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2009, 113, 3257–3263 Published on Web 02/23/2009
1,2-Dichloroethane in Haloalkane Dehalogenase Protein and in Water Solvent: A Case Study of the Confinement Effect on Structural and Dynamical Properties N. Arul Murugan* and Hans Ågren Department of Theoretical Chemistry, School of Biotechnology, Royal Institute of Technology, SE-10691 Stockholm, Sweden ReceiVed: September 30, 2008; ReVised Manuscript ReceiVed: February 4, 2009
The structural and dynamical properties of an individual molecule is much affected by changes in a surrounding solvent or protein environment. The focus of the present study is to investigate such changes between proteins and solvents, using as an illustrating example the structure and dynamics of 1,2-dichloroethane (DCE) within haloalkane dehalogenase (HAD) as protein and within water as solvent. We have studied DCE within HAD using Car-Parrinello molecular dynamics calculations in a quantum mechanics/molecular mechanics set-up. We find that the C-Cl bond length is shorter in HAD when compared to solution phase value, whereas the net atomic charges and dipole moment are significantly larger than the solution phase values. In contradiction to the usual trend that molecules in the vicinity of the proteins are less polar, we report the observation that the protein environment indeed polarizes the DCE solute more than the water solvent. Furthermore, within the protein environment we do not observe any conformational transition between gauche and trans conformers, and the DCE remains in the more polar gauche conformer during the entire simulation time scale. However, the trans conformer interconverts to the gauche conformer spontaneously within 0.4 ps, which clearly indicates that the trans conformer is unstable within the HAD protein. In contrast, the scatter diagram of total Kohn-Sham energy and dihedral angle between Cl-C-C-Cl atoms shows that the gauche and trans conformers have comparable energies in water. Overall, the present calculations show the within the protein not only the structure of DCE is altered but also that the conformational interconversion dynamics is affected very much. To investigate the confinement effect on the conformational equilibrium, we have also carried out force-field molecular dynamics calculations which show that the population of trans conformer is significantly lower within the protein when compared to that in water solvent. 1. Introduction The structural and dynamical properties of individual molecules are highly affected by the nature of the environment.1-4 A well-known classical example is 1,2-dichloroethane (DCE), which shows a remarkable solvent dependence of the conformational distribution.1,5-7 It exists in two major conformations, namely gauche (with dihedral angle between Cl-C-C-Cl, (60°) and trans conformations (with dihedral angle between Cl-C-C-Cl, 180°). Due to electrostatic interaction, the trans conformation is stabilized in the gaseous phase with respect to the gauche conformation.8 In solvents, the conformational distribution is highly affected depending upon the polarity. In a more polar solvent, such as water, the gauche conformer is stabilized with respect to the trans conformer, and the stability order is thus reversed when compared to the gas-phase.1,5-7 This behavior has been attributed to the difference in the dipole moments of these conformers. Because of dipole-dipole interaction, the gauche conformer (with a dipole moment of 3.5 debye) is more stabilized than the trans conformer (with a dipole moment close to zero) in polar solvents.7 Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations carried out on DCE in polar and nonpolar solvents have clearly shown * To whom correspondence should be addressed. E-mail: murugan@ theochem.kth.se.
