Primary Events of Photodynamics in Reversible Photoswitching

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Primary Events of Photodynamics in Reversible Photoswitching Fluorescent Protein Dronpa Xin Li,† Lung Wa Chung,† Hideaki Mizuno,§ Atsushi Miyawaki,§ and Keiji Morokuma*,†,‡ †

Fukui Institute for Fundamental Chemistry, Kyoto University, Kyoto 606-8103, Japan, ‡Cherry L. Emerson Center for Scientific Computation, Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States, and § Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan

ABSTRACT Reversible photoswitching fluorescent protein Dronpa can reversibly switch between fluorescent on-state and nonfluorescent off-state by two radiations. The primary events of photodynamics in Dronpa were elucidated by nonadiabatic ONIOM (CASSCF:AMBER) molecular dynamics simulations. All radiationless decay processes are found mainly to result from one bond flip of a bridge C-C bond of the chromophore in the protein, regardless of its protonation state or conformation, rather than hula twisting. In the off-state protein, trans-cis photoisomerization of the neutral trans chromophore takes place via a rotation around the imidazolinone ring. In the wild-type on-state protein, the anionic cis chromophore mostly remains planar for at least 20 ps. In contrast, in the H193T mutant on-state, faster decay via a rotation of the phenoxy ring or imidazolinone ring of the anionic cis chromophore was found, suggesting that flexibility of the chromophore and its immediate protein environment is the key to radiationless decays. SECTION Biophysical Chemistry

reen fluorescent protein (GFP) and its variants play a vital role in biological imaging and analysis.1-3 Recently, a new class of photoactivatable fluorescent proteins (FPs), for example, reversible photoswitching fluorescent proteins (RPFPs), have been developed.3-11 Remarkably, fluorescent on-state and nonfluorescent off-state in RPFPs can be reversibly switched by two different radiations.3,4 Such photochromism in RPFPs advances FP technology and can potentially result in some applications, such as optical data storage.12 The mechanism of this reversible process remains unclear, although protonation state, conformation, and nonplanarity of the chromophore, as well as intersystem crossing, were proposed to be involved.3,4,6,9 For the most promising Dronpa,4 anionic cis (Acis) and neutral trans (Ntrans) chromophores were suggested to be the on- and off-states, respectively. On the basis of the kinetic isotope effect and crystal structures,5,7,9 two mechanisms were proposed for photoactivation (routes 1 and 2 in Scheme 1). Our recent calculations supported the proposed on- and off-states13 and also found favorable τ-rotation (with the imidazolinone ring, I-ring) of Ntrans enhancing the acidity of the phenoxy ring (P-ring) in S1.13,14 As a result, a new mechanism (route 3 in Scheme 1) via photoisomerization coupled with excited-state proton transfer (ESPT) to nearby Glu144 was proposed.13 Herein, we report nonadiabatic CASSCF(6e,6o)/3-21G:AMBER molecular dynamics (MD) simulations on photodynamics in Dronpa.15 We will show that radiationless decay channels take place with the one bond flip (OBF) mechanism and that

G

r 2010 American Chemical Society

Scheme 1. Proposed Mechanism for Dronpa

excited-state dynamic processes can be modified by different protonation states and protein structures. As we will show, upon the excitation, the chromophore evolves from the Franck-Condon (FC) region to a roughly planar minimum region. In some trajectories, it then takes some time to reach a twisted minimum region and crossing point, where the potential energies in S1 and in S0 are slightly decreased and significantly increased, respectively, and the energy gap becomes very small to allow surface crossings (Figures 1 and S1-S3 (Supporting Information)). At the crossing, surface hopping to S0 takes place, and then, the Received Date: October 15, 2010 Accepted Date: November 9, 2010 Published on Web Date: November 12, 2010

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DOI: 10.1021/jz101419p |J. Phys. Chem. Lett. 2010, 1, 3328–3333

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Figure 1. Time evolution of the bridge dihedral angles and relative potential energies for (a) Ntrans and (b) Acis. A dashed vertical line shows electronic transition. Table 1. Computed Mean Lifetime (τ, ps) of the Excited State and Quantum Yield for Photoisomerization (Φphotoiso)

Chart 1

τNCa (Φphotoiso)

τCSb (Φphotoiso)

Ntrans

0.655 (0.33)

0.874 (0.20)

Ncisc

0.829 (0.00)

1.588 (0.00)

Ncisd Acis

2.695 (0.29) see notee (0.00)

3.008 (0.50) see notee (0.00)

Acis(H193T)

3.429 (0.00)

a

3.181 (0.00) c

Nonadiabatic crossing. Crossing seam. Cationic His193 and anionic Glu211. d Neutral His193 and Glu211. e Decay times are 2.79 (4.53), 12.04 (13.02), no NC (15.97), and 17.62 (17.71) ps for NC and CS (in parentheses) in four trajectories, but there is no decay in the other six trajectories.

