Molecular Understanding of Laccase Adsorption on Charged Self

School of Materials and Chemical Engineering, Hainan University, Haikou 570228,. China, P. R. China. *. Corresponding author. Tel./fax: +86 20 8711406...
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Article Cite This: J. Phys. Chem. B 2017, 121, 10610-10617

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Molecular Understanding of Laccase Adsorption on Charged SelfAssembled Monolayers Jie Liu,†,‡ Yun Xie,§ Chunwang Peng,† Gaobo Yu,†,∥ and Jian Zhou*,† †

School of Chemistry and Chemical Engineering, Guangdong Provincial Key Lab for Green Chemical Product Technology, South China University of Technology, Guangzhou 510640, P. R. China ‡ Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, P. R. China § Huizhou University, Huizhou 516007, P. R. China ∥ School of Materials and Chemical Engineering, Hainan University, Haikou 570228, P. R. China S Supporting Information *

ABSTRACT: Controlling the orientation of laccase on electrodes is crucial for the achievement of fast direct electron transfer. It is important to find a short pathway between the T1 copper site of laccase and a substrate during the laccase immobilization. In this work, we studied the adsorption orientation and conformation of Trametes versicolor laccase (TvL) on two kinds of charged selfassembled monolayers (SAMs), including NH2−SAM and COOH−SAM, by parallel tempering Monte Carlo and all-atom molecular dynamics simulations. TvL adsorbs on positively and negatively charged surface with “end-on” and “lying” orientation, respectively. On the positively charged surface, T1 copper site of TvL is closer to the surface. The orientation of TvL on positively charged surface is narrower than that on negatively charged surface. Thus, the positively charged surface is more conducive to the immobilization of TvL. The conformational changes of TvL on the charged surfaces are analyzed by RMSD, superimposed structures, dipole moment, gyration radius, and eccentricity. Results show that native structures of TvL are well preserved when it adsorbs on the charged surfaces. This work provides atomistic insight into the mechanism of TvL adsorption on charged surface and is helpful for the design and development of laccase-based electrodes.

1. INTRODUCTION

An efficient enzymatic electrode can be established by developing efficient electron transfer between the electrode and catalytic sites of the enzyme. To achieve the fast electron transfer, two schemes, including mediated electron transfer (MET)11 and direct electron transfer (DET),12 are usually adopted by many researchers. The difference between MET and DET is whether it needs redox mediators or not. Redox mediators usually make the design of enzyme electrode more complicated, which may induce higher overpotentials and reduce operational stability during the electrocatalytic process. Redox mediators usually are toxic.13 Compared with MET, DET avoids the use of redox mediators. To achieve DET, it is necessary to orient the redox center of enzyme toward the electrode. For laccase, it is crucial to shorten the distance between the T1 copper site of laccase and the substrate. Many researchers focused on the immobilization of laccases on different kinds of solid materials, such as carbon nanotubes,14−19 carbon electrode,20 titanium dioxide,21 nanofibrous membranes,8,22 mesopurous silica materials,23−26 gold electrodes/nanoparticles,13,27,28 and self-assembled monolayers

Laccase (EC 1.10.3.2), which is subordinate to a subclass of multicopper oxidases, is popular for its ability of catalyzing the oxidation of various aromatic substrates, and oxygen is usually required as a second substrate.1 Four copper ions within the laccase, classified as type-1 (T1), type-2 (T2), and type-3 (T3), are catalytic sites.2 Either T1 or T2 copper site contains a single copper ion while the T3 copper site consists of two copper ions. T1 copper site is the primary oxidation site which accepts the electron from the electrode. Then the electron is transferred to the T2/T3 trinuclear copper sites through an intermolecular electron-transfer mechanism and induces the reduction reaction of oxygen.3,4 With a high redox potential of the T1 copper site and catalytic efficiency of oxidizing pollutants, laccase has been widely used in biofuel cells, biosensors, and pollutant degradation.5−7 In practical applications, the reaction conditions (e.g., organic solution and pH) can induce the denaturation of free laccases, especially when mixed with reactants. It is also hard to recycle the free laccases from mixed reactants, which limits the industrialization of laccases. Through immobilization of laccase, the thermal stability, reusability, and suitable range of working pH as well as temperature are all enhanced.8−10 © 2017 American Chemical Society

Received: September 2, 2017 Revised: October 26, 2017 Published: November 2, 2017 10610

