly more polar charge distribution for the PBE0 density:the positive charge on the bridge moiety is 0.19 versus 0.13at the CASSCF level. Because of the large energy penalty

Table 1. Geometry Parameters at the Stationary Points for the Gas-Phase Cis-Trans Chromophore Isomerizationa

structure method C1P-C1B C1B-C1I τP τI

cis-isomer PBE0/6-31+G(d,p) 1.404 1.385 0 180CASSCF(12/11)/cc-pVDZ 1.406 1.383 0 180SOS-MP2/cc-pVDZ 1.415 1.397 0 180SA3-CAS(4,3)/DZPb 1.408 1.382 0 180

transition state PBE0/6-31+G(d,p) 1.365 1.458 0.3 88.2CASSCF(12/11)/cc-pVDZ 1.362 1.477 1.0 86.4SOS-MP2/cc-pVDZ 1.374 1.476 0.0 87.6

trans-isomer PBE0/6-31+G(d,p) 1.403 1.392 0 0CASSCF(12/11)/cc-pVDZ 1.402 1.395 0 0SOS-MP2/cc-pVDZ 1.414 1.405 0 0SA3-CAS(4/3)/DZPb 1.407 1.390 0 0

a Distances in Å and angles in degrees. b Ref 23.

Table 2. Cumulative Natural Charges on the Fragments of the HBDI molecule: the Phenyl and Dimethylimidazolin Ringsand the CH bridge Calculated with CASSCF(12/11) and DFT (PBE0 Functional)

CASSCF(12/11)/cc-pVDZ PBE0/6-31+G(d,p)

cis TS trans cis TS trans

Gas Phasephenyl –0.61 –0.20 –0.50 –0.59 –0.30 –0.57bridge 0.13 0.13 0.11 0.10 0.20 0.09imidazolin –0.52 –0.93 –0.61 –0.51 –0.90 –0.52

Solutiona

phenyl –0.85 –0.09 –0.91 –0.63 –0.19 –0.70bridge 0.14 0.08 0.11 0.13 0.16 0.13imidazolin –0.29 –0.99 –0.20 –0.50 –0.97 –0.43

a Calculated with QM/MM: EFP for the solvent-QM part interaction and TIP3P for the water-water interaction.

Figure 3. Frontier valence molecular orbitals of the HBDI anion in the cis-form (left) and at the transition state of the cis-transisomerization path (right).

1910 J. Chem. Theory Comput., Vol. 5, No. 7, 2009 Polyakov et al.

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

due to charge separation in the gas phase, small differencesin ionicity may produce a large effect. Another notabledifference is larger asymmetry in oxygen charges: at theCASSCF level the imidazolin oxygen is by 0.22e morenegative than that of the phenyl oxygen, whereas at the DFTlevel this difference is reduced to 0.15. This suggests a largercontribution of the second resonance structure in theCASSCF wave function, which can also contribute to theenergy difference.

More ionic character of the PBE0 density and the cusp-like shape of the profile (Figure 2) are due to the multicon-figurational character of the wave function at the TS, whichis not adequately described by DFT (or MP2). The CASSCFamplitudes show almost equal weights of the two dominantconfigurations, (HOMO)2 and (HOMO)1(LUMO)1 at the TS.The HOMO and LUMO at the TS differ considerably fromthose at the equilibrium geometry (shown in figure 3 of ref4), they become localized on the imidazolin and phenyl rings,respectively. Thus, (HOMO)2 and (LUMO)2 correspond tothe two charge-localized configurations, and their interactionresults in less ionic electron distribution. The ionicity is alsoreduced by (HOMO)1(LUMO)1.

A stability analysis of the Hartree-Fock wave functionat the TS shows a RHF-UHF (restricted and unrestrictedHartree-Fock) instability with a negative eigenvalue of-0.037. The DFT/PBE0 solution, however, proves to bestable, which means that using the symmetry brokenunrestricted solution to achieve better description of thebarrier will not be useful in this case. Therefore, a multi-configurational approach is necessary not only for describingexcited-state isomerization of the GFP-like chromophoresbut also for modeling isomerization in the ground state.

Although the CASSCF wave function is capable ofcapturing the multiconfigurational character of the wavefunction, it needs to be augmented by dynamical correlationto provide accurate energy differences. We included dynami-cal correlation correction via MRMP2 for the Etc and Ea

energies (at the CASSCF geometries). This yields anactivation energy of Ea ) 26.2 kcal/mol, which is 3.7 kcal/mol higher than that of the CASSCF result.

