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Role of Bulk Water Environment in Regulation of Functional Hydrogen-Bond Network in Photoactive Yellow Protein Koichi Tamura, and Shigehiko Hayashi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07555 • Publication Date (Web): 23 Nov 2015 Downloaded from http://pubs.acs.org on November 28, 2015

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The Journal of Physical Chemistry

Role of Bulk Water Environment in Regulation of Functional Hydrogen-Bond Network in Photoactive Yellow Protein

Koichi Tamura and Shigehiko Hayashi* Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan E-mail: [email protected] Phone: +81-75-753-4006. Fax: +81-75-753-4000

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Abstract

Photoactive yellow protein is a soluble photoreceptor protein involved in signal transduction for phototaxis.

A hydrogen-bond between the chromophore, p-coumaric acid (pCA), and a nearby

carboxyl group of Glu46 at the active site is known to play a crucial role in the formation of the signaling state in the photoactivation.

Since the hydrogen-bond at the active site as well as the

extensive conformational changes of the protein in the formation of the signaling state are considered to be controlled by water molecules, we theoretically examined influence of bulk water environment on the functionally important hydrogen-bond by means of molecular simulations.

Theoretical analysis of

potential energy profiles of the proton transfer between pCA and Glu46 with quantum mechanical/molecular mechanical (QM/MM) calculations revealed critical effect of electrostatic screening of bulk water on the electronic character of the hydrogen-bond.

Moreover, QM/MM free

energy geometry optimizations identified the water-penetrating state where Glu46 forming a putative low-barrier hydrogen-bond with pCA is hydrated by water molecules penetrating from bulk environment in addition to the water-excluded state which corresponds to X-ray crystallographic structures.

The

present results suggest that the water-penetrating state is a precursory conformational substate that leads to efficient formation of the signaling state.


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Introduction

Photoactive yellow protein (PYP) of Halorhodospira halophila is a 125-residue water-soluble cytosolic photoreceptor protein1 and considered to be involved in the negative photoaxis of H. halophila.2 PYP shows an overall α/β fold and includes a PAS core domain which is well preserved among sensor proteins and signal transduction proteins.3

The chromophore of PYP is a p-coumaric acid (pCA)4,5

covalently bonded to the side chain of Cys696 via thioester linkage.7 choromophore is deprotonated and exists as a phenolate anion.8

In the dark state, denoted pG, the

The chromophore is bound in a trans

configuration and its phenolic oxygen participates in a hydrogen bond network with neighboring Tyr42 and Glu46, and Thr50 (Figure 1a, b).9

The chromophore and its adjacent hydrogen bond network are

buried inside the hydrophobic core of the protein. The chromophore pCA and its counter-ion, Glu46, are shielded from bulk solvent via Arg52 and Ile49, respectively. PYP in pG absorbs a blue photon (the absorption maximum wavelength λmax = 446 nm) to drive a photocycle, which involves at least three major photo intermediates at ambient temperature (Figure 1c).10,11

The first red-shifted ( λmax = 465 nm) intermediate, pR, generates in nanoseconds after

photo-absorption, and later blue-shifted ( λmax = 355 nm) intermediates (pB’ and pB) appear on sub-millisecond to millisecond time scales.

The pB intermediate is rather long-lived and decays to the

initial pG state on a millisecond to second time scale.

Accordingly, pB is widely believed to be the

signaling state, although its transducer is not known.

In the transition from pG to pR upon



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photo-absorption, the trans-cis photoisomerization of the chromophore takes place.12–20

The

photo-induced conformational change at the chromophore then leads to the protonation of pCA and the deprotonation of Glu46 during the relaxation from pR to pB’.21–24

Given a spectroscopic observation

of similar kinetic behaviors of the protonation and the deprotonation, it is suggested that a proton transfer from Glu46 to pCA occurs.23 The formation of the signaling state, pB, after the protonation of pCA and the deprotonation of Glu46 in pB’ was suggested to involve extensive structural changes throughout the protein, which is sometimes referred to even as “partial unfolding”, by thermodynamic measurements,25,26 spectroscopic studies,23,27– 31

NMR spectroscopy,32,33 combined structural probes,34 pump-probe X-ray solution scattering,35 and

classical molecular dynamics (MD) simulations.36–39

Consistently, the solution NMR structure of pB

state of the PYP mutant whose N-terminal 25 residues are lacking revealed partially unfolded conformations.40

The structural changes upon the formation of pB are also known to be strongly

dependent on the surrounding environment.

For example, time-resolved X-ray crystallography

observed structural changes which are rather confined to a local site including the chromophore and the surrounding amino acids,16–18,41–43 presumably due to crystal packing that hampers large conformational changes of the protein.

Xie et al. performed FTIR spectroscopy on PYP in the crystalline lattice and no

significant structural changes were observed.23 Furthermore, it is also suggested that water environment surrounding the protein could affect the 4 ACS Paragon Plus Environment

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structure and dynamics in the photocycle.27,44

Hoff et al. examined the effect of reduced hydration on

the formation of pB by FTIR spectroscopy and observed that large conformational changes were diminished when the hydration level of PYP was reduced.27

van der Horst et al. also measured UV/Vis

spectra and kinetics of the photocycle of PYP in various degrees of relative humidity, and revealed that, in relatively low humidity environment, the lifetime of pR was prolonged while the rate of pB formation was lowered.44

The observations clearly indicate that water molecules play a role in the large structural

changes in the formation of the signaling state. The role of water molecules in the functional process is considered to be tightly correlated with the changes in the protonation states of the chromophore and its counter ion, Glu46.

Water molecules can

penetrate into the protein to solvate the newly formed anionic carboxylate of Glu46 in the buried hydrophobic environment in the formation of pB and in turn induce a crack in the protein that leads to the large structural changes referred to as “protein quake”.23

Note that pKa’s of the groups undergoing

the protonation and the deprotonation can also be strongly modulated by water molecules as well as the surrounding polar groups.

A recent neutron diffraction study for a crystalline PYP shows that pCA and

Glu46 form a low-barrier hydrogen bond (LBHB) where the proton is shared nearly equally by these residues.45

Furthermore, the authors claimed that Arg52 which is placed on the surface of the protein is

deprotonated.