DOI: 10.1021/acs.jpcb.7b08738 J. Phys. Chem. B 2017, 121, 10610−10617

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The Journal of Physical Chemistry B (SAMs).29−32 Arzola et al.30 investigated the adsorption of laccase on bare, hexadecanethiol and highly ordered pyrolytic graphite (HOPG) modified gold electrodes, and both electrochemical response and catalytic activity can be retained by SAM modified electrodes. Gutiérrez-Sánchez et al.13 covalently combined gold nanoparticles with laccases and assembled these compounds into porous graphite electrodes. They found that the DET reaction can be enhanced by controlling the orientation of laccase on functionalized gold nanoparticles, and the ordered adsorption of laccase on electrode with T1 copper site oriented close to the electrode was significant for fast DET occurring. Most researchers focused on the laccase loading capacity of the nanomaterials and the optimization of reaction conditions (e.g., pH, temperature, solvent, et al.).8,15,24,33−36 However, the charged property of surface which can affect the ordered orientation of adsorbed enzymes also plays a key role in the enzyme immobilization.13,37−39 Olejnik et al.4 found that the electrocatalytic current value of laccase adsorbed on positively charged surface was stronger than that on negatively charged surface. They mentioned that the orientation of laccase on electrode surfaces played a dominant role in the electrocatalytic current. Up to now, atomistic-level information about the orientation of laccase on charged surfaces has rarely been explored. It is difficult for experiments to directly detect the orientation of a single enzyme on a substrate. Molecular simulation provides an efficient avenue to investigate molecular level interactions between proteins and solid surfaces.40−46 The preferred orientation of proteins on surfaces could hardly be caught by single conventional molecular dynamics (MD) simulation. Thus, it is necessary to perform a Monte Carlo (MC) simulation first, which can catch the optimized configuration of proteins adsorbed on surfaces quickly and save computational resources, before long-time MD simulations.47 In our previous work, we developed a parallel tempering Monte Carlo (PTMC) simulation, which can fast and efficiently predict the preferred orientation of a protein on a charged surface48 and provide the initial configurations for the later long-time MD simulations.49−51 To the best of our knowledge, the accurate orientation of laccase on charged surfaces and its effect on DET between laccase and electrode have not been studied yet. In this work, the combination of PTMC and all-atom molecular dynamics (AAMD) simulations were performed to study the orientation and conformational changes of laccase on charged SAMs. The orientation of T1 copper site within the laccase was analyzed in detail. Electrocatalytic current differences of laccase adsorbed on charged surfaces, which were found in experiment,4 will be illustrated theoretically. This work can provide a better understanding of laccase immobilization on electrodes.

Figure 1. Crystal structure of TvL and its three domains. D1, D2, and D3 represent domain 1, domain 2, and domain 3, respectively. Green beads represent the copper clusters (i.e., T1 and T2/T3 copper sites).

three domains in TvL are shown in Figure 1. In both PTMC and AAMD simulations, the relative positions of copper atoms within TvL are kept as its crystal positions. As TvL is in its oxidized state, the coppers in TvL are assigned with the charge of +2 e. The lysine and arginine residues along with the Nterminal of TvL were protonated, whereas the glutamic acid and aspartic acid along with the C-terminal were deprotonated. The protonation state of histidine was calculated by the PDB2PQR and APBS algorithms52,53 with CHARMM force field. The net charge of TvL is −4 e. 2.1. PTMC. The PTMC48 simulations were performed to get the preliminary orientation of TvL on charged surfaces. In order to keep the basic structure of protein, united-residue model41 (i.e., each residue is reduced to a sphere centered at its α-carbon) was used. During PTMC simulations, the protein backbone was maintained rigid, and SAM surface was treated as a flat surface. TvL can freely rotate and translate around its center of mass during the whole PTMC simulation. To ensure enough sampling of PTMC algorithm, we plotted the interaction energies between TvL and charged surfaces over electric dipole orientation and hydrophobic dipole orientation in Figure S1. In our previous work,54 we proposed that the orientation was controlled by both electric and hydrophobic dipoles. Sometimes the electric dipole played a dominant role, and sometimes the other. As can be seen in Figure S1, for TvLs adsorption on both charged surfaces, electric dipole dominates the adsorption. Thus, in section 3, electric dipole is discussed in detail. The force field parameters were taken from our previous works.48 Both van der Waals (vdW, Uvdw) and electrostatic interactions (Uele) were considered. In total, 40 000 000 MC cycles were performed; the former 20 000 000 MC cycles were used for system equilibration while the remaining cycles were used for result analysis. We adopted five replicas, at 310, 350, 400, 500, and 700 K, to ensure sufficient energy overlap between neighboring replicas and to allow for the acceptance of configuration swaps. Each swap was carried out every 500 cycles. The final results were taken as the initial orientation for further AAMD simulations.49,51,55 2.2. AAMD. AAMD simulations were performed by GROMACS 4.5.4 with CHARMM force field.56 The carboxyl(COOH-) and amino- (NH2-) terminated SAMs on Au(111) with √3 × √3 structures are simulated as the model charged surfaces. There are totally 224 thiol chains and 2016 gold

2. SIMULATION DETAILS The high-resolution crystal structure of Trametes versicolor laccase (TvL) which is in its oxidized, copper-complete state2 (PDB-ID: 1GYC) was obtained from the RCSB (www.rcsb. org). TvL structure is a monomer and organized in three sequentially arranged domains (i.e., domain 1, domain 2, and domain 3, see Figure 1). The T1 copper site is located in domain 3. The trinuclear copper cluster, constructed by T2 and T3 copper sites, is embedded between domains 1 and 3, and both domains provide residues for the coordination with copper atoms.2 The secondary structures, copper clusters, and 10611