The CASSCF results represent an improvement over DFT;however, there is still a considerable discrepancy betweenthe theoretical (22-26 kcal/mol) and experimental (15.4 kcal/mol) values for Ea, as noted by the authors of experimental

studies.12,13,17 Below we demonstrate that this discrepancyis resolved when solvent effects are taken into account.

3.2. Cis-Trans Isomerization Pathway in AqueousSolution: Continuum Solvation Models. Continuum sol-vation models32 provide a reasonable starting point inmodeling ground-state isomerization in aqueous solution. Atfirst, two versions of the polarized continuum model (PCM),D-PCM33 and C-PCM,34 were applied to optimize theequilibrium geometry parameters of the cis- and trans-isomers and the TS configuration and to compute the relativeenergies ∆Etc and Ea at the PBE0/6-31+G(d,p) level. Bothmodels produced similar results: ∆Etc ) 2.1 (D-PCM) and2.3 kcal/mol (C-PCM); Ea ) 33.5 (D-PCM) and 34.0 kcal/mol (C-PCM). The geometry parameters and the energiesare close to the gas-phase values obtained with the sameDFT model. These results are fairly stable with respect tothe basis set: upon expanding it to 6-311++G(2d,p), Ea and∆Etc change by less than 1.5 and 0.7 kcal/mol, respectively.Table 3 summarizes solvent effects on Etc and Ea computedusing different approaches.

Next, we considered a new version of the continuumsolvation model, SVPE,35 which has recently been imple-mented in GAMESS(US),29 for the single point calculationsof relative energies ∆Etc and Ea at the gas-phase geometryparameters. At the PBE0/6-31+G(d,p) level, we obtained aconsiderable reduction of the activation energy compared tothe PCM results: Ea ) 24.6 kcal/mol, while the energy ∆Etc

) 2.1 kcal/mol was almost the same as in the PCM model.According to a comment by Chipman,35 the improvementsintroduced in SVPE do affect the reaction barrier estimatedwith the continuum solvation models, especially for chargedsubstrates. The large solvent effect on Ea (9.1 kcal/molreduction relative to the gas-phase value) is consistent withthe more ionic character of the TS (see Table 2).

Finally, we combined the SVPE model with the adequatedescription of the electronic structure of the solute and carriedout the calculations of relative energies with the CASSCF(12/11)/cc-pVDZ description for the chromophore. The quanti-ties, ∆Etc ) 2.6 and Ea ) 9.9 kcal/mol, can now be directlycompared to the experimental free-energy difference of 2.3and 15.4 kcal/mol.12 The reduction of about 15 kcal/mol inEa when going from DFT to CASSCF results within theSVPE solvation model is consistent with the correspondingreduction (13 kcal/mol) in the gas-phase calculations.

Table 3. Cis-Trans Energy Difference ∆Etc and the Energy Barrier Ea for the Cis-Trans Isomerization of the HBDI AnionCalculated at Various Levels of Theory for the Chromophore Molecule and Solvent

method ∆Etc, kcal/mol Ea, kcal/mol

chromophore solvent gas phase solution shift gas phase solution shift

PBE0/6-31+G(d,p) 2.3 34.5D-PCM 2.1 -0.2 33.5 -1.0C-PCM 2.3 +0.0 34.0 -0.5SVPE 2.1 -0.2 24.6 -9.9QM/MMa 5.0 +2.7 26.0 -8.5

CASSCF(12/11)/cc-pVDZ 3.5 22.5SVPE 2.6 -0.9 9.9 -12.6QM/MMa 2.1 -1.4 11.1 -11.4

MRMP2/cc-pVDZ 3.7 26.2

a Calculated with QM/MM: EFP for the solvent-QM part interaction and TIP3P for the water-water interaction.

Protein Chromophore: Cis-Trans Isomerization J. Chem. Theory Comput., Vol. 5, No. 7, 2009 1911

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

Moreover, DFT and CASSCF agree in the magnitude of thereduction of Ea due to solvent (9.1 and 12.6 kcal/mol,respectively). Thus, the absolute value of Ea is overestimatedby DFT due to the multiconfigurational character of theelectronic structure at the TS geometry, which is correctlycaptured by CASSCF.