On the other hand, hybrid quantum mechanical/molecular mechanical (QM/MM)

calculations for PYP with a protonated Arg52 in the isolated condition, i.e., in the gas phase, showed that 5 ACS Paragon Plus Environment


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the hydrogen bond is not an LBHB.46

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ONIOM study for PYP in the isolated condition further

examined influence of the protonation state of Arg52 on the hydrogen bond and concluded that there could be LBHB if Arg52 is deprotonated.47

Nadal-Ferret et al. investigated influence of bulk water

environment on the hydrogen bond network by QM/MM simulations and reached a conclusion that LBHB can be formed if PYP is in the isolated condition and Arg52 is deprotonated, whereas LBHB is absent if PYP is solvated and Arg52 is protonated.48

Lastly, more recent ONIOM(MC_QM:MM)

calculations for PYP with deprotonated Arg52 in the isolated condition reproduced the experimentally observed distance of LBHB.49

Those studies thus suggested that electrostatic environment generated

by the surrounding water molecules as well as Arg52 can significantly affect the pKa of pCA and Glu46, leading to the electronic and geometric changes of the functionally important hydrogen bond network. In the present study, we investigated influence of water environment on the physico-chemical nature of PYP by means of molecular simulations.

The effect of water molecules examined is twofold. First,

we analyzed effect of electrostatic field of bulk water molecules on energetics of proton transfer between pCA and Glu46.

Since pCA and Glu46 which form LBHB are not very far from bulk water

environment, electrostatic field produced by bulk water molecules is expected to exert an influence on the hydrogen bond.

Potential energy curves computed by QM/MM calculations for the proteins with

and without bulk water molecules revealed non-negligible effect of electrostatic field of the surrounding environment on the characteristics of the hydrogen-bond. 6 ACS Paragon Plus Environment

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Second, we examined behavior of water molecules inside the protein in bulk water environment. Unlike the protein in a crystalline environment from which the structural information on the hydrogen bond network in atomic resolution was obtained, it can undergo large structural fluctuation among conformational substates in bulk water environment. A possibility of redistribution of water molecules inside the protein accompanied by the thermal fluctuation of the protein structure in bulk water environment therefore needs to be examined, although occupation of water molecules inside the protein in the crystallographic structures was not observed.

For the purpose, we performed QM/MM free

energy geometry optimizations by QM/MM reweighting free energy self-consistent field (QM/MM RWFE-SCF) method,50,51 which combines QM/MM geometry optimization of the electronically active moiety with MD simulations of the protein in water environment, and thus allows one to examine dynamic behavior of the water molecules inside the protein in thermal fluctuation and its effect on geometric and electronic characters of the functionally important hydrogen-bond network.

We found

two distinct conformational substates and, in one of the two substates, water molecules penetrate into the protein and alter the character of the hydrogen-bond.

The results indicate that the water molecules play

a pivotal role in controlling the hydrogen-bond network that governs the formation of the signaling state.



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Computational Methods

Two types of QM/MM were employed in the present study.

In general, QM/MM method is a hybrid

method in which a reactive part of the system (QM part) is treated with accurate but costly quantum mechanics (QM), while the other surrounding part (MM part) is treated with empirical but efficient molecular mechanics (MM) force field.

For analysis of electrostatic effect of bulk water environment,

we employed a conventional QM/MM potential energy geometry optimization method and computed potential energy curves of proton transfer between pCA and Glu46.

For examination of redistribution

of water molecules, on the other hand, we performed QM/MM RWFE-SCF calculations50,51 by which geometry of the QM part is optimized on a mean-field free energy surface constructed with statistical samples of the MM part generated by MD simulations. The method searches free energy minima of conformational substates by iterative calculations of QM/MM geometry optimizations and MD samplings.

The iterative procedure, called the sequential sampling, is continued until simultaneous

convergences of QM geometry and statistical samplings are achieved.

Since protein’s characteristic

slow fluctuation and relaxation can be taken into account by long MD simulations of the MM part, one can search (quasi-)stable states on an extensive free energy surface. implemented QM/MM codes was used.

MD simulations.

GAMESS52 with locally

Details of the QM/MM calculations are described later.

We performed MD simulations with a classical force field for setup of the QM/MM 8 ACS Paragon Plus Environment

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AMBER12 was utilized for MD simulations.53

systems and QM/MM RWFE-SCF calculations.

Force field parameters of pCA were determined as follows.

Geometry of an isolated deprotonated

trans pCA was optimized with the DFT B3LYP functional.54

The initial coordinates was taken from a

crystallographic structure45 (PDB ID: 2ZOI) and the convergence criterion was set to be 1.0 × 10-4 a.u. for the largest component of the gradient. optimization. chromophore.55

6-31+G** basis set was employed for the geometry

RESP module of AMBER12 was used to determine RESP charges for the The restraint parameters were set to be 5.0 × 10-4.

parameters were taken from GAFF.56

Bonded and Lennard-Jones

For the system setup for QM/MM calculations of the protein

with deprotonated Arg52, partial charges of the deprotonated arginine were obtained with the same procedures above, except for the basis function (6-31G**). We constructed the simulation systems with a crystallographic protein structure of PYP45 (PDB ID: 2ZOI).

Missing hydrogen atoms of PYP seen in the crystallographic structure were added with LEaP

module of AMBER12.

The protein was immersed in the rectangular box (72 × 73 × 76 Å3) filled with

TIP3P water molecules.57

Five and six sodium ions were added to neutralize the systems with

positively charged protonated Arg52 (pArg52) and neutral deprotonated Arg52 (nArg52), respectively. Figure S1a in the Supporting Information (SI) depicts the simulation box. Periodic boundary condition was employed for MD simulations and QM/MM RWFE-SCF calculations.

The Amber ff12SB

parameter set was employed for force field of the protein except for that of the choromophore described 9 ACS Paragon Plus Environment


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above.

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The protonation state of titratable side chain was determined following observations of the

neutron diffraction experiment.45

Accordingly, Glu46 and His108 were protonated, and the protonation

states of the other titratable groups are set to be the standards ones at the neutral pH. systems with pArg52 and nArg52 were constructed.

Both of the

The simulation systems were first potential

energetically minimized with the steepest decent method and the conjugate gradient method. temperature was raised by 50 K every 100 ps until 300 K. temperature reached 200 K. after the heating.

Then,

Pressure was maintained to 1 bar after

Equilibrium MD simulations for 1 ns with NVT condition were performed

Temperature and pressure were maintained with Langevin bath (collision frequency

of 2 ps-1) and Berendsen’s method, respectively.58

Long range part of electrostatic interactions was

calculated with particle mesh Ewald method.59 Short-range non-bonded interactions were cut off at 8 Å.

Equations of motion were integrated with Brünger-Brooks-Karplus (BBK) method.60,61

bonds including hydrogen atoms were constrained by SHAKE/RATTLE method.62,63 of freedom of water molecules were fixed by SETTLE.64

Length of

Internal degrees

The integration time step was set to be 2 fs.

