Characterizing the Structures, Spectra, and Energy Landscapes

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Characterizing the Structures, Spectra and Energy Landscapes Involved in Excited-state Proton Transfer Process of Red Fluorescent Protein LSSmKate1 Fasheng Chen, Qiao Zeng, Wei Zhuang, and WanZhen Liang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b04708 • Publication Date (Web): 01 Sep 2016 Downloaded from http://pubs.acs.org on September 6, 2016

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The Journal of Physical Chemistry

Characterizing the Structures, Spectra and Energy Landscapes Involved in Excited-state Proton Transfer Process of Red Fluorescent Protein LSSmKate1 Fasheng Chen,† Qiao Zeng,† Wei Zhuang,‡ and WanZhen Liang∗,† State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, and Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China, and State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China E-mail: [email protected]

∗ To

whom correspondence should be addressed Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, and Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡ State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China † State

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Abstract By applying the molecular dynamics (MD) simulations and quantum chemistry calculations, we have characterized the states and process involved in the excited-state proton transfer (ESPT) of LSSmKate1. MD simulations identify two stable structures in electronically ground state of LSSmKate1, one with a protonated chromophore and the other with a deprotonated chromophore, thus leading to two separated low-energy absorption maxima with a large energy spacing as observed in the calculated and experimentally-measured absorption spectra. The proton transfer is induced by the electronic excitation. When LSSmKate1 is excited, the electrons in the chromophore are transferred from the phenol ring to the N-acylimine moiety, the acidity of a phenolic hydroxyl group is thus enhanced. The calculated potential energy curves exhibit the energetic feasibility in the generation of the fluorescent species in LSSmKate1 and the exact agreement between the calculated and the experimentally-measured values of large Stokes shift further provides a solid theoretical evidence for the ESPT process taking place in photo-excited LSSmKate1. The molecular environments play a significant role on the geometries and absorption/emission energies of the chromophores. Overall, TD-ω B97X-D/MM provides a better description to the optical properties of LSSmKate1, although it always overestimates the excitation energies.


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1. INTRODUCTION Fluorescent proteins (FPs) have been widely used in biochemical and biomedical fields. 1–3 Genetically encoded FPs allow the specific and targeted labeling of proteins, organelles, cells, tissues, and organisms. This characteristic makes FPs superior over synthetic organic dyes for imaging applications in vivo since FPs require no additional enzymes and cofactors except for molecular oxygen to form a chromophore once they are expressed. 4–8 One desired property of FPs is a large Stokes shift (LSS), which is especially useful in fluorescence cross-correlation spectroscopy 9–11 and single-FP biosensors. 12–17 LSS indicates a difference between excitation and emission maxima larger than 100 nm, and is usually generated by FP chromophore transformation between a neutral state and an anionic state. 18–23 An important family of FPs, the red FPs (RFPs), have drawn a significant amount of attention since they provide new potential to study biological processes at levels ranging from single molecules to whole organisms. Namely, red-shifted absorption and fluorescence result in reduced autofluorescence, lower light-scattering, and lower phototoxicity at longer wavelengths. 24–27 These properties make RFPs superior probes for in vivo imaging in deep tissues and for imaging approaches that require low background noise including single-molecule super-resolution techniques. 28–30 Two mutants of the red FP mKate 31,32 with LSSs, LSSmKate1 and LSSmKate2, have been developed by Piatkevich et al. 33,34 These two RFPs have absorption/emission maxima at 463/624 nm (2.68/1.99 eV) and 460/605 nm (2.70/2.05 eV), respectively. 34 Crystallographic and mutagenesis analysis, as well as isotope and temperature dependencies, suggested that an ESPT 35–40 is responsible for the LSSs observed in LSSmKates. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 and 1.5 Å, respectively, revealed possible ESPT pathways involving a hydrogening bond formed between the chromophore hydroxyl and Glu160 in LSSmKate1, and a hydrogen-bonding network composed of the chromophore hydroxyl, Ser158, and Asp160 in LSSmKate2, as shown in Figure S1 in the Supporting Information (SI). To investigate ESPT pathways of LSSmKate proteins, pH-dependent spectroscopic measurements at room temperature have been conducted. The results indicate that at pH’s lower than 11, the protein exhibits a strong absorption centered at 460 nm (2.70 eV), and at pH 11, an additional weak absorption peak appears at 579 nm (2.14 eV), which has been attributed to the absorption of LSSmKate with the deprotonated form of the chromophore. Excitation with 579 nm wavelength light at pH 11 resulted in a red emission with a maximum at 628 nm (1.97 eV), which is close to the emission maximum of 624 nm (1.99 eV) of the LSSmKate1 protein. 33 Other similar experiments 41–43 have also been carried out and confirmed the pH-dependent spectral behaviors. To further investigate whether ESPT is responsible for the LSS of LSSmKates or not, the isotope and temperature dependencies of the steady-state emission spectra have also been determined. With