10.1021/jp808647c CCC: $40.75
that the gauche conformer is more stabilized in water,9 whereas the trans conformer population was relatively larger in chloroform, which is nonpolar.9 A careful analysis on the solvent dependence of the charge distribution and dipole moments indicated that the solute molecule is highly polarized in a water solvent.9 The net atomic charges were almost twice in magnitude when the solute molecules were placed in water solvent instead of chloroform solvent.9 It is an interesting aspect to look into the effect of a change from a solvent to a protein on the charge distribution and dipole moment of the solute molecule. In the present work we have investigated the role of a protein, namely haloalkane dehydrogenase (HAD), on the structure and dynamics of a substrate or solute molecule, DCE. The study is motivated by the importance to understand where proteins are placed in the polarity scale when compared to solvents and to learn how much the proteins polarize the solute. We have therefore calculated the charge distribution and dipole moment of DCE in HAD and have made a comparison to the corresponding values for DCE in water. Another important motivation behind the present calculation is the experimental observations that the reactions that are not feasible in solvents in ambient conditions can be carried out easily within the proteins. To explain these observations, one should understand the structural changes occurring in a molecule within a protein. 2009 American Chemical Society
3258 J. Phys. Chem. B, Vol. 113, No. 11, 2009 HAD, a globular R/β protein stems from Xanthobacter autotrophicus GJ10. It serves as an environmental protectant and plays an important role in detoxification of many halogensubstituted alkanes, which are hydrolyzed to alcohol with the help of water, which is a cosubstrate in the protein.10 It has a molecular mass of 35 kDA and is made of 310 amino acid residues.11,12 The structure of HAD has two domains, the first domain is formed by residues 1-144 and 230-310. The second domain is made of the residues between 156 and 229.11 Between these two domains there exists a cavity of size 112 Å3.13 This cavity is predominantly made of hydrophobic residues such as Glu56, Trp125, Phe128, Leu262, Leu263, Phe164, Phe172, Trp175, Phe222, Pro223, and Val226. The cavity also includes charged residues such as Asp124 and His289.11,14 In the crystal structure, the cosubstrate water molecule is hydrogen-bonded to the oxygen atom of Asp124. Our aim was to investigate the structure of DCE within this cavity. The time scale for the hydrolysis reaction to happen is much larger than the time scale we can study using the Car-Parrinello molecular dynamics technique. Moreover, we are interested in the protein confinement induced structural and dynamical changes in DCE rather than studying its enzyme-assisted actual reaction mechanism for the hydrolysis reaction. We have adopted a hybrid QM/ MM Car-Parrinello molecular dynamics (CPMD)15,16 technique that can model the HAD-DCE system more accurately than usual force-field-based MD techniques. It allows us to model the system in an isothermal-isobaric condition (or in an NPT ensemble) similar to the experimental condition. The DCE is treated in a more accurate QM level, whereas the protein, HAD, and the solvent (water in the present case) are treated using MM force fields. The employed calculational technique with CPMD in a QM/ MM setup has successfully been used to study many solute-solvent systems.17-21 The QM/MM implementation takes care of the charge evolution in the solute molecule based on the instantaneous electric field due to the solvent or protein environment, so the solvent or protein-environment induced changes in the charge distribution and dipole moment can be easily studied. Furthermore, this technique does not suffer from the inaccuracy of empirical force-fields, which are commonly applied in MD calculations.22 However, due to the computational expense of these calculations, we have limited the study to a time scale around 175 ps. To investigate the confinement effect on the conformational equilibrium we have also performed force-field MD calculations for DCE in water and for DCE confined within HAD protein. MD calculations do not account for the polarization of solute molecule due to the dynamic solvent environment or protein environment. However, when we are dealing with the situation when molecular processes only are rare events, the MD simulations are efficient and give good insight of the equilibrium rotamer or conformer distributions. Due to the computational expense associated with the CPMD calculations (which can be carried out for a maximum time scale of a few hundreds of ps), we have to rely on the MD calculations to address the confinement effect on conformational equilibrium of DCE in water and in HAD protein. In the literature, there are many reports on the behavior of molecules and atomic clusters within a confinement. Many experimental and theoretical reports show drastic differences in the confined molecular properties when compared to those of bulk.23 In addition to the structural quantities, dynamical quantities such as diffusivity have shown to be remarkably different in mesoporous materials.24,25 The differences are attributed to the reduced dimensionality and large interface