Protein atoms were fixed at their initial coordinates (i.e., coordinates of the crystallographic structure) during the minimization and the MD simulations for the system setup, while the fixation is removed in the MD simulations for statistical sampling.

MD calculations with AMBER12 were performed on

GPUs with SPFP mode.


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QM/MM potential energy geometry optimization.

Starting from the last snapshots of the MD

simulations for the system setup described above, temperature was lowered by 50 K every 100 ps until 10 K.

Protein coordinates were also fixed during the annealing.

The QM regions included the side

chain of Tyr42, Glu46, Thr50, Arg52 (protonated and deprotonated ones, respectively), and pCA. Sphere clusters that consist of all atoms within 30 Å from the center of mass of the QM region were taken from the last snapshots of the cooling MD simulations.

The sphere clusters contained the entire

protein and 3,347 water molecules for the system with pArg52 and 3,339 water molecules for the system with nArg52 (Figure S1b in SI).

QM/MM potential energy geometry optimizations were performed

with a method described previously.65

Dangling bonds at the boundaries of the QM and MM regions

were capped with hydrogen atoms.

Restraint parameters for the restrained electrostatic potential

(RESP) charge operators65 were set to be 0.01, except for those of boundary atoms and the boundary carbon atom of chromophore, which were set to be 0.05 and 0.1, respectively. functional was employed.66

The DFT M06-2X

Basis functions for atoms consisting the hydrogen bond network were

6-31++G**, 6-31+G** for the chromophore, and 6-31G** for others, respectively. atoms, 6-31G basis functions were employed.

For the boundary

Gradient tolerance for the QM optimization was set to

be 0.001 a.u., while that for the MM optimization was set to be 0.0001 a.u.

We optimized geometries

of all of the atoms of the QM and MM regions, except for the outermost layers of 5 Å which were fixed during the optimization. 11 ACS Paragon Plus Environment


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QM/MM RWFE-SCF free energy geometry optimization.

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The optimized structure from the

conventional QM/MM calculation was embedded in the last snapshot from the MD simulation for the system setup, and the first cycle of MD simulations of the sequential sampling was launched from the conformation.

The QM region was fixed during each cycle of the MD samplings. NVT condition

was employed for the MD samplings.

In each cycle of the sequential sampling, a MD trajectory

calculation for 3 ns was performed, and the last 2-ns part was used as the MM ensemble (20,000 samples).

After the 40th sequential sampling, the length of the MD trajectory calculation was switched

to 15 ns and the last 10-ns part was used as the MM ensemble (100,000 samples).

The procedure and

parameters of the QM/MM geometry optimizations in the cycles of the sequential sampling were the same as those for the QM/MM potential energy geometry optimization described above, except for treatment of long-range electrostatic interactions with Ewald method in QM/MM RWFE-SCF method.

Free energy evaluation.

Free energy difference between two distinct free energy minima defined by

two different geometries and electron densities of the QM part, denoted as R A , R B and q A , q B , respectively, was evaluated by a procedure described previously.51

The total free energy is written as

0 ∆FQM/MM = ∆EQM + ∆FQM-MM,MM

(1)

0 where ∆EQM is the energy difference coming from the QM Hamiltonian and ∆FQM-MM,MM is the free

energy difference originating from the QM-MM interaction and the MM part. 12 ACS Paragon Plus Environment

The former was directly

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obtained from the RWFE calculations, while ∆FQM-MM,MM was evaluated with Bennett acceptance ratio (BAR) method of free energy perturbation (FEP).67–69

For the calculation of FEP, the geometry and the

charges of the QM part were divided into 20 discrete points (N = 20) as, q ( i ) = λi q B + (1 − λi ) q A

(2)

R ( i ) = λi R B + (1 − λi ) R A ,

λi =

i , i = 1, 2,L, N − 1. N −1

(3)

Conformational samples of the MM parts at each point were obtained by a MD trajectory calculation for 50 ns; 400,000 samples taken from the trajectory of the last 40 ns were employed for the evaluation of free energy difference between the neighboring two points.

The MD simulation at each point was

started from the last snapshot of the MD trajectory calculation at the previous point. the MD trajectory calculation is therefore 1 µs.

The total length of

The forward (i.e., i is incremented starting from the

state A) and the backward (i is decremented starting from the state B) calculations were performed to judge the statistical convergence.

The number of water molecules N ( t )

Evaluation of the number of water molecules around Glu46.

at time t that coordinate to Glu46 was calculated with a smoothed step function as,70

N (t ) =

10

Nw

1 − ( di ( t ) / d 0 )

i =1

( di ( t ) / d 0 )

∑1−

20

.

(4)

Here, N w is the number of water molecules in the system, di ( t ) is the distance between the center of 13 ACS Paragon Plus Environment


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geometry of the i th water molecule and that of Glu46 at time t , and d0 is the threshold value which was set to be 5 Å.

Evaluation of vertical excitation energy.

Vertical excitation energies were calculated with three

different levels of theory, i.e., configuration interaction singles (CIS), time dependent density functional theory (TDDFT), and second order extended multi-configurational quasi-degenerated perturbation theory71 (XMCQDPT2) at the free-energetically optimized structures with the mean field electrostatic potentials.72

For the excitation energy calculations with the former two methods which are less

time-consuming but less accurate, we employed the same QM system and the same basis functions as those used for the free energy geometry optimization.

On the other hand, a smaller QM system

consisting of only pCA and Glu46, and smaller basis functions, 6-31G**, were employed for the calculations with XMCQDPT2 method as the method is too expensive to treat larger QM systems although the method is much more accurate than the former two ones since XMCQDPT2 method can explicitly deal with multi-configuration and dynamic electron correlation.

To evaluate the electrostatic

potentials in XMCQDPT calculations, we employed the free energetically optimized geometries and the effective charges of Amber force field for the atoms in the region which was a part of QM region in the free energy optimization and was then treated as a part of MM region in XMCQDPT2 calculations. CAM-B3LYP73 functional was employed for TDDFT calculations. 14 ACS Paragon Plus Environment

Complete active space (CAS) for

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XMCQDPT2 calculations consists of 12 electrons in 11 valence π orbitals of pCA. averaged CASSCF wavefunctions were refined by XMCQDPT2 calculations.


Three-state


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RESULTS

Electrostatic effect of bulk water environment. We performed QM/MM potential energy geometry optimizations for the sphere cluster systems including bulk water molecules (Figure S1b in SI) to investigate its electrostatic effect on the functionally important hydrogen bond between pCA and Glu46. First, we optimized the QM/MM systems with pArg52 (positively charged protonated Arg52) and nArg52 (neutral deprotonated Arg52), respectively, from the X-ray crystallographic structure (PDB ID: 2ZOI).