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the above experiments, the possible photocycles for LSSmKates have been finally suggested by Piatkevich and coworkers. 33 There are neutral (A) and anionic (B) ground-state form of the chromophore (with absorption maximums at wavelengths of 460 nm and 579 nm, respectively). The equilibrium between these two forms of chromophores is controlled by a hydrogen bonding network that permits proton transfer between the RFP chromophore and its neighboring side chains. The protein with the protonated and deprotonated forms of the chromophores can be slowly interconverted in the electronic ground state, but the process is much faster in the excited state. The process of interconversion goes through an intermediate state (I), which is electronically similar to state B, but environmentally similar to state A. 44,45 Although the above assumption by Piatkevich and co-workers has been supported by their experimental results to some extent, the details of the photocycle of LSSmKates are not clear and the structures of A , B and I states have not been identified. The target of this work is to characterize the geometric and electronic structures of those states. Both the classical MD simulations and quantum-chemistry calculations will be employed to identify the equilibrium conformations. The differences existed in the ground and excited-state geometric and electronic structures arisen by the different structural forms of the chromophores will be shown. Meanwhile, to show the energetic feasibility of the proposed ESPT mechanism involved in the generation of the fluorescent species in LSSmKate1, we scan the potential energy curves involved in ESPT process. Due to limited experimental tools, theoretical study becomes extremely useful for exploring the ESPT processes and the LSS mechanism in these FPs. 46–49 However, previous theoretical investigations were hindered by the lack of computational tools to accurately calculate the ground and excited-state geometries and the Stokes shift, especially when the atomic details of the protein environment are required to be taken into account. Therefore, there are limited reports about the energy landscape of a whole FP. 50,51 There are a few quantum chemical studies of considerably simplified model systems for RFP. For example, Nadal-Ferret et al. 52 performed excitation energy calculations on the chromophore with an increasing number of atoms surrounding it, and compared those results with excitation-energy calculations on an ensemble of structures obtained from a MD simulation of the whole protein. The environment effect on the absorption spectra has been revealed. However, a study on the emission process of the whole protein is still missing. Similarly, Randino and co-workers 53 focused on the LSSmKate2 protein and carried out a molecular dynamics simulation in which a series of excited states were calculated and analyzed based on different snapshots. The final spectrum is obtained as an average of all the individual spectra. Unfortunately, they only calculated the protein with the neutral form of the chromophore and didn’t provide the possible chromophore transformation between a neutral state and an anionic state. Randino and co-workers 54 computed the potential energy curves of the LSSmKate1 and LSSmKate2 proteins in large cluster models including the surrounding of the chromophores for the respective reaction 4 ACS Paragon Plus Environment

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coordinates, but they did not predict a minimum in the product side for LSSmKate1. In their calculations, they defined the reaction coordinates by shifting different protons from donor to acceptor groups, retaining all the remaining coordinates frozen. However, the actual system is not rigid, and can relax both in the ground and excited states away from crystallographic structure. It is thus essential to involve the relaxation processes to bring about such a minimum. Here we revisit the LSSmKate1 system using the latest state-of-the-art approaches. Our group has successfully implemented the analytic excited-state energy gradient and Hessian in the framework of time-dependent density functional theory (TDDFT) in Q-Chem quantum chemistry software. 55–57 Very recently, we have coupled TDDFT with conductor-like PCM (CPCM) 58,59 and molecular mechanics (MM) to investigate the excited-state properties of molecules in condensed phases and implemented the analytic energy gradient and Hessian of TDDFT/C-PCM and TDDFT/MM excited states into the locally-modified Q-Chem software package. 60–62 Those successful implementations allow us to take advantage of the Q-Chem/CHARMM interface 63–66 to characterize the details of photocycles for the LSSmKates. The paper is organized as follows. The computational details for the MD simulation and the hybrid QM/MM method will be provided in Section 2. The environmental effect on the geometries and excitation energies of LSSmKate1 with protonated and deprotonated chromophores, the equilibrium fluctuation effect on the absorption spectra, and PEC scans will be shown in Section 3. Concluding marks will be given in Section 4.

2. COMPUTATIONAL DETAILS In order to reflect dynamic characteristics, we performed molecular dynamics simulations to collect snapshot configurations from the canonical ensemble (NVT) using the CHARMM software package. 67 The CHARMM22 force field 68,69 and the TIP3P water model 70 were employed for the protein and water molecules, respectively. It is noted that the force field parameters for the RFP chromophore are not available in the CHARMM22 force field as the RFP chromophore is a non-standard amino acid residue, and we obtained most of the missing parameters and the double bond parameters involving nitrogen and carbon atoms from the works of Thiel’s 71 and Lluch’s groups, 52 respectively. The crystal structure obtained from X-ray diffraction experiments only provides a given configuration at low temperature and without bulk solvent molecules. However, what we are more concerned about is the case when the proteins in vivo are surrounded by the solvent molecules, which refers to waters in most cases. Solvation water molecules were constructed by the CHARMM software package to simulate the protein in vivo. The targeted protein was solvated by water