Letters effects.26 The confinement effect has an important industrial application as many reactions can be catalyzed within the mesoporous materials.27 Zeolites and inorganic mesoporous materials were described as solid solvents, as the electronegative oxygen framework can polarize the absorbed molecules like any polar solvents.28 Here, we make a detailed investigation on the confinement effect due to a protein environment on the molecular geometry, charge distribution, and dipole moment of DCE. There are only few theoretical investigations on HAD, the focus has mostly concerned the reaction mechanism for the HAD-assisted enzymatic hydrolysis reaction of DCE. Soriana et al. studied the nucleophilic displacement of a chloride anion of DCE in HAD.29 Silberstein et al. performed free energy calculations on HAD and other similar proteins to investigate different binding sites suitable for various substrates.30 The chlorine kinetic isotopic effect for the dehalogenation reaction in HAD was investigated by Lewandowicz et al.31 Also, there exists MD calculations on enzyme-substrate complexes and for the transition state.32 The dehalogenation reaction of DCE catalyzed by HAD has also been investigated using a combined QM/MM MD simulation technique, and the results have been compared with the reaction occurring in water.33,34 In relation to these studies, the present investigation looks into the confinement effect on the substrate molecule, DCE. 2. Computational Details The initial structure for the DCE/HAD system has been taken from the X-ray crystal structure report by Verschueren et al.11 The structure was refined to 1.9 Å resolution and includes the DCE substrate bonded within the cavity of HAD. The protein data bank (PDB) number for the DCE/HAD substrate-enzyme complex is 2DHC.35 Figure 1 shows the HAD-DCE system, where HAD is shown in the ribbon display mode and the substrate DCE is shown in space-fill display mode. We have solvated the DCE/HAD system in 9666 water molecules, which also include the protein water molecules. First, we have carried out MD calculations at ambient conditions using the SANDER module of AMBER software.36 We have used the TIP3P forcefield37 for the description of water, whereas for the protein the parm9938 force field has been employed. For the DCE molecule, the generalized AMBER force field39 has been used. The equilibrated structure from the MD simulations have been used as the initial structure for the CPMD40 calculations. In our present calculations, we have used the Becke, Lee, Yang, and Parr (BLYP) gradient corrected functional41,42 and the TroullierMartins norm conserving pseudopotentials.43 Here, the electronic wave function is expanded in a plane wave basis set, and the cutoff used was 80 Ry. We have used 5 au as the time step for the integration of the equation of motion and 800 amu as the fictitious electronic mass. The calculations were carried out in a QM/MM setup,44,45 where the solute molecule (here the DCE molecule) is treated at the density functional theory level, and the entire protein and solvents (here water) are treated with a MM force-field (either TIP3P or parm99). The interaction between the QM and MM systems involves electrostatic, shortrange repulsion and long-range dispersion interaction terms (using the empirical van der Waals parameters). The CPMD calculations involve the following three procedures: (1) quenching run: the electronic and ionic temperature of initial structure is quenched to remove any hot spot in the system due to inaccurate molecular starting geometry; (2) scaled temperature run: a short run using temperature-scaled dynamics carried out to bring the system to the required temperature and pressure; (3) nose run: in this run, the system is kept to interact with a
Letters
J. Phys. Chem. B, Vol. 113, No. 11, 2009 3259
Figure 1. The structure of DCE (in gauche form) in HAD.
Nose-Hoover thermostat at the required temperature. The Nose thermostat mimics the real system connected to a heat-bath to maintain the system temperature. The total time scale for the Nose production run is around 175 ps. The results are compared to our earlier results reported for DCE in water,9 where the total time scale was around 120 ps. In addition to the CPMD calculations, MD simulations of total time scale 3.5-4 ns were carried out for DCE in water and in HAD protein to investigate the confinement effect on conformational equilibrium. 3. Results and Discussions The conformational state of DCE can conveniently be discussed in terms of the dihedral angle between the atoms Cl-C-C-Cl, which will be referred as φ. When the angle φ is 180°, DCE is said to be in trans form, and when the angle is (60° it is in the gauche state. The dihedral angle, 300°, is equivalent to -60°, which means a gauche conformation. Figure 2 shows the time evolution of the angle, φ as a function of the time step for DCE/HAD (referred to as system-I) along with the same quantity calculated for DCE/water (referred as systemII). The results for the DCE/water system have been taken from our previous manuscript.9 Figure 2 clearly shows that within the protein cavity, DCE remains in the gauche conformation, but in the case of the water solvent there occurs infrequent conformational transitions between the gauche and trans conformers of DCE. Overall, the computational results show that the protein confinement effect results in lifting the conformational equilibrium between gauche and trans conformers as we see in water. The absence of the interconversion between gauche and trans conformers in HAD protein can be due to any of the following reasons: (1) The gauche conformer is stabilized more than the trans conformer due to the charge-dipole interaction with the Asp124 residue. (2) The conformational transition from
Figure 2. Dynamical evolution of dihedral angle, φCl-C-C-Cl of DCE in HAD and in water.