energy gap increases immediately. Different protonation states of chromophore (neutral and anionic) or/and protein environment change dynamic features of the excited-state chromophore, that is, lifetime and structural changes of the bridge C-C bonds (R5 and R6) as well as torsions (τ, τH, φ, and φH) (Chart 1). As shown in Table 1 and Figure 1, for the neutral trans chromophore, Ntrans, of the off-state Dronpa undergoes ultrafast radiationless decay exclusively via the τ-rotation (i.e., I-ring). The computed mean lifetime of the excited state (S1) is about 0.66 and 0.87 ps via nonadiabatic crossing (NC) and crossing seam (CS),16 respectively. In addition, trans -cis photoisomerization of Ntrans was found to occur with the calculated quantum yield (Φphotoiso) of about 0.20-0.33, depending on the crossing modes (i.e., NC or CS).16 On the other hand, the neutral cis chromophore, Ncis, of the on-state Dronpa requires a longer time to decay via the τ-rotation. In contrast to the above-discussed neutral forms, the anionic


b

cis chromophore, Acis, of the on-state Dronpa remains in S1 and roughly planar for at least 20 ps in 6 of 10 trajectories. In the other four trajectories the φ-rotation (i.e., P-ring) on S1 takes place, to result in a twisted structure where the internal conversion to S0 occurs. Remarkably, in all trajectories for the H193T mutant, Acis decays faster than that in the wildtype (WT) via mostly φ-rotation (and in a small fraction via τ-rotation), which nicely explains nonfluorescence in the mutant experiment.6 The φ-rotation does not lead to isomerization of the chromophore, and the τ-rotation after hopping to S0 regenerates the cis chromophore at the end due to steric hindrance; these lead to a zero quantum yield for photoisomerization of Acis both in WT and H193T. It should be noted that every decay occurred via the φ- or τ-rotation, which is the so-called OBF process. Thus, irrespective of the protonation state or conformation of the chromophore,

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torsions involving Hbr (τH or/and φH, Figure S6 (Supporting Information)) are found to be the major event immediately after hopping to S0 in most cases, in which the potential energy in S0 decreases and the energy gap increases rapidly. Moreover, in most crossings of the neutral chromophores (Ntrans and Ncis), the torsion τ is rotated by about 80-100° (see Figure S7 (Supporting Information)), while the magnitude of φ-rotation is small. The crossings of Acis in WT involve the φ-rotation by about 80° and a wide distribution of τ. Here, a larger rotation of τH (30-50°) was also found, indicating some degrees of pyramidization.26,27 Acis in the H193T mutant can undergo either the φ- or τ-rotation but not simultaneous φ- and τ-rotation (see Figure S7 (Supporting Information)). Although all crossings mainly involve a rotation of one bridge C-C bond, involvement of some rotation of the other bridge C-C bond and slight pyramidization at two carbons bonded to the CH part should further bring two energy surfaces closer. In addition, the very fast movement of Hbr after the hopping keeps the shape and size of chromophore not much altered for comfortable fit in the protein. Therefore, the above factors and electrostatic interactions with the protein (see below) make the simultaneous twisting of the I-ring and P-ring (i.e., the HT pathway) unnecessary in the protein.2,17,19,20 Notably, the photoinduced rotations trigger large and different charge redistribution of the P- and I-rings (Figure 3).13,14,17,18 Therefore, in all crossing regions, electrostatic interaction and polarization from the protein stabilize S1 relative to S0 by about 20-28 kcal/mol and thus reduce the energy gap to promote crossing (Figure 3 and Table S3 (Supporting Information)). For instance, in the internal conversion via the τ-rotation of Ntrans and Ncis, the protein environment favors S1 over S0 by about 20.4 and 25.4-28.0 kcal/mol, respectively. The major contributors are the nearby charged residues; for example, the negatively charged I-ring (I-) is stabilized by Arg91 (in Ntrans and Ncis) and Arg66 (Ncis), as well as the positively charged P-ring (Pþ) by Glu144 (Ntrans). For the internal conversion via the φ-rotation of Acis in the WT and H193T mutant, the protein environment also promotes the crossings by stabilizing S1 relative to S0 by about 20.3 and 21.8 kcal/mol, respectively, in which I- is strongly stabilized by Arg66 and Arg91. On the other hand, a loss of electrostatic interaction of the I-ring with Arg66 and Arg91 should disfavor the τ-rotation of Acis in WT. For the τ-rotation in some trajectories of the H193T mutant, the negatively charged P-ring is stabilized by Arg66, Ser142, and Thr193. It should be noted that electrostatic stabilization on the crossing was found in a homogeneous water environment.18,21 The electrostatic stabilization in the heterogeneous protein environment also promotes crossing28 but furthermore modulates the crossing mode, for instance, no τ-rotation of Acis in WT. The photoinduced twisting of the bridge bond (i.e., τ- or φrotation for Acis and τ-rotation for Ncis) is intrinsically favorable even in the vacuum,14,17,18 and as discussed above, the proteins further promote the crossings via electrostatic interaction. In addition, additional factors discussed below should affect the lifetime of S1 and suppress, in particular, the τ- or φ-rotation of Acis in WT. Because the I-ring linked with the