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molecules with a sphere of radius 16.0 Å and the solvation water molecules were divided into inner and outer layers. The inner water layer includes water molecules that are within a sphere of radius 8.0 Å. The hydrogen atoms in the X-ray crystal structures were missing. We added these hydrogen atoms using the HBuild command within the CHARMM software package and assign most of the standard amino acids to their given ionization state in a neutral environment i.e. pH 7.0 by default. We first performed a classical energy minimization for the hydrogen atoms of the protein using steepest descent 72 (SD) and adopted-basis Newton-Raphson 73 (ABNR) methods with 200 and 500 steps, respectively. Then, to remove poor contacts between water molecules, multistep operations were performed. For the inner water layer, the following steps were performed: (1) the structures of inner water molecules were optimized with 200 SD steps and 300 ABNR steps, (2) the temperature was increased gradually from 0 to 300 K over a period of 30 ps with a 1 fs time step, (3) another 50 ps equilibration was carried out for further relaxation of the inner water layer, and (4) the interspace of the solvent layer was checked and filled up until only 50 extra water molecules were incorporated into the interspace of the protein. It should be cautioned that only the inner layer water molecules were minimized during the entire process; the coordinates of the protein were kept fixed to preserve a protein structure close to the original crystal one. As a result, the structure of the whole system involving protein and water molecules was minimized, heated and equilibrated step by step in the upcoming operations. These processes are similar to the multistep operations for the inner layer water molecules. Finally, an NVT ensemble was chosen for the MD simulation with a time duration of 40 ns. The outer water layer was kept inactive at any given time. To obtain an accurate description of LSSmKate1, we started from the structures obtained in the preliminary MM equilibration or X-ray crystal structure and perform the geometry optimization with QM/MM schemes. The QM region mainly includes the atoms marked in red as shown in the right panels of Figure 1 and is described by (TD-)DFT. In some QM/MM calculation, the QM region also includes the water molecules which bind with the phenolic oxygen of the RFP chromophore with hydrogening bonds (H-bonds). The (TD-)DFT/MM scheme is adopted within the locally-modified Q-Chem/CHARMM interface. 61 The current QM/MM scheme adopts an additive QM/MM framework and covers all the interactions between QM and MM atoms, including the electron-nuclear attractive potential, nuclear-nuclear repulsion, and van der Waals interactions. The link atoms are introduced to mimic the covalent bonds connecting QM and MM regions. QM region was filled by the addition of "junction" hydrogen atoms (link atoms) for the QM calculation. The link atoms did not have any bonded or non-bonded interactions with the atoms of MM region, but they did interact with the field of MM point charges. In order to avoid overpolarization effect, MM atom in position close to link atom was excluded from interaction of external charges to QM region. A detailed description about the QM/MM scheme has been given in our previous work. 61 In addition, in order to probe the S0 and S1 PECs with respect to the length of the oxygen-hydrogen 6 ACS Paragon Plus Environment

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bond in the hydroxyl groups involving RFP chromophore or Glu160, the (TD-)DFT/C-PCM model 60 was applied to optimize the ground- and excited-state geometries and calculate the ground and excited-state energies. The conventional DFT XC functionals B3LYP 74 and long-range-corrected DFT (LRC-DFT) functional ω B97X-D 75,76 with basis set 6-31+G(d) were employed.

3. RESULTS AND DISCUSSION 3.1. Ground-state Thermal Equilibrium and Averaged Absorption Spectra of LSSmKate1

2

0.6

O O

O(3) H(2)

N

O(1)

NH

N O

0.3

N O

1

protein chromophore O(3)-O(1)

4

H(2)-O(1)

3

H(2)-O(3)

2

0

2

4

6

Time /ns

8

10

0.9 0.6 0.3

protein chromophore

1

0.0

0.0 2

4

6

O1.

26

A

H

O

1

1

O(3) .85 H(2)

6

H

3

O(1)

0.9

H

4

H

8

O H

1 1.9 0O(3) .96 7 0.9 H

7

NH2

N

N

8

6

9

O

10

N

O

O

H

O

B

O

5

2

8 1.4 2.02

H(2)-O(1)

O

NH2 N

H(2)

O

N N

O(1)

= 4.11

6

0.9

H(2)-O(3)

H

O H

1.6

8 O(1)-O(3)

0.96

6

0

4

1.4

2

0.99

10

3

8

1.5

6

6

4

0.9

RMSD /angstrom

RMSD /angstrom

3

2

RDF /arbitrary unit RDF /arbitrary uint

Distance /angstrom

Time /ns

0

1.33

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O

H

Distance /angstrom

Figure 1: Left panels: RMSDs for the non-hydrogen atoms of the whole RFP and the chromophores + Glu160. Middle panels: RDFs of the distances among the atoms involved in the ESPT process of LSSmKate1. The solid and dashed lines are for the neutral and anionic forms of the chromophores, respectively. Right panels: The key parts of structures involved in the ESPT taken from two trajectories at 30 ns.

