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Thermal Isomerization of the Chromoprotein asFP595 and its Kindling Mutant A143G: QM/MM Molecular Dynamics Simulations Vladimir A. Mironov, Maria G. Khrenova, Bella L Grigorenko, Alexander P Savitsky, and Alexander V. Nemukhin J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 01 Oct 2013 Downloaded from http://pubs.acs.org on October 2, 2013

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The Journal of Physical Chemistry

Thermal Isomerization of the Chromoprotein asFP595 and its Kindling Mutant A143G: QM/MM Molecular Dynamics Simulations Vladimir A. Mironov,a,* Maria G. Khrenova,a Bella L. Grigorenko,a,b Alexander P. Savitsky,c Alexander V. Nemukhina,b a

Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, 1/3, Moscow, 119991, Russian Federation

b

N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4, Moscow 119334, Russian Federation c

A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, 119071, Russian Federation

*

[email protected] 1

ACS Paragon Plus Environment


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ABSTRACT

Chromoprotein asFP595 and its A143G variant called kindling fluorescent protein (KFP) are among the chronologically first species for which trans-cis chromophore isomerization has been proposed as a driving force of photoswitching. In spite of long-lasting efforts to characterize the route between protein conformations referring to the trans and cis forms of the chromophore the molecular mechanism of this transformation is still under debates. We report the results of computational studies of the trans-cis isomerization of the anionic and neutral chromophore inside the protein matrices in the ground electronic state for both variants, asFP595 and KFP. Corresponding free energy profiles (potentials of mean force) were evaluated by using molecular dynamics simulations with the quantum mechanical – molecular mechanical (QM/MM) forces. The computed free energy barrier for the cis-trans ground state (thermal) isomerization reaction is about 2 kcal/mol higher in KFP than that in asFP595. These results provide interpretation of experimental studies on thermal relaxation from the light-induced activation of fluorescence of these proteins and correctly show that the A143G mutation in asFP595 noticeably increases the life time of the fluorescence species.

Keywords: fluorescent proteins; thermal isomerization; QM/MM; ab initio molecular dynamics; potential of mean force

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INTRODUCTION Chromoprotein asFP595 and its A143G mutated variant called KFP1,2 are among the chronologically first representatives of the photoswitching fluorescent proteins for which cycling between trans and cis chromophore isomers has been proposed. The native form of asFP595 corresponds to the dark or “off”-state which can be transformed to the bright or “on”-state when exposed to the intense green light (λmax = 568 nm) irradiation.1,3–5 The “on”-state can be switched back by a flash of the blue light (λ = 450 nm),1,3,4 or by a thermal conversion within several seconds.1,3,4 In crystals, this cycle can be repeated several times with only a minor loss in fluorescence.3 In solution experiments, cycling between “off” and “on” states can be observed by varying pH in the range of neutral-alkaline values.6 These kindling properties of asFP595 can be enhanced when its monomeric A143G mutant (KFP) is considered.1,2 X-ray analysis of the crystals of another mutant (A143S) of asFP595 explicitly demonstrates that the chromophore isomerizes from trans to cis state upon absorption of green light.3 According to the current working hypothesis, photoactivation of asFP595 and KFP, initially containing the trans chromophore in the “off” state, leads to the trans-to-cis chromophore isomerization while thermal relaxation restores species with the trans form (Fig. 1).

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Figure 1. Trans- and cis-isomers of the asFP595 (or KFP) chromophore presumably involved in photoswitching. The inset illustrates the dihedral angles that can be used as reaction coordinates to characterize the isomerization pathway. In general terms, cis−trans photoinduced isomerization is considered as a key process in the photoswitching of several fluorescent proteins from the green fluorescent protein (GFP) family beyond asFP595 including, in particular, Dronpa, Padron, HcRed and other. 7–21 Due to a great importance of this process, intensive efforts have been undertaken to elucidate molecular mechanisms of switching between conformations with the trans or cis chromophores of fluorescent proteins both from experimental and theoretical sides. The pioneering papers traced back to the studies of the ground-state isomerization of a model GFP chromophore in aqueous solution by He et al.22 and to the semiempirical computations for the GFP chromophore in vacuo by Weber et al.23 and Voityuk.24 Since that, different methods of quantum chemistry were applied to describe such transformations in vacuo for 4