Figure 2. Dynamic changes of the bridge torsion angles (in degrees) against bond distance (R5 or R6, in Å) evolved from the Franck-Condon (FC) point in S1 (blue) hopping to S0 (pink) via nonadiabatic crossing (NC) or the crossing seam (CS). Dynamic change in S0 for Acis is shown in Figure S5 (Supporting Information).

OBF, not hula twisting (HT), takes place in the congested protein.2,17-20 (Figures 1, S2 and S4-S7 (Supporting Information)). OBF was suggested to be responsible for the decay in the gas phase and flexible water environment.14,18,21,22 Moreover, OBF was just observed experimentally in the synthetic GFP chromophore.23 Looking at details of dynamics and structural changes, as shown in our previous static calculations,13 excitation of Ntrans and Ncis to S1 is found to first induce a reversal of bond order of the bridge bonds (Figures S1-S2 (Supporting Information)) in the current MD simulations. Due to the weaker R5 bond in S1, the neutral chromophore specifically rotates around τ and τH (i.e., τ-rotation) to give a twisted intermediate (NTI) before reaching the crossing (Figure 1a and S2 (Supporting Information)). The bond distance difference between R5 and R6 is reduced in the twisted minimum region. After hopping to S0, the R5 and R6 bond distances become normal (i.e., short R5 and long R6) and are coupled with rapid changes of the bridge part to give the roughly planar form, which determines the final configuration of the chromophore (cis or trans). After that, the P-ring with the weak R6 bond rotates and finally becomes nearly coplanar. Such a qualitative feature of this dynamics (Figures 2 and S5 (Supporting Information)) is analogous to the two-state two-mode model.17,24 A very different photodynamics is found for Acis. After excitation of Acis in WT, R5 and R6 bonds are elongated to a similar extent, followed by changing torsions φ and φH by ∼90° (i.e., φ-rotation) coupled with bond order alternation (weaker R6 bond) before hopping to S0 (Figures 1b and S3 (Supporting Information)). However, this φ-rotation (with the phenoxy group) does not render cis-trans isomerization. For Acis in H193T mutant, the τ-rotation (around the R5 bond, in 3 of 10 trajectories) and the φ-rotation (around the R6 bond, in 7 trajectories) have similar photodynamics, except that the τ-rotation causes different alternation of the bridge bond order as well as the movement of Arg66 to the vacant site above the P-ring. In this case, Arg66 can form new interactions with the P-ring and Glu211, which somehow resembles the off-state Dronpa (Figures S4 and S9 and Movie S3 (Supporting Information)). However, steric repulsion with Thr59 blocks cis-trans isomerization, regardless of the momentum of the bridge hydrogen (Hbr). Interestingly, Hbr significantly changes its position and points either upward or downward in all simulations.25 The


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Figure 4. Side-view of superimposition of the chromophore and its nearby key residues at the Franck-Condon point (blue and purple) and crossing seam (red and orange) for (a) Ntrans in the off-state protein and (b) Acis in the on-state protein.

trajectories is statistically small, our simulation results demonstrate that the decay processes are generally faster in the H193T mutant than those in the WT. Therefore, elimination of strong π-stacking interactions of cationic His193 with Acis in the H193T mutant is shown to promote the decay, which is in agreement with nonfluorescence in expeiement.6 In other words, the rigidity of the immediate environment around the chromophore should suppress the rotation and thus hinder the internal conversion. Recently, a nonadiabatic QM/MM MD simulation on another RPFP, asFP595, using the same QM method, was reported.28 Similar to our findings, the neutral chromophores were found to undergo ultrafast decay via the τ-rotation. However, the zwitterionic chromophores, but not the anionic chromophores, were suggested to be responsible for the onstate.28,29 Such differences may be attributed to different chromophore structures and protonation states of the nearby residues. As in GFPand its variants, ESPTwas suggested to take place in Dronpa,2,5 possibly through a new route.13,30,31 We adopted a larger QM part (model ML, including Glu144 and a water molecule, see Supporting Information) in our simulations of the ESPT process. ESPT from the excited-state Ntrans in all trajectories occurred in about 100 fs to give excited-state Atrans. Atrans mainly (9 out of 10 trajectories) undergoes φrotation, which reduces the opportunity for trans-cis photoisomerization. Four of 10 trajectories reached the crossings within our affordable computation time (3-5 ps) and gave the ground-state trans chromophore. Our current MD simulations using a small active space and basis sets without dynamic correlation, unavoidable because of the computational cost of the large model ML, may favor the ESPT process over isomerization. To consider another scenario for ESPT after the τ-rotation, additional MD simulations with the large QM model were performed starting from the twisted form (NC) obtained from the τ-rotation of Ntrans. Only in 2 out of 10 trajectories ESPT was found to occur, coming out of excited-state NTI to give ATI, which was followed by hopping to S0. The trans-cis isomerization from ATI was found in one trajectory, and regeneration of the trans chromophore occurred in the other one. In both trajectories, after hopping to S0, the proton was transferred back to the anionic chromophore; this may be due to exclusion of the feasibility of proton transfer from the neutral Glu144 to the bulk in our simulations or/and the lack of dynamic correlation.32 In this