At first we characterize the ground-state thermal equilibrium and calculate the averaged absorption spectra of LssmKate1 by combining MD simulations and QM/MM calculations. To sample the trajectory using a model ground-state MM force field, two equilibrium MD simulations were performed: the first one starts from the LssmKate1 structure with the protonated RFP chromophore and deprotonated Glu160, and the second one starts with the deprotonated RFP chromophore and protonated Glu160. Two initial structures were prepared by manually transferring the proton be7 ACS Paragon Plus Environment

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Normalized Oscillator Strength


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neu-chro

1.0

neu-chro+glu

ani-chro

0.8

ani-chro+glu

0.6 0.4 0.2 0.0 200

300

400

500

600

700

Wavelength /nm

Figure 2: The statistically averaged absorption spectra of LssmKate1 with neutral and anionic chromophores, produced by TD-ω B97X-D/MM with the basis set 6-31+G*. 200 snapshot configurations were sampled from each trajectory with the time duration of 20 − 40 ns. To have a close comparison with the experimental absorption spectra, the overall calculated spectra have been redshifted 0.37 eV. The locations of the two bright green lines denote the experimentally-measured absorption maxima of LssmKate1 at pH 11. tween the RFP chromophore and Glu160. As described in Section 2, MD simulations were performed with a 1 fs time step under the canonical NVT ensemble. The root mean square deviations (RMSDs) of the whole and a part of the protein structure as shown in the left panels of Figure 1 demonstrate that the MD simulations have reached dynamic thermal equilibrium. After 10 ns of MD simulation, we find that the systems equilibrate and the resulting RMSDs reach a plateau. Therefore, the snapshot configurations taken from two trajectories with the time duration of 20−40 ns were collected for the statistical analysis of molecular spectra. Since we care more about the H-bond network formed between the chromophore and Glu160 in LSSmKate1, we show the distribution functions (DFs) for the distances and angles among the atoms involved in ESPT (see the middle panels of Figure 1 and Figure S2). From the radial DFs (RDFs) collected in the first trajectory, we find that the distribution of RH(2) −O(3) centers around 1.8 Å and the H-bond between H(2) and O(3) is maintained along the first trajectory. Along the second trajectory, we observe that the additional distributions appear around 4 ∼ 7 Å on the DFs of RO(1) −O(3) and RH(2) −O(1) , and the H-bond between H(2) and O(1) is not always kept. As time evolves, the protonated Glu160 rotates away from the phenol group of the chromophore, and a dramatic change appears in ̸ O(1) H(2) O(3) at 0.7 ns as shown in Figure S2. ̸ O(1) H(2) O(3) changes from the initial value close to 1800 to around 450 . At dynamic equilibrium of the second trajectory, the H-bond network formed among O(3) -H(2) -O(1) is fully destroyed and the LSSmKate1 structure


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goes to the more stable form in ground state, which is denoted by the ‘B’ form. The key parts of the structures taken from two trajectories at 30 ns are shown in the right panels of Figure 1, indicating that after two MD trajectories reach dynamic equilibrium, the structure in the first trajectory prefers to stay with the neutral form of the chromophore (the ‘A’ form) while the structure in the second trajectory prefers to stay with the anionic form of the chromophore (the ‘B’ form). Our MD simulations clearly reveal that there are two stable forms of RFP chromophores in the ground-state structure of LssmKate1, one with protonated chromophore and the other with the deprotonated chromophore, which provides an essential precondition for the subsequent proton transfer after the photoexcitation. The ‘I’ form, which refers to the structure with the H-bond bounded deprotonated chromophore and protonated Glu160, is unstable. As the second trajectory shows, the anionic state prefers to stay with the ‘B’ form indicated by the value of ̸ O(1) H(2) O(3) ∼ 450 . The proteins enclosed in water solution or other more complex physiological environments are dynamic and flexible due to thermal motion. Therefore, in order to preferably describe the excitedstate electronic structures of the complexes, the statistically averaged spectra have been calculated and demonstrated. After dynamic equilibrium, we extracted a snapshot every 100 ps from the last 20 ns’s MD simulation (from 20 to 40 ns of the trajectory) and total of 200 snapshot configurations were sampled from each MD simulation. Then TD-B3LYP/MM and TD-ω B97X-D/MM were employed to calculate the vertical excitation energies and the corresponding oscillator strengths for the singlet excited states of each configuration. Two different-sized QM regions are considered here: the first one only includes the RFP chromophore (marked in red in the right panels of Figure 1), and the other includes both the RFP chromophore and the side chain of Glu160 (marked in green in the right panels of Figure 1). It is noted that the QM region also includes the water molecules which bind with the phenolic oxygen of the RFP chromophore with H-bonds. The final spectrum is obtained as an average of all the individual spectra. The averaged absorption spectra calculated by TD-B3LYP/MM and TD-ω B97X-D/MM are displayed in Figure S3 and Figure 2, respectively. Clearly, the size of the QM region doesn’t have an obvious effect on the excitation energies, indicating that the electronic excitation mainly takes place within the RFP chromophore. However, the effect of DFT XC functionals is significant. ω B97X-D produces an energy difference of 101 nm (0.49 eV) between two absorption maxima when the RFP chromophore transforms between the neutral and anionic states, which is less than the experimentally-measured value of 119 nm (0.56 eV). 33 B3LYP significantly underestimates this energy gap. It seems that the underestimation of this energy gap comes from the discrepancy of single-reference TD-DFT approach in calculating the excitation energies of the anionic chromophore. TD-DFT overestimates the excitation energies for the anionic states too much compared with those for the neutral states. To further validate the computed energy differences, we performed additional calculations us9 ACS Paragon Plus Environment