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the GFP-like chromophore molecules in the ground and/or excited states.3,23–38 Several attempts to apply molecular dynamics (or molecular mechanics) methods with the classical force fields to describe the ground state chromophore’s isomerization reaction inside a protein are known.3,28,30 To the best of our knowledge the paper of Sun et al.18 is the only report on QM/MM calculations for the thermal trans-cis isomerization of the chromophore in HcRed, the red fluorescent protein.17,20 For the QM region the authors used the SelfConsistent-Charge Density Functional based Tight Binding (SCC-DFTB) method while energy in the MM subsystem was computed with the CHARMM force field parameters. The single-point energies of the cis and trans conformations and the corresponding transition states were recomputed in the DFT(B3LYP)/MM approximation at the SCCDFTB optimized geometry parameters. The anionic form of the chromophore was assumed in calculations. The computed energies of the trans conformations were higher than those of the cis conformations (between 6 and 12 kcal/mol depending on details of QM/MM minimization) while the potential barriers were between 40 and 60 kcal/mol. Because of the very high barriers obtained in simulations the authors conclude that “the two alternative cis and trans conformers are “frozen” in their positions during the process of maturation in HcRed”.18 In this work we computed free energy profiles for the chromophore isomerization in asFP595 and KFP in the ground electronic state by applying the methods of molecular dynamics with the QM/MM potentials (QM/MM MD). Beyond a significance of visualization of the trans-cis chromophore isomerization pathway inside photoreceptor proteins, these results also provide an interpretation of yet non-explained experimental data2,4 on different decays of fluorescence in asFP595 and KFP when illumination is turned 5



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off. The lifetime of the asFP595 fluorescent state is not very high (7 s) but that of KFP is considerably higher (~50 s).2,4 Analysis of the Arrhenius plot for the active state decay in KFP allowed Quillin et al.39 to estimate the activation energy of this process as 17.0 kcal/mol. As shown below the results of our QM/MM MD simulations are quantitatively consistent with the experimental data on thermal relaxation from the lightinduced activation of fluorescence in asFP595 and KFP as well as with the available structural experimental data.

MODELS AND METHODS Molecular dynamics with QM/MM forces To calculate the free energy profiles for the chromophore isomerization in the proteins we use the umbrella sampling method40,41 coupled with the weighted histogram analysis method42 (WHAM). This approach allows one to recover the potential of mean force (PMF) profile from relevant MD trajectories by subdividing the assumed reaction path to several overlapping “windows”. In each window an ad hoc biasing harmonic potential is applied and MD runs are performed. Complete PMF profile is recovered by using WHAM43–46. Isomerization reaction of the asFP595 chromophore in vacuo was considered previously by Olsen and Smith.34 Following their work we selected the reaction coordinate as a halfsum of two dihedral angles ~∠2- 2- 2- 2 and ~∠2- 2- 2- , as clarified in the inset in Fig. 1. More details including positions of the windows and parameters of biasing potentials are given in Supporting Information. Atomic coordinates of heavy atoms for asFP595 and KFP are known from the X-ray structures available in the protein data bank (PDB): 1XMZ,39 1XQM17 and (2A50-2A56)3. 6