gauche to trans forms involves a change in molecular volume that may be energetically unfavorable in the restricted dimension. In other words, there may exist a kinetic barrier between gauche and trans conformational states that does not allow the gauche form of the DCE molecule to explore the other minimum energy conformational state, trans. The calculated molecular volumes for the gauche and trans conformers are, respectively, 76.6 and 78.3 cm3/mol. These volumes are calculated using the Monte Carlo method (as implemented in gaussian0346) by integrating over the volume elements with an electron density of 0.001 electrons/bohr3 density. To find out the actual reason among these two, we have carried out one more calculation as described below. As it has been described in the Computational Details section, we have carried out a separate MD calculation for DCE/HAD in water. The only difference in the new run is that we have constrained the DCE molecule to be in trans conformational state (with dihedral angle φ equal to 180°). The
3260 J. Phys. Chem. B, Vol. 113, No. 11, 2009
Letters
Figure 4. (a) Scatter diagram of Kohn-Sham energy and dihedral angle, φCl-C-C-Cl of DCE. (b) A plot of 〈EKS(φ)〉 and φCl-C-C-Cl of DCE.
Figure 3. (a) Dynamical evolution of dipole moment and (b) φCl-C-C-Cl of DCE for the trans DCE/HAD system CPMD calculation.
total time scale for this constrained MD run is 620 ps. The equilibrated structure from this MD run has been used as the input for a new CPMD QM/MM run that again follows the quenching run, the scaling run, and the Nose run. During this CPMD run, we have followed the evolution of the conformational state of DCE molecule. Interestingly, we find that within 0.4 ps after the start of the Nose run, DCE interconverts from trans to gauche conformational state. Figure 3 shows the evolution of the dipole moment (Figure 3a) and dihedral angle (Figure 3b) as a function of time step during this run. This clearly explains the reason for the disappearance of the conformational transition of DCE in protein environment that the gauche conformer is highly stable. The second reason that the existence of a kinetic barrier between the gauche and trans conformational states is completely excluded since we observe the spontaneous or immediate interconversion of trans to gauche conformers. To understand the energetics of the gauche and trans conformer in water, we have plotted a scatter diagram between the dihedral angle and the total Kohn-Sham energy (EKS) corresponding to a partial trajectory. The EKS is the CPMD analogue of the classical potential energy. We can clearly see in Figure 4a that the conformers with dihedral angle 30° > φ > 100° (gauche conformers) occupy the region corresponding to smaller energy, and the conformers with dihedral angle 150° > φ > 230° (trans conformers) occupy the region of higher energy. The EKS is calculated by dividing the total Kohn-Sham energy by the number of solute and solvent molecules, which is 8892 in the present case. We have also calculated the average energies (〈EKS(φ)〉) of the gauche and trans conformers as a function of dihedral angle, which is shown in Figure 4b. The 〈EKS(φ)〉 for the gauche and trans conformers are, respectively, 52.1 and 51.5 kJ mol- 1. Although the energies calculated for the gauche and trans conformers are reliable, we are not able to comment on the accuracy of the energies calculated for the conformers with dihedral angle 100° > φ > 140° since the molecule spends less time in this configuration phase space. So we are not able to comment on the actual barrier height between these conformational states. Overall, the energies of gauche and trans conform-
Figure 5. Dynamical evolution of dipole moment of DCE in HAD and in water.