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ing another ab initio approach based on the scaled-opposite-spin second-order corrected configuration interaction singles, or SOS-CIS(D). 77 The SOS-CIS(D) calculations were performed with the Q-Chem program for the RFP chromophores. As reported in Table 1, for the anionic chromophore, the excitation energies produced by TD-DFT are always larger than those by SOS-CIS(D). SOSCIS(D) can yield more consistent results relative to the experimental value. For the neutral chromophore, the excitation energies calculated by TD-DFT and SOS-CIS(D) are nearly identical. Table S1 in the SI lists the calculated vertical excitation energies of low-lying excited states with the largest oscillator strengths for a few snapshots. It can be observed that the low-energy absorption maxima fluctuate, and the excitation from the ground state (S0 ) to the singlet excited state with the largest oscillator strength doesn’t always remain S0 → S1 , though the corresponding orbital transitions between the occupied and unoccupied molecular orbital (MOs) are the same (HOMO → LUMO). These results are perplexing at first glance, until the excitation-induced density difference involved in the absorption maxima are considered. From the attachment/detachment density analysis 78,79 depicted in Figure S4, all the excitations with the largest oscillator strengths are found to possess intramolecular charge transfer (ICT) characters, where the photoexcitation induces the electrons to migrate from the phenol ring to the N-acylimine moiety. The ICT character of the excited state explains why the LRC-DFT XC functional ω B97X-D produces a better result than B3LYP. The photoexcitation-induced ICT enhances the acidity of the phenolic hydroxyl group, which provides the possibility for intermolecular ESPT.

3.2. The Molecular Environmental Effect on the Geometries and Absorption/Emission Energies of Neutral and Anionic RFP Chromophores It is clear that the chromophore’s properties and dynamics are closely related to its molecular environment, and that studying the influence of the micro-environment on the chromophore’s absorption and fluorescence can give us direct and convincing evidence for changes in protein structure and function. 80–83 Here, we thus calculate the geometric and electronic structures of the RFP chromophore under different molecular environments. We put the neutral/anionic RFP chromophore or the chromophore attached by one water (marked in red and blue in the right panels of Figure 1) in vacuo, in water solution (described by the C-PCM), and in the cavity of the protein (described by MM), respectively. To optimize the ground and excited-state geometries of LSSmKate1 with QM/MM approaches, we start from three different initial conformations: the first and second ones, labeled as the ‘A’ and ‘I’ forms in Table 1, start from the crystal structure, differing in the H-bond network; the third one starts from the structure taken from the second trajectory at 30 ns (labeled as the ‘B’ form). The calculated main geometric parameters are shown in Figure 3, Figures S5−S9,


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Table 1: Calculated Dihedral Angles, Absorption/emission Energies (∆E) and Oscillator Strengths (in Parentheses) of Different Geometries with TD-ω B97X-D. The Label ‘SS’ Denotes the Difference between the Absorption and Emission Energies of Structures with the Neutral or Anionic RFP Chromophore, and ‘LSS’ Denotes the Difference of the Absorption and Emission Energies between the Structures with the Protonated Chromophore and the Deprotonated Chromophore. The Calculated Absorption and Emission Energies of the Chromophore with respect to the SOS-CIS(D) Approach are Shown behind the Slash.

neutral S0 anionic S0 S1 neutral S0 in vacuo S1 (optimized) anionic S0 S1 in vacuo neutral S0 (unoptimized) anionic S0 neutral S0 in water S1 (optimized) anionic S0 S1 Chrom. in water neutral S0 (unoptimized) anionic S0 ‘A’ S0 S1 in protein ‘I’ S0 (optimized) S1 ‘B’ S0 S1 in protein ‘A’ S0 (unoptimized) ‘I’ S0 neutral S0 in vacuo S1 (optimized) anionic S0 S1 in vacuo neutral S0 (unoptimized) anionic S0 neutral S0 in water S1 (optimized) anionic S0 S1 Chrom.+H2 O in water neutral S0 (unoptimized) anionic S0 ‘A’ S0 S1 in protein ‘I’ S0 (optimized) S1 ‘B’ S0 S1 in protein ‘A’ S0 (unoptimized) ‘I’ S0

Dihedral angles (in degree) 1-2-4-5 3-2-4-5 6-8-9-10 7-8-9-10 162.3 -19.7 -177.1 2.4