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These structures have been analyzed at different conditions (pH, temperature) and for slightly varying compositions of the protein (see discussion in Ref.(17)). In our simulations we used the coordinates of heavy atoms from the structure 2A503 for asFP595 and 1XMZ39 for KFP. Hydrogen atoms were added by assuming conventional protonation status of polar residues: negatively charged Asp and Glu and positively charged Arg and Lys. Model protein systems were inserted into the periodic water box of the size 60x70x60 Å3 containing ~8000 water molecules. Sodium and chlorine ions were added at physiological concentrations to maintain the neutral charge of the system. CHARMM47,48 force field parameters were used to describe the protein, water molecules (TIP3P) and ions. Forcefield parameters for the chromophore were taken the same as for the GFP chromophore49 with minor modifications (see Supporting Information). At preliminary stages we performed classical MD simulations by using NAMD.50 The lengths of trajectories were 5 ns with a 1 fs time step. The Langevin piston Nose-Hoover method was used to maintain temperature and pressure at 300 K and 1 bar respectively. Conditions of NPT ensemble were imposed at this stage only. All subsequent MD simulations were carried out under conditions of NVT ensemble with a periodic box sizes fixed at the relaxed values from NPT calculations. The CP2K package51 was used to perform QM/MM-based MD simulations. Forces and energies in the QM subsystem were calculated using the DFT/GPW approximation.52 The BLYP functional53 with empirical dispersion corrections,54 and QZV2P55 basis set with Goedecker-Teter-Hutter pseudopotentials56 were applied. The multigrid approach57 with the cutoff of the finest grid level of 350 Ry was utilized. The QM subsystem included the chromophore, the side chains of Lys67, Arg92, Glu145, Ser158, Glu195, His197, Glu215 7



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and several water molecules. The other parts of the protein and bulk water were described by the CHARMM force field parameters. Coupling between QM and MM subsystems was described within the customary electrostatic embedding scheme with the hydrogen link atoms. Integration time step 1 fs and temperature 300 K were applied. Canonical sampling through velocity rescaling58 (CSVR) thermostat or the Nosé-Hoover thermostat chain59 were used in QM/MM-based simulations. CSVR thermostat with small relaxation time was used only in the equilibration runs, while the production runs were performed with the Nosé-Hoover thermostat chain. Short equilibration runs (1-3 ps) were performed before every QM/MM-MD simulation to avoid significant overheating of the QM-subsystem. Flexible effective fragment QM/MM method To estimate energy difference between KFP structures with the cis and trans chromophore isomers we used the same computational protocol as in Ref. (60). To locate minimum energy structures we applied the flexible effective fragment QM/MM approach.61 In this method, interaction of the QM and MM subsystems is described in the effective fragment potential method,62 while interaction between the fragments is modeled by using conventional force field parameters. Optimization of geometry coordinates in the ground state was carried out by utilizing DFT with the PBE0 functional and 6-31G* basis set in the quantum mechanical (QM) subsystem and the AMBER force field parameters in the molecular mechanical (MM) subsystem. The PBE0 functional was selected for DFT calculations due to good results in geometry optimizations of organic molecules.63 To scan potential energy surfaces and to locate minimum energy structures the properly modified GAMESS(US)64,65 and TINKER66 programs were utilized. The QM subsystem included the entire chromophore molecule, the side chains Arg92, Ser158, His197, Glu215 8


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and two water molecules. The model system with the trans chromophore was optimized when starting from atomic coordinates of heavy atoms in the crystal structure 2A50;3 while the model system with the cis chromophore was initially created manually by rotating the chromophore inside the protein from its trans isomer and then re-optimized in QM/MM calculations. Thermodynamic properties Equilibrium constants Kc and rate constants k were evaluated from the computed free energy changes (∆A) and activation energies (ΔA‡ ) for the corresponding reactions by using the conventional formulae: ΔA RT ⋅ lnK

(1)

kT ΔA‡ k exp ! " h RT

(2)

To estimate changes in the pKa values upon trans-cis isomerization we used the thermodynamic cycle depicted in Fig. 2: ΔpK # pK # $%&'( pK # )*(

ΔA+,-. $%&'( ΔA+,-. )*( 2.303 ⋅ RT ΔA234567896 neutral > ΔA896723456 anion 2.303 ⋅ RT

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Figure 2. Thermodynamic cycle used to estimate changes in pKa upon chromophore isomerization. RESULTS We start from consideration of model systems with the anionic chromophore. For better visualization we show in Fig. 3 location of the most important molecular groups as obtained in the flexible effective fragment QM/MM optimization for KFP. According to these calculations the energy of the system with the cis chromophore is 11.5 kcal/mol higher than that with the trans chromophore. This conclusion is consistent with the experimental data showing that both asFP595 and KFP in the dark state contain the chromophore as the trans isomer.3 As shown elsewhere,60 the optical spectral bands computed for these model structures match experimental results, namely, the S0→S1 absorption wavelengths are 526 nm for trans-conformation and 551 nm for cis10