3.3. Cis-Trans Isomerization Pathway in AqueousSolution: Explicit Solvent Molecules. The relative energies∆Etc and Ea for the HBDI anion inside a cluster of watermolecules were computed using a QM/MM technique. Tobuild the starting point, the chromophore molecule at thegas-phase TS geometry was placed in a sphere of 200 watermolecules using the VMD computer program.42 Afterrunning short molecular dynamics trajectories at varioustemperatures, the energy was minimized using molecularmechanics with the CHARMM force field43 by keeping thesolute species frozen. Then the solvent outside the firstsolvation shell was removed with 49 water molecules com-pletely covering the chromophore remaining in the system. Thissaddle point structure was reoptimized for all geometric degreesof freedom by QM/MM using DFT with the PBE0 functionaland the 6-31+G(d,p) basis for the QM-part (chromophore),EFP36 for QM-solvent interactions, and the empirical TIP3Ppotential for water-water interactions. The steepest descentpathways taken in both directions from the optimized TS leadto the cis- and trans-isomers of the trapped chromophore(Figure 4). The energies obtained with this method, ∆Etc )5.0 kcal/mol and Ea ) 26.0 kcal/mol, are consistent with theDFT/SVPE calculations (Table 3).

The energies of the cis-, trans- and TS structures werealso calculated using CASSCF(12/11) for in the QM-part.The interactions between the chromophore molecule andsolvent and between the water molecules were handled withEFP and TIP3P, respectively. In line with the gas-phase anddielectric continuum model results, the activation energy islower at the CASSCF level relative to those of DFT: ∆Etc

) 2.1 kcal/mol, Ea ) 11.1 kcal/mol.

The relative energies can be further refined by includingthe dynamical correlation effects. In the gas phase, account-ing for dynamical correlation via MRMP2 raises the CAS-SCF activation barrier by 3.7 kcal/mol. Applying thatcorrection, we estimate the activation energy for thecis-trans isomerization of the GFP chromophore in aqueoussolution to be 14.8 kcal/mol. The remaining discrepancy withthe experimental value of 15.4 kcal/mol12 can be partlyattributed to the differences between the potential-energy andthe free-energy barriers. The present QM/MM model withonly 49 explicit water molecules is not sufficient fordescribing the statistical state in solution. This water solvationshell completely covers the chromophore molecule andaccounts for principal environmental effects, but precise free-energy values, along the isomerization pathway, should beestimated by using more appropriate condensed-phase mod-els. As shown, e.g., in ref 44, no considerable changes inconclusions are expected if the dielectric continuum modelis applied on top of the model with explicit water molecules.

4. Conclusion

Calculations of the cis-trans isomerization pathway requirewave functions that are flexible enough to reflect themulticonfigurational character of the transition state. CAS-SCF gives a qualitatively correct curve, whereas DFT andSOS-MP2 fail at the twisted geometries. The gas-phaseCASSCF value of the barrier height is 10 kcal/mol higherthan that of the experimental value; however, the inclusionof solvent effects brings it down to 9.9-11.1 kcal/mol, whichagrees well with 15.4 kcal/mol derived from experimentalmeasurements. Including dynamical correlation correctionyields 14.8 kcal/mol, which is within 0.8 kcal/mol from theexperiment. Good agreement between SVPE and QM/MMcalculations further supports the validity of our results. Alarge solvent effect on Ea is due to the more ionic characterof the TS, where the negative charge is localized onimidazolin ring, which is in contrast to the equilibriumstructure, where the negative charge is delocalized betweenthe two rings. Our calculations help to resolve previous

Figure 4. Cis-trans isomerization of the chromophore insidea cluster of water molecules. Relative energies of the station-ary points are computed with QM(CASSCF(12/11)/cc-pVDZ)/EFP/MM(TIP3P).

1912 J. Chem. Theory Comput., Vol. 5, No. 7, 2009 Polyakov et al.

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

disagreements between theory and experiment with respectto the GFP chromophore isomerization in an aqueoussolution.

Acknowledgment. We thank Alex Granovsky forgenerous help and valuable discussions of this work. Thiswork is supported by the joint grant from the U.S. CivilianResearch and Development Foundation (project RUC1-2914-MO-07) and the Russian Foundation for Basic Research (08-03-91104-AFGIR). I.P., B.G., and A.N. thank the SKIF-GRID program and SKIF-Siberia for providing computationalresources. E.E. and A.I.K. acknowledge the iOpenShellCenter for Computational Studies of Electronic Structure andSpectroscopy of Open-Shell and Electronically ExcitedSpecies (http://iopenshell.usc.edu) supported by the NationalScience Foundation through the CRIF:CRF CHE-0625419+ 0624602 + 0625237 grant as well as through the CHE-0616271 grant (AIK). A.I.K. is grateful to the Institute ofMathematics and its Applications in Minnesota for itshospitality and productive environment during her stay as avisiting professor.