Expt.a

a

176.0 176.1 177.8 176.6

-3.4 -3.6 -1.1 -3.0

-179.1 -179.6 -178.8 178.1

3.1 2.1 4.2 1.1

175.3 176.4 -175.9 -175.8

-4.4 -3.1 4.1 4.4

170.0 -178.3 172.1 173.7

-7.9 3.7 -5.2 -4.0

166.1 166.1 166.9 163.7 -174.3 -173.2

-12.7 -12.7 -9.6 -14.6 4.1 5.9

-155.8 -168.4 -157.9 -166.9 -157.4 -158.2

15.5 6.2 11.6 6.3 18.6 17.1

174.9 175.8 -177.0 175.9

-4.4 -3.8 2.8 -3.7

177.1 -180.0 176.4 178.0

-0.4 2.3 1.9 0.6

-174.5 179.4 -175.0 -175.1

4.9 -0.3 4.7 4.9

170.9 -177.9 172.2 173.2

-7.3 4.1 -5.0 -4.2

166.0 166.1 166.8 163.8 -174.2 -173.2

-12.9 -12.7 -9.8 -14.6 4.2 5.9

-155.7 -168.3 -157.7 -166.8 -157.3 -158.4

15.6 6.4 12.0 6.3 18.7 17.1

∆E(in eV) 2.70 33 2.14 33 1.97 33 3.37 (0.83)/3.53(1.30) 2.67 (0.79)/2.62(1.31) 2.55 (1.06)/2.01(1.54) 2.36 (1.02)/1.73(1.48) 2.81 (0.59, S3 ) 2.13 (1.01) 3.25 (1.01) 2.39 (1.01) 2.64 (1.28) 2.19 (1.29) 2.59 (0.91) 2.18 (1.08) 3.27 (0.95) 2.70 (0.95) 2.84 (1.10) 2.56 (1.08) 2.97 (1.27) 2.78 (1.24) 2.55 (0.75) 2.28 (1.02) 3.40 (0.83) 2.68 (0.78) 2.58 (1.07) 2.36 (1.01) 2.84 (0.60, S3 ) 2.15 (1.00) 3.28 (1.01) 2.41 (1.01) 2.70 (1.26) 2.20 (1.28) 2.60 (0.90) 2.21 (1.07) 3.28 (0.96) 2.70 (0.96) 2.84 (1.12) 2.56 (1.09) 2.97 (1.28) 2.77 (1.25) 2.56 (0.75) 2.28 (1.03)

The data taken from the crystal structure are listed as a reference.


SS

0.17

LSS

0.69-0.72 33,34

0.70/0.91 1.01/1.80 0.19/0.28

0.86 1.06 0.45

0.57 0.71 0.28 0.19

0.72 1.04 0.22

0.87 1.08 0.50

0.58 0.72 0.28 0.20


1.3 O 5 35 1.4 12 1.40 8 0 .459 4 . 1 1 7 1.46

1 1.3.400 90

1.395 1.349

A

1.5 1.5 16 19 56NH2 1.3 4 42 35 4 . . 3 1 1 44 1.

80 1.3 76 1.3

N

N

1.5 1.5 11 11

N

1.272 1.297

O

H 1.3 O 03 1 83 1.4 16 .391 2 1.4 1.44 5 1.46

1.3 1.3 97 99

N

1. 1.3 301 34

1.5 1.5 12 10

N

O

N 1.276 1.299

59 1.3 46 1.3

O(1) 80 1.2 79 1.2

1.5 1.5 17 17 59 NH2 1.3 37 58 4 . 1 40 1.3 1.4

1.466 1.444

1.374 1.375

=3.284 3.307 1.385 1.366

8 26 1. 271 1.

1.50 1.50 9 9

O(1)

5 NH2 35 5 35 1.

1.

O

7 37 1. 370 1.

1.307 1.341

72 1.4 8 4 1.4

N

1.51 1.51 5 6

1 1. .44 44 3 6

O 1. 1.39 1. 38 2 37 7 96 4 1.397 .439 4 3 1 64 1. .41 N 1.4 1

O(3) 0.9 H(2) 6 0.9 3 62

O

1.229 H 1.238

1.39 1.39 6 9

O(3) 0.9 H(2) 0.9 78 76

1.383 1.363

1.7 5 1.7 2 86

O

B

O

1.225 1.236

83 1.3 73 1.3

I

O 68 1.3 46 1.3

1.2 1.3 93 46

1.479 1.443

O 1.26 1.2 4 65 1.0 O(3) 1 00 1.5 H(2) .004 1.5 24 16 O(1) 0 5 1.3 38 1.3

1.368 1.369

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N

1.276 1.296

H

O

1.225 1.230

Figure 3: The key parts of the ground (in dark) and excited-state (in red) geometries optimized by (TD-)ω B97X-D/MM. Table 1 and Table S2. Table 1 and Table S2 also display the calculated absorption and emission energies by TD-ω B97X-D and TD-B3LYP, respectively. For comparison, the excitation energies of the crystal structure of LSSmKate1 taken directly from the PDB without geometry optimization are also calculated and demonstrated. It is obvious that the bond lengths of the optimized structures have the overall changes compared with those of the crystal structure, especially those involved in the imidazolinone ring of the RFP chromophore. Much larger deviations appear in the dihedral angles of the bare RFP chromophore. After geometry optimization, the two rings within the RFP chromophore tend to stay in the same plane and thus the extent of the entire conjugation is enhanced. The situation is different when the RFP chromophore is set into the protein micro-environment. The discrepancy between the optimized geometries by QM/MM approaches and the crystal structure is largely decreased compared with the bare RFP chromophores in gas phase and water solution. It is clear that the protein micro-environment restricts the rotation of two rings within the RFP chromophore, resulting in a more consistent result with the experimental data. The geometric changes definitely have impact on the electronic structures. We thus calculated the absorption and emission energies of low-lying singlet excited states for the unoptimized and optimized structures with different DFT XC functionals. It is obvious that the excitation energies 12 ACS Paragon Plus Environment