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conformation (the experimental values are 568 and 575 nm respectively4). Experimental emission S1→S0 wavelength (610 nm5) is also well reproduced computationally for the cisconformation (609 nm).60

Figure 3. Fragments of QM/MM optimized structures for KFP with the anionic chromophore. The system with the trans-isomer is shown in the left panel, the higher energy system with the cis isomer is shown in the right panel. Carbon atoms are colored in green, oxygen in red, nitrogen in blue, sulfur in yellow; selected distances between heavy atoms are given in Å. The free energy profiles connecting conformations with the trans and cis anionic chromophore for KFP (blue curve) and asFP595 (red curve) as computed in the QM/MM MD approach are shown in Fig. 4. First of all we note an agreement of these calculation results with the experimental data2,3 and consistency of both theoretical approaches (QM/MM and QM/MM MD) applied in this work: all results predict that the system with the trans anionic chromophore is the lowest energy structure. The computed free energy difference between cis and trans conformations in KFP as shown in Fig. 4, 11.8 kcal/mol, well correlates with the potential energy 11



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difference 11.5 kcal/mol computed with the QM/MM method in spite of large differences in the electronic structure methodologies, force field parameters, treatment of entropic contributions, and so forth.

Figure 4. Calculated free energy profiles for chromophore isomerization: red curve – asFP595 with the anionic chromophore, blue curve – KFP with the anionic chromophore, dark green curve – asFP595 with the neutral chromophore. Left side of the reaction coordinate corresponds to the trans isomer, right side – to the cis isomer. Another encouraging result is a fair agreement of the computed free energy barrier for thermal deactivation of the “on” state (corresponding to the system with the cis chromophore) for KFP, 20.7 kcal/mol, and the experimentally determined Arrhenius activation energy of 17.0 kcal/mol39 derived from the Arrhenius plot for the fluorescence 12


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decay in KFP. Consistently with the experimental results2 the free energy barrier of cistrans isomerization of the chromophore is lower for the asFP595 variant (19 kcal/mol) than that for the KFP variant (20.7 kcal/mol). Analysis of the isomerization pathways allows us to comment on geometry changes. The isomerization coordinates (1/2(τ+φ)) at the barrier for both proteins are fairly close: 105° and 102° for KFP and asFP595, respectively. Significant deviation of the angle from 90° is an illustration of the Hammond’s postulate, according to which a transition state of endothermic reaction should resemble better products than reagents. The minimum energy configurations suggest slightly non-planar conformations for both trans and cis isomers. These deviations are smaller in KFP (+3° and +8° for trans and cis isomers respectively) than in asFP595 (+7° and -9°). A noticeable difference between geometry configurations with the cis-chromophore for asFP595 and the A143G mutant (KFP) can be explained by Ala143 hindrance in the former case. The computed isomerization profile for asFP595 with the neutral form of the chromophore is also shown in Fig. 4 as the dark green curve. According to the QM/MM MD calculations the energy difference 5.2 kcal/mol between cis and trans conformations is considerably less than in the case of the anionic chromophore (11.8 kcal/mol). Importantly, energy barriers for both trans to cis and cis to trans isomerization reactions are much higher (by about 20 kcal/mol) than those for the systems with the anionic chromophore. Therefore, the ground state cis-trans isomerization reaction is prohibited at room temperature for proteins with the neutral chromophore. For this reason we did not compute the isomerization energy profile of KFP with the neutral chromophore: a single

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A143G mutation cannot change dramatically the energy barrier as shown for the systems with the anionic chromophore. Table 1. Calculated energy differences of conformations ΔA896723456 , the corresponding

equilibrium constants Kc, thermal activation energies ΔA‡896723456 , rate constants k, and lifetimes τ1/2 compared to the experimental data τ1/2 (exp). Model system