Molecular structures of the QM parts in both of the systems with pArg52 and nArg52 slightly

deviated from that in the X-ray crystallographic structure (Figure S2 in SI).

RMSDs of heavy atoms in

the QM regions with respect to those of the X-ray crystallographic structure were 0.16 Å and 0.26 Å for the systems with pArg52 and nArg52.

Marked differences were observed at Thr50 and Arg52 in both

of the systems, and the differences were larger in the system with nArg52.

Inter-oxygen distances of

the hydrogen bond network around the chromophore did not largely change by the optimization; the deviations from those in the X-ray crystallographic structure were found within 0.01–0.04 Å (Table 1). Overall, given the resolution of the X-ray crystallographic structure (1.25 Å) and the computational accuracy of the method employed in the geometry optimization, both of the QM/MM optimized structures are in good agreement with the X-ray crystallographic one. Next, the molecular nature of the hydrogen-bond between pCA and Glu46 was examined through QM/MM calculations of potential energy curves of proton transfer between them. 16 ACS Paragon Plus Environment

We employed a

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reaction coordinate of Rc = r1 − r2 shown in Figure 2a, where r1 and r2 are distance between the oxygen of Glu46 and the transferring proton and that between the oxygen of pCA and the proton, respectively.

QM/MM potential energies along the reaction coordinate were calculated by the

QM/MM geometry optimizations (Figure 2b).

We found that potential energy profiles of the proton

transfer from Glu46 to pCA in bulk water environment for both of the systems with pArg52 and nArg52 are largely endothermic and thus indicate that Glu46 is stably protonated without formation of LBHB. The increase of the potential energy upon the proton transfer for the system with pArg52 is slightly larger than that for the system with nArg52, showing Coulombic stabilization of the negatively charged pCA by the positively charged pArg52 in the initial state where pCA is deprotonated and Glu46 is protonated, respectively. In order to examine electrostatic effect of bulk water molecules, potential energy profiles along the proton transfer paths obtained above for QM/MM systems without bulk water molecules were calculated (Figure 2b).

We found that the increases of potential energy along the proton transfer are significantly

reduced without bulk water environment. reduction than that with pArg52.

Especially, the system with nArg52 undergoes a larger

Consequently, potential energy of the final state of the proton transfer

where Glu46 accepts the proton from pCA is close to that of the initial state, suggesting a possibility of formation of LBHB. We further analyzed the electrostatic effect of bulk water molecules by decomposing the potential 17 ACS Paragon Plus Environment


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energy curves into several components.65

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The total QM/MM potential energy EQM/MM is expressed by

a sum of three components as EQM/MM = E0 + EQM-MM + EMM

(5)

where E0 is the expectation value of the electronic Hamiltonian of the QM part, EQM-MM is the QM-MM interaction, and EMM is the potential energy of the MM region.

EQM-MM component is

further decomposed into contributions from protein and bulk water environment including water molecules and ions.

Figure 2c,d depict potential energy curves of the decomposed components.

The

potential energy curves solely including the bare QM energies show steep increase along the proton transfer paths.

The increases are then significantly reduced by the QM-MM interaction of the protein

without bulk water environment.

Finally, the QM-MM interactions of the bulk water environment

contribute to increase of the potential energy curves along the proton transfer and thus compensate the reduction by the QM-MM interaction of the protein. The behavior of compensation clearly indicates that the main electrostatic effect of the bulk water environment is screening of the preorganized strong electrostatic field of the protein. We also mapped electrostatic potentials coming from the bulk water molecules onto the QM molecules (Figure 2e).

In the case of the system with nArg52, the screening of the bulk water

molecules produce largely positive electrostatic potentials in the QM region.

Especially, the positive

electrostatic potentials on pCA are significantly larger than the other region.

On the other hand, the


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positive electrostatic potentials on Arg52 as well as pCA are reduced in the system with pArg52 due to strong hydration of bulk water molecules to the positively charged pArg52.

Because of the weakening

of the positive electrostatic potentials of the bulk water molecules on pCA, difference in the electrostatic potentials between Glu46 and pCA, i.e., the proton donor and acceptor, respectively becomes small, explaining the smaller electrostatic effect of the bulk water environment for the system with pArg52 than that with nArg52 observed in Figure 2b.

Penetration of water molecules from bulk. We next investigated dynamic behavior of water molecules inside the protein by classical MD and QM/MM RWFE-SCF simulations.

In the present study, we only

report results of the system with pArg52, and not those of the system with nArg52, since we found in preliminary simulations that more elaborate treatments are required for simulations of the system with nArg52. Note that it does not mean that possibility of the deprotonation of Arg52 is ruled out.

Study

on the system with nArg52 will be reported in the future. In the crystallographic structures of dark state,9,13,14,45,74–78 water molecules are not observed around the active site inside the protein.

However, transient penetration of water molecules to the active site

around Glu46 inside the protein in bulk environment was frequently observed in classical MD simulations (Figure S3b in SI), although the overall structure of the protein was kept intact as indicated by small Cα-RMSD with respect to the X-ray structure (Figure S3a in SI). 19 ACS Paragon Plus Environment

The observation indicates


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that thermal fluctuation in bulk water environment allows the protein to transiently bring water molecules inside the protein and thus to modulate electrostatic environment inside the protein. However, the classical MD simulation using a fixed charged molecular model is not capable of capturing electronic changes of the highly chemically active site around pCA and Glu46 that are expected to couple with the modulation of electrostatic environment.

We therefore carried out QM/MM

RWFE-SCF geometry optimizations which permit one to describe the electronic changes at the active site induced by the modulation of electrostatic environment due to penetration of water molecules from bulk in thermal fluctuation. In QM/MM RWFE-SCF, iterative sequential sampling is performed until description of mean field potential with reweighting scheme becomes valid (see Computational Methods).

The free energy

geometry optimization required 58 cycles of sequential sampling with MD trajectory calculations for 390 ns in total.

During the geometry optimization, Cα-RMSD with respect to the X-ray structure

increased only slightly (Figure 3a), indicating that the overall structure of the protein was almost maintained, and is comparable to that of the classical MD simulation (Figure S3a in SI).

On the other

hand, behavior of water molecules penetrating into the inside of the protein underwent drastic changes in QM/MM RWFE-SCF simulations (Figure 3b-e).