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and the relative order of the excited-state energy levels are sensitive to the DFT XC functional, and molecular environment. The excitation energies of optimized structures are appreciably higher than those of unoptimized ones. With the optimized structures, all the absorption maxima as listed in Table 1 correspond to electronic transitions from S0 →S1 (the main MO changes are from HOMO to LUMO), while with unoptimized structures, the absorption maxima may not always correspond to S0 →S1 and in some cases the corresponding MO changes are mainly from HOMO−1 →LUMO as shown in Table S2. For the unrelaxed structures with the neutral chromophore in water solution and in a protein environment, both TD-ω B97X-D and TD-B3LYP yield lower-lying absorption maxima than the experimental value of 2.70 eV, while for the unrelaxed structures with an anionic RFP chromophore, both functionals produce more consistent absorption maxima relative to the experimental value of 2.14 eV. 33 In the end, the calculated energy gap between the absorption maxima of unrelaxed structures with neutral and anionic chromophores is smaller than the experimental value of 0.56 eV. 33 On the contrary, all the excitation energies of optimized structures are overestimated. The difference between the absorption maxima of the ‘A’ and ‘I’ forms produced by the TD-ω B97XD/MM approach is about 0.44 eV. TD-B3LYP/MM still significantly underestimates this value although the optimized LSSmKate1 structures are adopted. If the proposed ESPT really takes place and LSS is generated by FP chromophore transformation between a neutral state and an anionic state, the experimentally-measured Stokes shift should be equal to the energy difference between the absorption and emission maxima of LssmKate1 with the neutral and anionic forms of the chromophore, respectively. Table 1 indicates that the calculated LSSs (Eabs,‘A′ − Eem,‘I ′ ) by TD-B3LYP/MM and TD-ω B97X-D/MM are about 0.63 eV and 0.71 − 0.72 eV, respectively, which agrees well with the experimentally-measured LSS of 0.69 − 0.72 eV. This agreement provides theoretical support to the previously experimentallysuggested LSS mechanism, which is generated by FP chromophore transformation between a neutral state and an anionic state. We also calculated the geometries and absorption/emission energies of the ‘B’ form, which is the dynamic equilibrium conformation of LSSmKate1 with the anionic chromophore. We note that the energy difference between the absorption maxima of the ‘A’ and ‘B’ form, and the energy difference between the absorption maximum of the ‘A’ form and the emission maximum of the ‘B’ form largely deviate from the experimental values. It seems that the experimentally-observed fluorescence of LSSmKate1 emits from the excited ‘I’ structure. In order to inspect the polarization effect of the protein environment on the QM region, the QM region was extracted from the optimized QM/MM structure and the calculated energy gap between the absorption and emission was 0.93 eV by TD-ω B97X-D/MM (see Table S3). Through this, we can see that the protein environment has both a mechanical and electronic effect on the spectra of the RFP chromophore. 13 ACS Paragon Plus Environment


3.3 Energy Landscapes Involved in ESPT Process

4 Relative energy /ev

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S0

a)

S1

S0 S1

b)

S0 S1

c)

3 O

O

2

O O

H(2)

O(3) H(2)

H

O(1) H

O(1)

H

H

O(3)

O(3)

O

1

H

H

H(2)

O(1)

O H

H

O

H

O

0 0.8

1.2 1.6 0.8 1.2 1.6 0.8 1.2 Distance (O(1)-H(2)) /angstrom

1.6

Figure 4: S0 and S1 potential energy curves vs the O(1) −H(2) bond length produced by the (TD)ω B97X-D/C-PCM approach. Here, three different structural configurations are calculated. Next we analyzed the ESPT process involved in the photocycle. Since it is difficult to obtain the excited-state anionic structure with respect to the excited-state geometry optimization by starting from the initial structure with the protonated chromophore and deprotonated Glu160, we scanned the ground and excited-state PECs by varying the bond length of O(1) −H(2) through the constrained ground and excited-state geometry optimizations. At each optimization, only the value of RO(1) −H(2) was constrained and the other structural parameters were allowed to relax. Here, the (TD-)DFT/C-PCM approach with B3LYP and ω B97X-D functionals was used. To reveal the role of the rigorous hydrogening bond network near the RFP choromophore and Glu160 on the ESPT process, three different-sized structural configurations, as shown in Figure 4, were calculated, differing from each other by the number of waters attached to the RFP chromophore or Glu160 with hydrogening bonds. Those waters are present in the x-ray crystal structure of LSSmKate1. The results calculated by (TD-)ω B97X-D/C-PCM are shown in Figure 4, and those by (TD-)B3LYP/CPCM are shown in Figure S10 in SI. Figure 4 and Figure S10 in the SI show that the PECs of both S0 and S1 are relatively flat. The PECs of S0 are much more flat than those of S1 . The flat PECs provide an essential precondition 14 ACS Paragon Plus Environment