Supporting Information Available: Optimized geom-etry of the HBDI anion and the anion in water, the naturalpopulation analysis, the imaginary frequency mode at theTS, and the CASSCF natural orbitals figures. This materialis available free of charge via the Internet at http://pubs.acs.org.

References

(1) Tsien, R. Y. Annu. ReV. Biochem. 1998, 67, 509.

(2) Zimmer, M. Chem. ReV. 2002, 102, 759.

(3) Stepanenko, O. V.; Verkhusha, V. V.; Kuznetsova, I. M.;Uversky, V. N.; Turoverov, K. K. Curr. Protein Pept. Sci.2008, 9, 338.

(4) Epifanovsky, E.; Polyakov, I.; Grigorenko, B. L.; Nemukhin,A. V.; Krylov, A. I. J. Chem. Theory Comput. 2009; DOI:10.1021/ct903187f.

(5) Henderson, J. N.; Remington, S. J. Physiology 2006, 21, 162.

(6) Chen, R. F. Arch. Biochem. Biophys. 1977, 179, 672.

(7) Steiner, R. F.; Albaugh, S.; Nenortas, E.; Norris, L. Biopoly-mers 1997, 32, 73.

(8) Babendure, J. R.; Adams, S. R.; Tsien, R. Y. J. Am. Chem.Soc. 2003, 125, 14716.

(9) Silva, G. L.; Ediz, V.; Yaron, D.; Armitage, B. A. J. Am.Chem. Soc. 2007, 129, 5710.

(10) Constantin, T. P.; Silva, G. L.; Robertson, K. L.; Hamilton,T. P.; Fague, K.; Waggoner, A. S.; Armitage, B. A. Org. Lett.2008, 10, 1561.

(11) Ozhalici-Unal, H.; Pow, C. L.; Marks, S. A.; Jesper, L. D.;Silva, G. L.; Shank, N. I.; Jones, E. W.; Burnette, J. M.;Berget, P. B.; Armitage, B. A. J. Am. Chem. Soc. 2008, 130,1260.

(12) He, X.; Bell, A. F.; Tonge, P. J. FEBS Lett. 2003, 549, 35.

(13) Hager, B.; Schwarzinger, B.; Falk, H. Monatsh. Chem. 2006,137, 163.

(14) Loos, D. C.; Habuchi, S.; Flors, C.; Hotta, J.; Wiedenmann,J.; Nienhaus, G. U.; Hofkens, J. J. Am. Chem. Soc. 2006,128, 6270.

(15) Yang, J.-S.; Huang, G.-J.; Liu, Yi.-H.; Peng, S.-M. Chem.Commun. 2008, 1344.

(16) Nienhaus, K.; Nar, H.; Heiker, R.; Wiedenmann, J.; Nienhaus,G. U. J. Am. Chem. Soc. 2008, 130, 12578.

(17) Dong, J.; Abulwerdi, F.; Baldridge, A.; Kowalik, J.; Solntsev,K. M.; Tolbert, L. M. J. Am. Chem. Soc. 2008, 130, 14096.

(18) Voityuk, A. A.; Michel-Beyerle, M.-E.; Rosch, N. Chem.Phys. Lett. 1997, 272, 162.

(19) Weber, W.; Helms, V. J. A.; McCammon; Langhoff, P. W.Proc. Nat. Acad. Sci. U.S.A. 1999, 96, 6177.

(20) Levine, B. G.; Martinez, T. J. Annu. ReV. Phys. Chem. 2007,58, 613.

(21) Schafer, L. V.; Groenhof, G.; Klingen, A. R.; Ullmann, G. M.;Boggio-Pasqua, M.; Robb, M. A.; Grubmuller, H. Angew.Chem., Int. Ed. 2007, 119, 536.

(22) Schafer, L. V.; Groenhof, G.; Boggio-Pasqua, M.; Robb,M. A.; Grubmuller, H. PLoS Comput. Biol. 2008, 4,e1000034.

(23) Olsen, S.; Smith, S. C. J. Am. Chem. Soc. 2008, 130, 8677.

(24) Andresen, M.; Wahl, M. C.; Stiel, A. C.; Grater, F.; Schafer,L. V.; Trowitzsch, S.; Weber, G.; Eggeling, C.; Grubmuller,H.; Hell, S. W.; Jakobs, S. Proc. Nat. Acad. Sci. U.S.A. 2005,102, 13070.

(25) Adamo, C.; Barone, V. J. Chem. Phys. 1999, 110, 6158.

(26) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. ReV. B:Condens. Matter Mater. Phys. 1996, 77, 3865.

(27) Dunning, T. H. J. Chem. Phys. 1989, 90, 1007.