Figure 3. Schematic changes of the electronic structures of chromophores and protein environments at crossings, models M0 (Ntrans), M2 (Ncis), MA0 (Acis in the WT), as well as MA1 (Acis in the H193T mutant).

protein's backbone is relatively rigid and not very movable, movements of the P-ring and bridge CH going from FC to the crossing (NC or to CS) become more essential (Figures 4 and S8-S10 (Supporting Information)), regardless of the τ- or φrotation. In addition, in synch with the moving chromophore during the rotations, residues (e.g., Thr59, Val157, Met159, Phe173, and His193) around the P-ring and bridge CH part change their positions to avoid steric repulsion. Therefore, a more spacious or/and more flexible environment around the P-ring and bridge CH should be preferable for the decays. In this connection, some mutants, for example, M159T, were recently shown to increase switching kinetics but lower fluorescence quantum yield.7,8 Moreover, His193 in the WT can hold Acis to stay planar by forming π-stacking interactions (electrostatic and van der Waals) (Figure S9 (Supporting Information)). Replacement of the histidine by smaller and more flexible threonine would allow the bridge bonds of the chromophore to rotate more easily, which leads to the radiationless decay in our simulations. Although the number of


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regard, proton wires connecting Glu222 (a proton acceptor for ESPT) to the surface in GFP were recently proposed, in which the proton may leak outside.33 The recent classical MD simulations also suggested another route for proton uptake (via Ser158 and buried water molecules) and release (from Glu145 and His197 to Asp78) for asFP595.29 Overall, our QM/ MM MD simulation results suggest that the ESPT process could occur, but it would not occur before the τ-rotation for the trans-cis isomerization in the photoactivation process. Calculations of potential energy surfaces for the ESPT process using a more accurate method (such as the CASPT2 method with a larger active space and a larger basis set) are in progress and will be reported in due course. On the other hand, the reaction mechanism for the rare onto-off conversion starting from the excited-state on-site Acis remains unclear. Energetically unfavorable τ-rotation has to be involved, regardless of the protonation state in S1.14 In order to overcome this high barrier for the cis-trans isomerization, a very hot Acis or more flexible immediate environment may have to be formed via stepwise absorption of more than a single photon and energy dissipation (see Scheme S2 (Supporting Information)). In summary, the primary events of photodynamics in Dronpa were theoretically elucidated for the first time. OBF is generally responsible for all crossings. Trans-cis photoisomerization from Ntrans occurs via the τ-rotation, in which ESPT may occur, but may not be indispensable for the isomerization step. Comparing WT with H193T, the flexibility of both the chromophore and its immediate protein environment, particularly around the phenoxy ring and bridge CH part, is the key to radiationless decay. Further theoretical studies on the reaction mechanism of Dronpa are in progress and will be reported.

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SUPPORTING INFORMATION AVAILABLE Computational Methods, Tables S1-S6, Figures S1-S14, Schemes S1-S2, Movies S1-S3, and the complete citation for ref 6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author: *To whom correspondence should be addressed. E-mail: morokuma@ fukui.kyoto-u.ac.jp. Phone: þ81-75-711-7843. Fax: þ81-75-781-4757. (14)

ACKNOWLEDGMENT L.W.C. acknowledges the Fukui Institute Fellowship. This work is partly supported by the Japan Science and Technology Agency with a Core Research for Evolutional Science and Technology (CREST) grant in the Area of High Performance Computing for Multiscale and Multiphysics Phenomena and Research Center for Computational Science, Okazaki, Japan. The computational resource at the Research Center of Computer Science (RCCS) at the Institute for Molecular Science (IMS) is also acknowledged.

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Primary Events of Photodynamics in Reversible Photoswitching

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