ers are comparable, and the gauche conformer is more stable than the trans conformer by only 0.6 kJ mol-1. It is here important to notice that the thermal energy, RT is around 2.49 kJ mol-1 at ambient conditions. The calculated standard deviations in energy are 0.06 and 0.07 for the gauche and trans forms, respectively. Figure 5 shows the time evolution of the dipole moment of DCE in HAD and in water. This displays a feature similar to the time evolution of φ. It is clearly shown that the gauche conformers in HAD and in water have different magnitudes for the dipole moment. In the later part of the manuscript, we will analyze the origin for the increase in dipole moment for gauche conformer in HAD. In both cases of water and HAD, it is seen that the conformer with φ ) 60 and 300° have the same magnitude for the dipole moment. Figure 6 displays the dipole moment distribution for systems I and II. In system-I, the dipole moment distribution is unimodal whereas in system-II the dipole moment distribution curve is bimodal in nature. The peak appearing close to 0 debye in system-II is due to the trans conformer, and the peak appearing beyond 3.0 debye is due to the gauche conformer. An interesting observation is that the dipole moment of the gauche conformer has a larger magnitude in the case of system-I. As we have earlier reported the dipole moment of the gauche or trans conformer increases with the polarity of the solvent.9 In the case of chloroform solvent, the dipole moments for these conformers were significantly lower when compared to the water solvent.9 Surprisingly, the dipole moment for the gauche conformer is larger when it is confined within the protein than when it is in water. The water solvent remains as a highly polarizing solvent with a dielectric constant
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J. Phys. Chem. B, Vol. 113, No. 11, 2009 3261 TABLE 2: Average Dipole Moment and D-RESP Charges for the Gauche Form
Figure 6. Dipole moment distribution of DCE in HAD and in water.
TABLE 1: Average Bond Distances for the Gauche Form system
RClsC
RCsC
RHsH
DCE in HAD DCE in water
1.875 1.880
1.507 1.512
1.094 1.096
of 83.47 To our surprise, the protein seems to be more polarizing than water, as we see from the dipole moment (which is the first moment of the charge distribution) of the gauche conformer. The average dipole moment of the gauche conformer is 5.1 debye in HAD protein, whereas it is 4.5 debye in water. This is a striking observation when compared to the literature reports on the “biological water”.48,49 The biological water is the water bound to the protein, which is reported to be less polar than the bulk water.50 In other words, a water molecule bound to a protein is less polarized than a water molecule in water. We have also studied a few organic molecules such as adenosine51 and nile red52 in water and within protein, and we find that the molecular dipole moment of these molecules are smaller in protein than in water solvent. The dipole moments for adenosine in water and adenosine in beta-aspartate methyl transferase protein are, respectively, 3.9 and 2.8 debye.51 The dipole moments for nile red in water and in beta-lactoglobulin protein are, respectively, 24.0 and 16.0 debye.52 Contrary to these studies and literature reports on biological water, we observe in the present case of DCE in HAD the opposite feature, where the organic molecule is more polar in protein than in water. The reported molecular dipole moment is calculated using the relation n
µ¯ )
∑ qijri i)1
where the summation runs over all the atoms in the DCE molecule. Here, qi is the D-RESP charge on the ith atom with the atomic coordinate, jri. The increase in dipole moment of the gauche conformer within the protein might be due to two evident reasons: (1) either the CCl bond lengths are increased or (2) the actual net atomic charges are increased. To understand the origin for the increase in dipole moment for the gauche conformer within the protein cavity, we have calculated the bond lengths for the CsCl, CsC, and HsH bonds, see list in Table 1. The bond lengths are compared to the corresponding values for gauche DCE in water. As we can see from the table, the CsCl and CsC bond lengths are relatively small for DCE in the case of the protein environment. The variance (σ), standard deviation (σ) and standard error ((σ)/(N)) are calculated for the bond lengths as an average from 100 blocks of the trajectory.22 The values for variance vary between 0.000 016 and 0.004 761, whereas
system
DCE in HAD
DCE in water
µ, debye
5.1
4.5
atom
q, (DCE in HAD)
q, (DCE in water)
Cl C H1 H2
-0.262 0.042 0.129 0.090
-0.241 0.035 0.123 0.083