From the beginning of MD samplings in QM/MM

RWFE-SCF geometry optimization, transient water penetration from bulk environment to the active site around pCA and Glu46, which was also seen in the classical MD simulations described above (Figure 20 ACS Paragon Plus Environment

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S3b in SI), was observed.

However, around 50 ns, a water molecule transiently intruding into the

active site was captured by a strong hydrogen-bond with Glu46 (Figure 3b), and triggered formation of a single file water chain with 3–4 molecules accompanied by rotation of the sidechain of Ile49 which opens the channel to Glu46 (Figure 3c-e).

The newly found state with the single file water chain

penetrating from bulk environment to the active site, which we call the water penetrating state hereafter, was observed to be stable during the rest of MD samplings in QM/MM RWFE-SCF geometry optimization for ~300 ns (Figure 3a, b).

The stability of the water penetrating state was further

confirmed by an 800-ns classical MD simulation starting at the last snapshot of the QM/MM RWFE-SCF geometry optimization with fixed charges and coordinates of the optimized QM regions (Figure S4).

The protein backbone was stable and the water molecules kept penetrating inside the

protein during the simulation.

RMSD of the heavy atoms in the QM region with respect to those of the

X-ray crystallographic structure was 0.47 Å (Figure S5a in SI), which is slightly larger than those of the structures obtained by the QM/MM potential energy geometry optimization.

The deviation comes

from the fact that the QM region was optimized on a free energy surface of the system in the bulk water environment which is significantly different from the crystalline one.

Nevertheless, the deviation is

still small and thus the overall QM geometries in those two environments are qualitatively the same. The marked difference in the behavior of the water molecules between the trajectories obtained by the classical MD simulation and QM/MM RWFE-SCF one originates from coupling between the electronic 21 ACS Paragon Plus Environment


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structure of the active site and the electrostatic modulation of the transient penetration of water molecules around the active site, which the classical MD simulation cannot take into account.

Figure 4

shows changes in inter-atomic distances and RESP charges of the hydrogen-bond between pCA and Glu46 during QM/MM RWFE-SCF geometry optimization.

One can clearly discern that the

characteristic hydrogen-bond between pCA and Glu46 becomes significantly stronger after the formation of the penetrating single file water chain at ~58 ns (20th cycle of the sequential sampling). During the free energy geometry optimization, the O-O distance between pCA and Glu46 shrank by 0.13 Å from 2.60 Å to 2.47 Å, and the O-H bond distance of Glu46 elongated by 0.05 Å from 1.01 Å to 1.06 Å (Figure 4a-c).

The strengthening of the hydrogen-bond accompanied partial negative charge

migration from pCA to Glu46 (Figure 4d-f), which was stabilized by the hydration of the penetrating water chain.

A water-excluded conformational substate. As mentioned above, the penetration of water molecules was not found in crystallographic structures of dark state.9,13,14,45,74–78

It is therefore expected that a

conformational substate where the penetrating water molecules were excluded exists.

We thus

attempted to model the water excluded conformational substate by QM/MM RWFE-SCF free energy geometry optimization as well.

First, we obtained an initial structure of the free energy geometry

optimization by a conventional QM/MM potential energy optimization with a constraint which fixed the 22 ACS Paragon Plus Environment

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O-O distance between pCA and to be 2.7 Å, i.e., the distance longer by 0.1 Å than that fully optimized (2.6 Å).

We carried out the QM/MM RWFE-SCF optimization with the constraint of the fixed O-O

distance from the initial structure, and then the system was fully free energetically optimized with the QM/MM RWFE-SCF calculation without a constraint.

The numbers of the sequential sampling were

22 (66 ns) for the QM/MM RWFE-SCF optimization with the constraint and 2 (6 ns) for that without the constraint.

During the optimization processes, no water molecule was captured by Glu46 due to the

weaker interaction attributed to the elongated hydrogen-bond, and consequently a free energy minimum of the conformational substate where water molecules were excluded from the active site of pCA and Glu46 was successfully found.

The stability of the water-excluded state was confirmed by an 800-ns

classical MD simulation as was done for the water-penetrating state above (Figure S6).

Table 1 shows

the geometric parameters of the hydrogen-bond network of the active site in the optimized structure. The O-O distance between pCA and Glu46 was 2.68 Å, which was significantly larger than that of the X-ray structure (2.56 Å) and in the water penetrating state (2.47 Å).

RMSD of the heavy atoms in the

entire QM region with respect to those of the X-ray crystallographic structure was 0.34 Å (Figure S5b), indicating that the deviation of the optimized QM structure in the water excluded state from the X-ray crystallographic one is comparable to that in the water penetrating state, 0.47 Å (Figure S5a).

Characterization of the hydrogen-bond by vibrational analysis. 23 ACS Paragon Plus Environment

The nature of the hydrogen-bonds


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between pCA and Glu46 in the water-penetrating state is remarkably different from that in the water-excluded one.

Since the difference in the hydrogen-bond strength is expected to be detected by

infra-red (IR) spectroscopy, we performed normal mode analysis of the QM region on the free energy surface,51 and characterized the hydrogen-bond in terms of vibrational frequency.

We focused on the

vibrational frequency of C=O stretching mode of Glu46, which was assigned by a previous Fourier-transform (FT)-IR measurement.21

Given a scaling factor of 0.940 for vibrational frequency

computed with M06-2X/6-31+G**,79 the vibrational frequency of C=O stretching mode in the water-excluded state was calculated to be 1732 cm-1.

The frequency for the potential energetically

optimized QM/MM structure (Figure S1b) where the hydration of Glu46 is absent was found to be 1731 cm-1, which is similar to that in the free energetically optimized water-excluded state.

The frequencies

of ~1730 cm-1 are within a typical frequency region of C=O stretching mode of a protonated carboxyl group.

On the other hand, the frequency in the water-penetrating state was calculated to be 1653 cm-1,

which is greatly down-shifted from that in the water-excluded one due to its LBHB character.

Thus,

the frequency of C=O stretching mode of Glu46 is a sensitive indicator of the hydrogen-bond character.

Photoabsorption energy calculations. We examined excitation energies of the water-excluded state and the water-penetrating one to evaluate effect of their difference in the hydrogen-bond between pCA and Glu46 on the visible spectrum. We performed excitation energy calculations with CIS, TDDFT, and 24 ACS Paragon Plus Environment

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XMCQDPT2 methods (see Computational Methods).

The excitation energy of the water-excluded

state was computed to be consistently smaller than that of the water-penetrating state, although the magnitude of the shift (16–38 nm) varies depending on the condition of the calculation (Table 4).

Free energy difference between conformational substates.