(28) Granovsky, A. PC GAMESS. http://classic.chem.msu.su/gran/gamess/index.html (accessed April 27, 2009).

(29) Schmidt, M. W.; Baldridge, K. K. J. A.; Boatz, S. T.; Elbert;Gordon, M. S.; J. H.; Jensen, S.; Koseki; Mastunaga, N.;Nguyen, K. A. S.; Su, T. L.; Windus; Dupuis, M.; Montgom-ery, J. A. J. Comput. Chem. 1993, 14, 1347.

(30) Shao, Y.; Molnar, L. F.; Jung, Y.; Kussmann, J.; Ochsenfeld,C.; Brown, S.; Gilbert, A. T. B.; Slipchenko, L. V.; Levchen-ko, S. V.; O’Neil, D. P.; Distasio, R. A. R. C., Jr.; Lochan,Wang, T.; Beran, G. J. O.; Besley, N. A.; Herbert, J. M.; Lin,C. Y.; Van Voorhis, T.; Chien, S. H.; Sodt, A.; Steele, R. P.;Rassolov, V. A.; Maslen, P.; Korambath, P. P.; Adamson,R. D.; Austin, B.; Baker, J.; Bird, E. F. C.; Daschel, H.;Doerksen, R. J.; Drew, A.; Dunietz, B. D.; Dutoi, A. D.;Furlani, T. R.; Gwaltney, S. R.; Heyden, A.; Hirata, S.; Hsu,C.-P.; Kedziora, G. S.; Khalliulin, R. Z.; Klunziger, P.; Lee,A. M.; Liang, W. Z.; Lotan, I.; Nair, N.; Peters, B.; Proynov,E. I.; Pieniazek, P. A.; Rhee, Y. M.; Ritchie, J.; Rosta, E.;Sherrill, C. D.; Simmonett, A. C.; Subotnik, J. E.; Woodcock,H. L.; Zhang, W.; Bell, A. T.; Chakraborty, A. K.; Chipman,D. M.; Keil, F. J.; Warshel, A.; Herhe, W. J.; Schaefer, H. F.;Kong, J.; Krylov, A. I.; Gill, P. M. W.; Head-Gordon, M.Phys. Chem. Chem. Phys. 2006, 8, 3172.

(31) Ponder, J. W. TINKER-Software Tools for MolecularDesign. http://dasher.wustl.edu/tinker/ (accessed May 7, 2009).

(32) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. ReV. 2005, 105,2999.

(33) Cossi, M.; Barone, V. J. Chem. Phys. 1998, 109, 6246.

(34) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995.

(35) Chipman, D. M. J. Chem. Phys. 2006, 124, 224111.

Protein Chromophore: Cis-Trans Isomerization J. Chem. Theory Comput., Vol. 5, No. 7, 2009 1913

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448

(36) Gordon, M. S.; Freitag, M. A.; Bandyopadhyay, P.; Jensen,J. H.; Kairys, V.; Stevens, W. J. J. Phys. Chem. A 2001,105, 293.

(37) Adamovic, I.; Freitag, M. A.; Gordon, M. S. J. Chem. Phys.2003, 118, 6725.

(38) Nemukhin, A. V.; Grigorenko, B. L.; Topol, I. A.; Burt, S. K.Phys. Chem. Chem. Phys. 2004, 6, 1031.

(39) Grigorenko, B. L.; Rogov, A. V.; Nemukhin, A. V. J. Phys.Chem. B 2006, 110, 4407.

(40) Weinhold, F.; Landis, C. R. Chem. Edu.: Res. Pract. Eur.2001, 2, 91.

(41) NBO 5.0. Glendening, E. D.; Badenhoop, J. K.; Reed, A. E.;Carpenter, J. E.; Bohmann, J. A.; Morales, C. M.; Weinhold,F. NBO 5.0; Theoretical Chemistry Institute, University ofWisconsin: Madison, WI, 2001.

(42) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics1996, 14, 33.

(43) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. S.;Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4, 187.

(44) Nemukhin, A. V.; Topol, I. A.; Burt, S. K. J. Chem. TheoryComput. 2006, 2, 292.

CT9001448

1914 J. Chem. Theory Comput., Vol. 5, No. 7, 2009 Polyakov et al.

Dow

nloa

ded

by U

NIV

OF

SOU

TH

ER

N C

AL

IFO

RN

IA o

n A

ugus

t 18,

200

9Pu

blis

hed

on J

une

25, 2

009

on h

ttp://

pubs

.acs

.org

| do

i: 10

.102

1/ct

9001

448