∆A896723456 , kcal/mol

Kc

∆A‡896723456 , kcal/mol

k, s-1

τ1/2, s

τ1/2 (exp)*, s

wt-asFP595, anionic chromophore

7.3

4.7·10-6

19.0

8.7·10-2

8.0

7

KFP, anionic chromophore

11.8

2.5·10-9

20.7

4.7·10-3

148

~50

wt-asFP595, neutral chromophore

5.2

1.6·10-4

38.8

3.5·10-16

2.0·1015

no data available

* Experimental data correspond to fluorescence state thermal deactivation.2

The calculation results are summarized in Table 1 showing the energy differences, the equilibrium constants Kc, rate constants k, and life-times τ1/2. We note an almost perfect agreement between the computed (8 s) and experimental (7 s) values of the life time for asFP595 with the anionic chromophore. The results for KFP (148 s vs ~50 s) are quantitatively less accurate, although consistent with the experimental data.2 We note that a theoretical value closer to the experimental life-time for KFP could be obtained if the activation energy for the cis-trans thermal isomerization would be about 0.5 kcal/mol lower. Uncertainties of 0.5 kcal/mol in calculated energy differences on the energy surfaces are typical for the applied methodology. From the qualitative side, simulations correctly 14


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show that the A143G mutation in asFP595 noticeably increases the life-time of the fluorescence state. Importance of changes in the protonation state of the chromophore upon photoswitching was recently demonstrated for the protein Dronpa.19 The results of present simulations (Fig. 4) allow us to evaluate differences in pKa values ascribed to the chromophore in the trans and cis forms. To this goal we used the thermodynamic cycle shown in Fig. 2. The difference in the relative energies of cis-isomers (2.1 kcal/mol) is consistent with the conclusion that the trans-chromophore is more acidic than the cis-isomer with the pKa difference of 1.52 units. Previous computational study67 for the chromophore in vacuo D.#EC reported this difference as 1.42 pKa units (pK BC 8.38). It is believed # 6.96 and pK #

that protein environment can significantly affect the acidity of aminoacid residues.68–70 However, in our case, the phenolic oxygen in the chromophore is similarly surrounded by predominantly polar groups in both cis- and trans-conformations forming hydrogen bonds with Ser158 and the crystallographic water molecule (see Fig. 3). Consequently, it is not unexpected that the effect of the protein matrix on pKa values is almost similar for both chromophore isomers.

DISCUSSION Light-controlled modification of properties of proteins of the GFP family is of crucial importance for numerous imaging applications. Cis−trans isomerization of the chromophore presumably coupled with changes in the protonation state of the latter are considered as key processes in the photoswitching of photoactivatable proteins. However, as cited, e.g., in the recent paper of Warren et al.,19 the process of such interconversion 15



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remains poorly characterized. We believe that the computed energy profiles for the chromophore isomerization in asFP595 and KFP in the ground electronic state (Fig. 4) provide a contribution to this topic. Both QM/MM MD and QM/MM approaches place the protein conformations with the trans chromophore lower in energy than those with the cis chromophore in agreement with the experimental data. We also found that the free energy barriers of thermal cis to trans isomerization of anionic chromophore are consistent with the observed2 life-times of fluorescent state assuming that the transition state theory is applicable. In case of the neutral chromophore the computed activation free energy barrier (around 40 kcal/mol) does not match experimental observations. The results obtained in this work as well as in our previous simulations for asFP595 and KFP6,60,67 demonstrate that majority of observed photophysical properties of these proteins may be explained by considering conformations with the anionic chromophore. In this respect, a hypothesis on asFP595 fluorescence from the conformations with the zwitterionic chromophore71,72 does not seem convincing. On the contrary, consideration of structures with the neutral chromophore explains certain features of asFP595 and KFP. Species with the neutral chromophore are characterized by a significantly higher isomerization barrier, than those with the anionic form (Fig. 4). Similar results were obtained long ago for the gas-phase GFP chromophore by using semiempirical quantum chemistry methods.23 This can be easily explained in terms of Lewis structures: the CB2-CG bond order (see inset to Fig. 1) is nearly double in the vicinity of transition state, which corresponds to the quinonelike electronic structure of the six-membered ring. Protonation of the quinoid oxygen is energetically unfavorable and corresponds to the high isomerization barrier for neutral chromophore on the ground state. The calculated free energy profiles (Fig. 4) indicate that 16