In order to evaluate populations of the two

conformational substates, i.e., the water-penetrating state and the water-excluded one, we obtained free energy difference between them by FEP calculations with BAR method51 (see Computational Methods for details).

In the FEP calculations, the geometry and the charges of the QM part were gradually

transformed from one state to the other.

We performed the forward (i.e., transformation from the

water-excluded state to the water-penetrating one, denoted E→P) calculation and the backward (P→E) one, and found in the both directions that the seemingly small changes of the electronic and geometric structures of the QM part reproducibly led to the conformational changes of the protein and the water molecules in the MM region associated with the transition between the water-penetrating state and the water-excluded one.

Figure 5 shows temporal changes of the number of water molecules around Glu46.

During the gradual change of the QM part between the water-excluded state and the water-penetrating one in the FEP calculations reversibly induced the formation and the exclusion of the water-penetrating chain, respectively.

The observation indicates the tight coupling of the electronic and geometric

structures of the active site with the characteristic behavior of the water molecules in the conformational 25 ACS Paragon Plus Environment


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substates. Table 2 summarizes the energetics associated with the transition between the conformational substates. The free energy difference between the substates was evaluated to be ~2.5 kcal/mol.

Discrepancy

between the free energies calculated in the forward and backward directions is small (0.4 kcal/mol), indicating sufficient convergence of the statistical sampling in the free energy calculations.

The

sufficient statistical convergence also justifies the choice of the sequence of virtual intermediate states that connects two end states in the FEP calculation; although the present choice of the sequence (Eqs. 2 and 3) is somewhat artificial compared with a natural sequence, e.g., the minimum free energy path, one can choose an arbitrary sequence when statistical samples available for the calculation are so sufficient that the sequence can be regarded as a quasi-static process. The components of the free energy difference indicate that, in the formation of the water-penetration state, large free energy stabilization by the QM-MM interaction originating from the hydration of the water-penetrating chain compensates large increase of the QM internal energy.

The free energy

calculation showed that the water-penetrating state is slightly more stable than the water excluded one. However, the water-penetrating state is considered to be overstabilized in the present calculations due to a possible overestimation of the stabilization of the hydration of the water-penetrating chain to the partially anionic carboxyl group of Glu46 with the present treatment of the QM-MM interaction employing a point charge approximation.65,80

Taking into account the possible overstabilization of the 26

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water-penetrating state, therefore, those substates are expected to be free energetically close. Unfortunately, their relative stability cannot be determined due to the poor treatment of the QM-MM interaction.

A more accurate treatment of the QM-MM interaction,80 yet with sufficient statistical

sampling, would be necessary to definitively determine the free energy difference and the populations of the conformational substates.



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DISCUSSIONS

The QM/MM potential energy calculations of the reaction profile of the proton transfer from Glu46 to pCA in the present study (Figure 2) systematically showed that the nature of the functionally important hydrogen-bond between Glu46 and pCA strongly depends on the environment of the protein and the protonation state of Arg52. studies.46–49 studies.

The results are, roughly speaking, consistent with those of previous

Table 3 compares the geometric parameters of the hydrogen-bond obtained in the previous

In the bulk water environment, the potential energy of the proton transfer in the system with

pArg52 steeply increases (Figure 2b), suggesting formation of the normal hydrogen-bond where the proton is localized at Glu46.

The formation of the normal hydrogen-bond in the system with pArg52 in

the bulk water environment was also observed in a QM/MM MD simulation by Nadal-Ferret et al.48 and ONIOM calculations by Kanematsu and Tachikawa.49

In the absence of the electrostatic field of the

bulk water environment, the protonation state of Arg52 makes a marked difference in the nature of the hydrogen-bond.

The proton was suggested to be still localized at Glu46 in the system with pArg52, as

suggested in the previous studies of QM/MM calculations for the system in the isolated condition,46–49 although the increase of the potential energy is somewhat attenuated.

In the case of the system with

nArg52, on the other hand, the increase of the potential energy is largely reduced, and consequently LBHB could be formed when the quantum effect of the proton is taken into account as suggested by Kanematsu and Tachikawa.49 28 ACS Paragon Plus Environment

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The present analysis revealed that the strong dependence of the hydrogen-bonding character on the surrounding environment of the protein originates from screening of the electrostatic field generated by polar groups on the surface of the protein.

Because the protein is relatively small and the active-site is

close to the protein surface, the electronic structure of the active site is largely affected by the electrostatic field of the surface polar groups.

The bulk water environment screening the strong

electrostatic field of the surface polar groups therefore needs to be explicitly taken into account for the analysis of the electronic and geometric structure of the active site in water solution.

It would also be

necessary to explicitly consider the effect of the surrounding proteins in the protein crystals to examine the geometric structure of the active site, especially the formation of LBHB, observed in the diffraction experiments.45

The electrostatic field generated by the surface polar groups is expected to be

complexly modulated by heterogeneous environment in the protein-protein interface in the crystalline packing structure. The QM/MM RWFE-SCF free energy geometry optimizations which allow one to simulate explicit correlation of electronic and geometric changes of the QM region with large and slow conformational changes of the protein in bulk water environment identified the conformational substate of the water-penetrating state in addition to the substate of the water-excluded one which corresponds to the crystallographic structure.

Although hydration of Glu46 as seen in the water-penetrating state was

previously observed for the photoactivated signaling state, pB, where the photoisomerization of pCA 29 ACS Paragon Plus Environment


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and the proton is already translocated from Glu46 to pCA,36,37,81,82 the present simulations showed that the hydration of Glu46 can also occur in a conformational substate of the ground resting state, pG, through the coupling with the formation of the LBHB-like hydrogen bond between pCA and Glu46.

It

should be noted that the very short oxygen-oxygen distance observed is only an indirect phenomenon associated with LBHB formation, and the calculation of the nuclear delocalization of the proton is necessary to completely demonstrate the existence of LBHB.48,49

Unfortunately, the evaluation of the

quantum nuclear effect in addition to taking into account the thermal fluctuation of the protein surroundings requires a calculation of the free energy surface of the reaction coordinates, and thus is a computationally formidable task.

We therefore leave the evaluation for the future study.

The free energies of those substates are suggested to be similar, although their populations could not definitively be determined due to limitation of the computational accuracy.

A FT-IR measurement

showed a vibrational signal of C=O stretching mode of Glu46 at 1740 cm-1 in pG,23 which is in good agreement with the calculated frequency in the water-excluded state, 1732 cm-1, and is much higher than that in the water-penetrating state, 1653 cm-1. populate at least partially in water solution.