the standard deviation varies between 0.004 and 0.069, and the calculated standard errors are on the order of 10-4. Table 2 displays the D-RESP53 charges calculated for the Cl, C, and H atoms for DCE in protein and in water. We can clearly see from the values of charges the DCE is more polarized in the protein, leading to the increased dipole moment. To ensure the correctness of the results presented we have also calculated the standard error for the D-respective charges and dipole moments. The standard error is defined as (S)/(N), where S is the standard deviation for a particular quantity, and N is the number of points used in the averaging. The errors associated with the Drespective charges (in going from hydrogen to chlorine atoms) are between 1.2 × 10-4 and 2.4 × 10-4. Also, the error associated with the calculated dipole moment is on the order of 10-3 for the gauche conformer in HAD and in water. So, we have the interesting result that overall HAD lies beyond water in the polarity scale. Similar to the case of zeolites, the HAD protein can be considered as a flexible solid solvent due to this polarizing nature. This may be an important reason why the reactions that are not feasible at ambient conditions in solvents can be carried out with in a protein environment.33,34 3.1. Confinement Effect on Conformational Equilibrium. In this subsection, we discuss the confinement effect on the conformational equilibrium of DCE based on the force-field MD calculations. Because the molecular processes such as conformational interconversion or segmental motion are rare events, the MD simulations are very useful in these cases. Due to the kinetic barrier associated with the interconversion, a short run may result in a wrong conformational distribution at equilibrium. So, the MD calculations are handier tools than the CPMD technique when we are interested in calculating the conformational distribution. So, based on the MD calculations, we have addressed the confinement effect on the conformational distribution and rate of interconversion. Figure 7 shows the distribution of the dihedral angle, φ, for DCE in HAD and in water. It clearly shows that the population of trans conformer is relatively low in HAD protein compared to that in water solvent. Also, a zero
Figure 7. Dihedral angle distribution for DCE in HAD and in water as obtained from MD simulations.
3262 J. Phys. Chem. B, Vol. 113, No. 11, 2009
Letters References and Notes
Figure 8. Time evolution of conformational state of DCE in HAD and in water as obtained from MD simulations.
population in the curve around φ ) 120° suggests a significant barrier for conformational transition in the case of DCE in HAD protein. By integrating the curve up to φ ) 120° we calculate the percentage population of gauche conformer as 90% in the case of water solvent, whereas it is 97.5% in the case of DCE within HAD protein. To understand the confinement effect on the rate of interconversion, we have also plotted the time evolution of the dihedral angle, φ. Figure 8a shows φ as a function of time for DCE in HAD protein, and Figure 8b is for DCE in water. Figure 8 clearly shows that the number of conformational jumps is lesser in the case of DCE within HAD when compared to that in the case of water solvent. Also, in the case of HAD protein, DCE in trans form immediately bounces back to the gauche conformer, whereas in water solvent it also remains in the trans conformational state. Overall, the DCE mostly remains in the gauche conformational state in both HAD protein and in water. However, the trans conformational state is not completely prohibited in the case of water, and the percentage population of trans state is almost 10%, whereas in HAD protein it is only 2.5%. Altogether, MD simulations also suggest that the trans conformer is relatively less stable in HAD protein when compared to water solvent. 3.2. Conclusions. We have investigated the molecular geometry, charge distribution, dipole moment, and conformational distribution of DCE within the HAD protein and in water solvent. We find that the structural properties are very much altered by the protein environment when compared to the water solvent. We find that the protein environment polarizes the DCE molecule more than the polar water solvent, which is evident from the computed D-RESP charges. Our study indicates that within the HAD protein the interconversion dynamics is considerably affected. This study also explains that the extrapolarization of the organic molecules in the protein environment can be a possible reason for the reduction in the activation energy for many reactions that are not feasible in the ambient condition and in ordinary solvents. Also, the results based on MD simulations suggest that the conformational equilibrium can be affected by the protein confinement. Both CPMD and MD simulations suggest that the trans conformer is relatively less stable in HAD protein when compared to that in water solvent. Acknowledgment. N.A.M. acknowledges the Wenner-Gren foundation for financial support. This work was supported by a grant from the Swedish Infrastructure Committee (SNIC) for the project “Multiphysics Modeling of Molecular Materials”, SNIC 023/07-18.
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