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for possible ESPT. Except for the second structural configuration, the others show one obvious local minimum in the PECs of S0 . It is clear that in the ground state the proton of the targeted oxygen-hydrogen bond prefers to bind with the hydroxyl group of the RFP chromophore. In the second structural configuration, one additional water molecule is bounded to the phenolic oxygen. This additional water seems to stabilize the structure with the anion chromophore, so that two local minima in the S0 PEC of the second structural configuration are observed. The situation changes when more waters are involved as Figure 4(c) shows. In the excited-state PECs of all three structural configurations, the anionic states are always more stable than the neutral states. The proton prefers to stay away from the hydroxyl group of the RFP chromophore and bind with the carboxylic group of Glu160 in the S1 . That is to say, upon photoexcitation the proton in the hydroxyl group of the RFP chromophore can easily transfer to Glu160. Once the proton completes the migration in the electronic excited state, the ESPT process is finished. Our calculated PECs provide theoretical support to the generated mechanism of LSS.

4. CONCLUDING REMARKS By combing classical MD simulations, quantum-chemistry geometry optimizations, and electronic structure calculations, we have performed a thorough study on the structures and properties of LSSmKate1. Both static (geometric optimization) and dynamic (MD simulation) approaches have been used to characterize the geometries of LSSmKate1. (TD-)DFT or (TD-)DFT coupled with PCM and MM approaches have been used to calculate geometries and electronic structures. The effects of thermal equilibrium fluctuation and molecular environment on absorption/emission energies of chromophores have been revealed. Meantime, by applying the constrained ground and excited-state geometry optimization approach, we scan the S0 and S1 PECs involved in the ESPT process. The following conclusions can be reached. (1) MD simulation reveals that RFP LSSmKate1 can possess two stable structural configurations in the ground electronic state: one with the protonated chromophore (the ‘A’ form) and the other with the deprotonated chromophore (the ‘B’ form), thus leading to two separated low-energy absorption maxima with a large energy spacing of 0.56 eV as observed by the pH-dependent experiment at pH 11. (2) The structures and properties of RFP chromophores are sensitive to molecular environment and the computational methods. Comparing the calculated Stokes shifts and absorption-maxima difference with the experimental values, TD-ω B97X-D/MM provides a better overall description of optical properties of LSSmKate1 although it always overestimates excitation energies, especially for structures with anionic chromophores. The agreement between the calculations and experi-



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mental measurements provides solid theoretical support to the previously suggested mechanism of LSS, which is generated by FP chromophore transformation between a neutral state and an anionic state. (3) At 1.0 < RO(1) −H(2) < 1.8 Å, all the S0 and S1 PECs of three structural configurations are relatively flat. In S0 , the proton involved in ESPT prefers to bind with the phenolic hydroxyl group of the RFP chromophore, while in S1 the proton prefers to stay away from the hydroxyl group of the RFP chromophore and bind with the carboxylic group of Glu160. The proton transfer is induced by electronic excitation. When LSSmKate1 is excited, the photoexcitation induces electrons to migrate from the phenol ring to the N-acylimine moiety, leading to the enhancement of the acidity of the phenolic hydroxyl group, which provides the possibility for intermolecular ESPT. The ICT character of the excited state also explains why the LRC-DFT ω B97X-D produces a better result than B3LYP. (4) Based on our calculations, we suggest the following conformational changes. The change from ‘A’ to ‘I’ is mainly a protonation change, whereas the change from ‘I’ to ‘B’ is a slow conformational change. In the ground electronic state, both the ‘A’ and ‘B’ conformations can exist, however ‘A’ is more stable than ‘B’. Upon light illumination, ‘A’ is excited, the proton in the excited state leaves the phenolic oxygen to bind with Glu160, and ‘I’ is formed. ‘I’ emits the experimentally-observed fluorescence. In the meantime, a slow conformational change occurs. Glu160 starts to rotate, the hydrogen-bonding network involved in ESPT process breaks down, and ‘I’ may become ‘B’ as the second trajectory shows.

AUTHOR INFORMATION Corresponding Authors ∗ E-mail: [email protected]; phone: +86-592-2184300. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial supports from National Science Foundation of China (Grant Nos. 21290193, 21373163, and 21573177) are gratefully acknowledged.


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Supporting Information Available The structures and spectra calculated by (TD-)B3LYP, the chosen bond lengths and angles vs the MD simulation time with different force field parameters, the detached and attached densities are shown. This material is available free of charge via the Internet at http://pubs.acs.org/.

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Figure 5: TOC graphic.


Characterizing the Structures, Spectra, and Energy Landscapes

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