The water-excluded state is therefore suggested to On the other hand, the calculated frequency of the

water-penetrating state, 1653 cm-1, is considerably down-shifted from that of the water-excluded state. Unfortunately, a clear signal corresponding to the vibrational mode with the down-shifted frequency cannot be seen since a large band of amide I and pCA vibrations is present in the frequency region 30 ACS Paragon Plus Environment

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around 1650 cm-1,23 although a slight depletion of difference absorbance from pG one in the pR-to-pB’ transition in the frequency region around 1650 cm-1, which possibly represents existence of a mode in pG and thus can be a candidate for the down-shifted mode, was observed.

It is therefore suggested that

a detailed spectroscopic examination around this frequency region may clarify the extent to which the water-penetrating state is involved as a conformational substate in pG. We also calculated the excitation energies of the water-excluded state and the water-penetrating one to examine their properties of visible photoabsorption spectrum.

The red shift of the water-excluded state

obtained by the calculations (Table 4) originates from less stabilized and more delocalized negative charge of the phenolate anion of pCA in the electronically ground state due to the weakened hydrogen-bond with Glu46.

The mechanism of the red shift is suggested to be the same as that for an

experimentally observed red shift of E46Q mutant by ~15 nm,83–87 because pKa of the amide group of the sidechain of glutamine is much higher than the carboxyl group of glutamic acid and thus the former is expected to form a weaker hydrogen-bond with pCA than the latter.

The weaker hydrogen-bond in

E46Q is consistent with the higher pKa of pCA in the mutant observed experimentally.85,87,88 Unfortunately, the present calculations did not provide conclusive results of the magnitude of the difference in the excitation energy between the water-excluded state and the water-penetrating one, which was found to vary in 16–38 nm depending on the condition of the calculation (Table 4), due to difficulty in accurate calculations of the excitation energy of the present system. 31 ACS Paragon Plus Environment

In the case of CIS and


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TDDFT calculations, the absolute excitation energies are largely overestimated compared to the experimental one presumably due to lack of multi-configurational description.

On the other hand,

XMCQDPT calculations improved the absolute excitation energies to some extent because of the explicit description of multi-configuration and dynamic electron correlation, although the energies are still slightly overestimated.

However, the difference in the excitation energy between the

water-excluded state and the water-penetrating one might be overestimated due to an artifact coming from the smaller QM size. If the difference in the excitation energy lies below 20 nm, the two components of absorption from the water-excluded state and the water-penetrating one could not clearly be distinguished since the difference is much smaller than the full width of half maximum of the absorption band, ~60 nm.83–87 Nevertheless, a narrower band width of E46Q spectrum by ~500 cm-1 for the half band width87 might be explained by the mixing of the two components of absorption in the spectrum of the native protein as the mutation is expected to modulate the population of the two conformational substates.

On the other

hand, if the difference in the excitation energy is ~40 nm which is comparable to the band width, given the experimental evidence that the absorption band consists of a single peak and the evidence by the FTIR measurement that the water-excluded state is populated, it would be concluded that the absorption band mainly originates from the water-excluded state and the population of the water-penetrating state is minor.

More extensive excitation energy calculations will be necessary to determine the populations of 32 ACS Paragon Plus Environment

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the conformational substates. The finding of the water-penetrating conformational substate provides an insight into the functional conformational changes of PYP in the photocycle.

The present simulations demonstrated that the

transition between the two substates involving penetration and exclusion of the water molecules in the active site is strictly coupled with the change of the electronic and geometric character of the hydrogen-bond between pCA and Glu46.

The water-penetrating state can therefore be considered to

contribute to efficient formation of pB’ where the protonation of pCA and the deprotonation of Glu46 take place without large conformational changes of the protein.

Xie et al.23 argued that an unstable

buried negative charge of Glu46 in a hydrophobic interior generated without large conformational changes in pB’ drives hydration of the buried charge accompanied by large protein conformational changes referred to as “partial unfolding” for the signaling in the following formation of pB.

A

question here arises: how is such an unstable buried charge generated without large conformational changes in the formation of pB’ which is kept quasi-stabe on millisecond time scale (Figure 1c)?

The

unstable deprotonation state in a hydrophobic environment is apparently expected to hardly form spontaneously because of its instability.

Although the induction of the unstable charged state is

obviously related with the conformational changes of pCA upon photoisomerization, molecular mechanism of the efficient formation of the charged state of Glu46 without large conformational changes in the formation of pB’ is still elusive. 33 ACS Paragon Plus Environment


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The existence of the water-penetrating conformational substate where the partial hydration of Glu46 is brought about revealed in the present study, together with the concept of the dynamic energy landscape established for the enzymatic catalysis of dihydrofolate reductase,89,90 provides a clear molecular mechanism of efficient modulation of the protonation state of Glu46.

In the view of the dynamic

energy landscape, a conformational substate existing in a chemical state in an enzymatic turnover cycle becomes the main conformational state in the following chemical state so that the turnover cycle of the enzymatic chemical reactions associated with large conformational changes of the protein proceeds efficiently.

In the case of PYP, the free energy of the conformational substate of the water-penetrating

state capable of partially hydrating Glu46 and reducing its proton affinity without large protein conformational changes was found to be as low as that of the water-excluded state even in pG with the chromophore in the trans conformation, which is considered to be the inactive conformation preventing further development of the formation of the charged state of Glu46.

Upon the photoisomerization of

the chromophore, the protein can use the low free energy water-penetrating conformational substate to efficiently reduce the proton affinity of Glu46 with the local conformational perturbation at the chromophore, eventually leading to the quasi-stable formation of the deprotonated Glu46 in pB’ even without large conformational changes.

The water-penetrating conformational substate is therefore

suggested to enable an efficient formation of pB’ and thus to play a role in connecting between the largely energy compensating events of the local deprotonation of Glu46 in the hydrophobic interior and 34 ACS Paragon Plus Environment

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the extensive conformational changes of the protein for the signaling.

Unfortunately, such a proposed

conformational substate is hardly detected experimentally because it populates only partially and/or transiently.

More elaborated molecular simulations will be needed to atomistically elucidate the

functional processes concerted across an extensive range of spatial scales in the future study.



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CONCLUSIONS

We performed molecular simulations of PYP to examine influence of bulk water environment on the functionally important hydrogen-bond between pCA and Glu46 in the active site.