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it is much easier for neutral chromophore to undergo isomerization through the anionic transition state than through the neutral one. It is worth noting, that experimental isomerization energies for the neutral and anionic forms of GFP chromophore in solution22 are almost equal. The authors proposed22 that a single ground state isomerization pathway must exist for both these protonation states. This pathway could correspond to anionic transition state. It is important to note, that two types of transition states (TS) are possible for the double bond isomerization,73 namely, with homolytical or heterolytical double bond cleavage. Those correspond to the diradical (TSDR) or charge-transfer (TSCT) electronic structures, respectively. For the isomerization reaction of retinal it was shown73 that both TS were located on the opposite sides of S0/S1 conical intersection, and TSCT was lower in energy than TSDR. In the current work based on the closed shell DFT calculations we studied only the TSCT type. We can assume that TSDR is probably higher in energy than TSCT (like in the case of retinal); however, this assumption requires an additional study by using higher level multireference methods. Finally, the computed pKa difference between the cis and trans chromophore isomers serves as an additional proof for the proposed isomerization mechanism of photoswithcing. Indeed, an additional 445 nm peak appears in the absorption spectrum upon kindling.4 According to the theoretical71,74,75 and experimental studies,76 only neutral chromophore’s form can absorb at this spectral region. As follows from our results, the trans-to-cis photoisomerization leads to increase of neutral chromophore concentration which is observed experimentally. The experimental spectrum of effectiveness of active state deactivation by the blue light shows maximum at 450 nm;4 it definitely corresponds to a 17



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small peak at 445 nm in absorption. Therefore our findings also provide an additional support for the hypothesis that a most probable candidate for the photochemical quenching of asFP595’s active state fluorescence is the neutral chromophore.4,35,36

CONCLUSION Novel aspects of this work are that we are able to characterize the free energy profiles for the trans-cis isomerization of the chromophore of the fluorescent proteins of the GFP family inside the protein matrices. We consider the isomerization reaction in the ground electronic state providing interpretation of experimental data on thermal deactivation of the fluorescence state in asFP595 and KFP. We relate the experimental observation that the asFP595 → KFP mutation noticeably increases the life time of fluorescence state to the computationally derived conclusion that the barrier for the cis-trans ground state isomerization reaction is 1.7 kcal/mol higher in KFP than that in asFP595. Our conclusions favor a mechanism that describes majority of photophysical properties of asFP595 and KFP by considering protein structures with the anionic chromophore.

ACKNOWLEDGEMENTS This study was partially supported by the Russian Foundation for Basic Research (projects 11-03-01214, 13-03-00207) and by the Program on Molecular and Cell Biology from the Russian Academy of Sciences. M.G.K. acknowledges support from the stipend of the President of Russian Federation and from the “Dynasty” foundation. We acknowledge

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the use of supercomputer resources of the M.V. Lomonosov Moscow State University and of the Joint Supercomputer Center of the Russian Academy of Sciences.

Supporting Information Available: details of umbrella sampling simulation and force field parameters of the chromophore are presented. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES

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For Table of Contents Only

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Figure 1. Trans- and cis-isomers of the asFP595 (or KFP) chromophore presumably involved in photoswitching. The inset illustrates the dihedral angles that can be used as reaction coordinates to characterize the isomerization pathway. 257x152mm (299 x 299 DPI)



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Figure 2. Thermodynamic cycle used to estimate changes in pKa upon chromophore isomerization. 259x157mm (299 x 299 DPI)


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Figure 3. Fragments of QM/MM optimized structures for KFP with the anionic chromophore. The system with the trans-isomer is shown in the left panel, the higher energy system with the cis isomer shown in the right panel. Carbon atoms are colored in green, oxygen in red, nitrogen in blue, sulfur in yellow; selected distances between heavy atoms are given in Å. 490x191mm (299 x 299 DPI)



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Figure 4. Calculated free energy profiles for chromophore isomerization: red curve – asFP595 with the anionic chromophore, blue curve – KFP with the anionic chromophore, dark green curve – asFP595 with the neutral chromophore. Left side of the reaction coordinate corresponds to the trans isomer, right side – to the cis isomer. 164x115mm (300 x 300 DPI)


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For Table of Contents Only 70x48mm (300 x 300 DPI)


Thermal Isomerization of the Chromoprotein ... - ACS Publications

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