The theoretical

analyses of the potential energy profile of the proton transfer between those groups obtained by the QM/MM calculations revealed that the electrostatic field generated by the polar groups on the surface of the protein can largely alter the potential energy profile, and electrostatic screening of bulk water environment surrounding the polar groups reduces the electrostatic effect of the surface polar groups. Furthermore, the QM/MM free energy optimization with QM/MM RWFE-SCF method identified the water-penetrating state where Glu46 forming a putative LBHB with pCA is hydrated by water molecules penetrating from bulk water environment, in addition to the water-excluded state which corresponds to the X-ray crystallographic structure, in the ground resting state, pG. are suggested to be free energetically close.

Those conformational substates

Given the tight coupling between the hydration of Glu46

and the formation of a putative LBHB demonstrated by the present simulations, the water-penetrating is suggested to play a role in efficient formation of the signaling state in the photoactivation.


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Supporting Information Available Supplementary table S1 and figures S1–S6. This material is available free of charge via the Internet at http://pubs.acs.org. Acknowledgments This work was financially supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (25104004 and 25291034), the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. Some computations were performed at the Research Center for Computational Science, Okazaki, Japan. created with VMD.91


Molecular figures were


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Page 45 of 54

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Table 1. Geometric parameters of the hydrogen bond network in the active site (unit in Å)†. E46 – pCA

Y42 – pCA

Y42 – T50

QM/MM(pArg52)a

2.60

2.51

2.87

a

2.55

2.53

2.90

b

RWFE(pArg52, P)

2.47

2.57

2.82

RWFE(pArg52, E)c

2.68

2.50

2.75

explt. (PDB ID: 2ZOI)

2.56

2.52

QM/MM(nArg52)

†

Inter-oxygen distances between indicated residues are depicted. b

QM/MM optimizations. water-penetrating state, P).

2.91 a

Results from the conventional

Results from the QM/MM RWFE-SCF optimization (the c

Results from the QM/MM RWFE-SCF optimization (the

water-excluded state, E).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 54

Table 2. Free energy differences between the water-excluded (E) and the water-penetrating state (P) and their energy components. (unit in kcal/mol). 0 ∆EQM

∆FQM-MM,MM

∆FQM/MM

forward (E → P)

+11.7

–14.0

–2.3

backward (P → E)

–11.7

+14.4

+2.7


Page 47 of 54

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Table 3. Comparison of hydrogen bonding distances (Å) in PYP in the dark state determined by previous studies and the present one.

OGlu46–OpCA

OGlu46–HGlu46

HGlu46–OpCA

Arg52

Environment

deprotonation

of PYP

1

2.57

1.00

1.58

no

isolated

2

2.56

1.05

1.51

yes

isolated

3

2.56

1.05

1.51

no

isolated

4

2.56

1.05

1.51

yes

isolated

5

~2.9

N.A.

N.A.

no

solvated

6

2.49

1.06

N.A.

yes

isolated

7

2.52

1.04

N.A.

no

isolated

8

2.47

1.08

N.A.

yes

isolated

9

2.60

1.01

1.59

no

solvated

10

2.55

1.03

1.53

yes

solvated

11

2.47

1.06

1.42

no

solvated

12

2.68

1.00

1.69

no

solvated

13

2.56

1.21

1.37

yes

crystal

1

Saito and Ishikita.46

2–3

Results obtained with single point energy calculations by Hirano and Sato.47

4

Results obtained with single point energy calculations by Nadal-Ferret et al.48

5

Results obtained with QM/MM-MD simulation by Nadal-Ferret et al.48

Average

values for OGlu46–XGlu46 and XGlu46–OpCA distances are not available. 6–8 9-12

System 2-dp (6), System 2-p (7), and System 3-dp (8) in Kanematsu and Tachikawa.49 Results from the present study.

Results obtained from the QM/MM potential energy

calculations (9–10) and the QM/MM RWFE-SCF simulations (11–12). 13

X-ray/neutron diffraction data (PDB ID: 2ZOI).45



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Table 4. Vertical excitation energies of the water-penetrating state and the water-excluded state (eV). Excitation energy CISc TDDFT(CAM-B3LYP)c XMCQDPT2 Experiment a b

d

Shifta,b

Water-penetrating state

Water-excluded state

4.24

4.11

0.13 (+20)

3.47

3.37

0.10 (+16)

3.11

2.85

0.26 (+38)

c

2.78 (446 nm)

Shifts of excitation energies of the water-penetrating state from that of the water-excluded state. Values in parentheses are wavelength shifts (nm) on the assumption that the absorption maximum of the water-excluded state is 446 nm.

c

The QM system is the same as that used for QM/MM RWFE-SCF free energy geometry optimization.

d

The QM system consists of only pCA and Glu46.

e

Meyer.1


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Figure 1. a A crystallographic structure of PYP in pG (PDB ID: 2ZOI). The protein backbone is drawn in transparent yellow ribbon representation. Heavy atoms of the chromophore and side chains of Tyr42, Glu46, Thr50, and Arg52 are depicted in stick representation. Carbon, oxygen, nitrogen and sulfur atoms are colored in cyan, red, blue and yellow, respectively. b Schematic picture of the hydrogen-bond network in the active site. c A simple description of the photocycle of PYP.



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Figure 2.

a Definition of the reaction coordinate of the proton transfer Rc .

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b Potential energy

curves of the proton transfer. c,d Energy components of the potential energy curves. The full potential energy curves were represented by blue lines (protein+bulk water+ion). Results of the systems with pArg52 (c) and nArg52 (d) are shown. e Electrostatic potentials on QM atoms generated by MM water molecules. QM atom indexes are tabulated in Table S1.


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Figure 3. a Temporal changes of Cα-RMSD with respect to an X-ray structure (PDB ID: 2ZOI) during the sequential samplings of the free energy geometry optimization. b Temporal changes of the number of water molecules around Glu46 during the sequential samplings. c Protein structure before the free energy optimization. d Protein structure after the free energy geometry optimization. Protein backbone is shown in yellow ribbon representation. Ile49 is depicted in vdW sphere representation and colored according to the atom types. Residues near Ile49 are depicted in vdW sphere representation and colored according to the residue index. In panel d, water molecules are depicted in ball-and-stick representation (oxygen atoms are colored in red and hydrogen are in white). e A snapshot of the water penetration. pCA and Glu46 are shown in stick representation, and water molecules are drawn in vdW representation.



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Figure 4. a-c Changes of hydrogen bond distances during the sequential samplings of the free energy geometry optimization. d-f Changes of RESP charges of HE2 (d), OE2 (e), and OE1 (f) of Glu46 during the sequential samplings. g Definition of the atom name.


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Figure 5. Temporal changes of the number of water molecules around Glu46 in the forward (a) and the backward (b) free energy calculations.



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for Table of Contents use only


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Role of Bulk Water Environment in Regulation of ... - ACS Publications

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