DOI: 10.1021/acs.jpcb.7b08738 J. Phys. Chem. B 2017, 121, 10610−10617

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The Journal of Physical Chemistry B atoms. Fifteen thiol chains were deprotonated/protonated, representing COOH−SAM and NH2−SAM with the surface charge densities of −0.05 and +0.05 C·m−2, respectively. The surfaces have dimensions of 6.99 × 6.92 nm2. During AAMD simulations, the sulfur atoms of thiol chains along with gold atoms were fixed. The potential parameters for SAMs were from the CHARMM force field for lipids.56 Hydrogen atoms within TvL were added by GROMACS 4.5.4.57 TvL was initially put above the surface with the closest distance of 0.5 nm. Water molecules, which were described by TIP3P water,58 were added into the single protein box with the dimension of 6.99 × 6.92 × 6.00 nm3. Water molecules within 0.28 nm of TvL and SAMs were removed to avoid overlaps of coordinates.47 Counter ions, including Na+ and Cl−, were added to keep the systems neutral. TvL in the bulk solution was also simulated as a reference system. Each system was initially optimized by the steepest descent method. During this step, TvL was kept rigid as its crystal structure. Then, NVT-MD simulations were performed at 310 K, and the time step was 2 fs. The Nose-Hoover thermostat59,60 with a time constant of 0.5 ps was adopted to control the temperature. Bonds containing hydrogen atoms were constrained by the LINCS algorithm.61 A cutoff distance of 1.0 nm was used to calculate the LJ interactions, and the 3dc particle mesh Ewald method62 was adopted to evaluate the electrostatic interactions. For systems including flat surfaces, only x and y directions were considered in periodic boundary conditions, and two hard walls were added at both top and bottom of the box.63 In order to study the early adsorption of TvL on charged surfaces, a 100 ns AAMD simulation was performed. To confirm the convergence of simulations, the evolution of rootmean-square deviation (RMSD) versus simulation time is plotted in Figure S2. It can be found that RMSD reached equilibrium in all systems after 60 ns. Thus, 100 ns simulation was sufficient for the simulation of TvL adsorption. TvL in the bulk solution was simulated in the same simulation parameters as mentioned above, except for the periodic boundary conditions. As there is no flat surface in bulk system, the periodic boundary conditions with three-dimensional directions were used. The visual molecular dynamics (VMD) program64 was used for structure visualization.

Figure 2. Adsorption configuration of TvL on charged SAMs simulated by PTMC (shown in parts (a) and (b)) and AAMD (shown in parts (c) and (d)). The blue and red planes in (a) and (b) are presented for positively and negatively charged surface, respectively. The surfaces terminated with blue groups and red groups in (c) and (d) refer to NH2−SAM and COOH−SAM, respectively. The orange arrow indicates the direction of the dipole. Copper sites are represented in green beads. The value of d indicates the distance between T1 copper site and the SAM surface.

3.1. Orientation of T1 Copper Site. The adsorption configurations of TvL on both charged SAMs (i.e., COOH− SAM and NH2−SAM), simulated by PTMC and AAMD, are shown in Figure 2. TvL adsorbs on NH2−SAM with the “endon” orientation (i.e., the principal axis of protein relatively parallel to the surface normal), whereas on COOH−SAM, the “lying” orientation (i.e., the principal axis of protein is relatively perpendicular to the surface normal) is found. The orientation trends obtained from AAMD are consistent with those from PTMC, which indicates that the PTMC algorithm is suitable for predicting orientation of proteins. From Figure 2, it can be seen that the dipole direction of TvL is almost parallel to the surface normal when it adsorbs on NH2−SAM. Because TvL has a net charge of −4 e, the orientation of TvL on NH2−SAM is induced by the electric dipole, which is consistent with what we had found in our previous works.37,38,49,51,54,55,65,66 For TvL adsorption on negatively charged surface, as enzyme has the same charge sign with the surface, the orientation is controlled by the distribution of charged patches above the protein surface. The key factor which should be emphasized in DET-based laccase electrode is an ordered orientation of laccases on electrode with the T1 copper site close to the surface.4,13,17 The distances between T1 copper site and top atoms of surfaces are noted in Figure 2. It is obvious that when TvL adsorbs on NH2−SAM, the distance is shorter. Thus, controlling the orientation of laccase by a positively charged surface is more conducive for the DET between laccase and the electrode. Olejnik et al.4 found that the electrocatalytic current value of laccase on a negatively charged surface was significantly smaller than that on a positively charged surface, although the amount of adsorbed enzyme was similar. They deduced that it may be caused by the distance between the electrode and the T1 copper site.4 In this simulation study, we present more detailed orientation information on laccase on charged surfaces. It is consistent with experiment results that the positively charged surface is favorable for the DET-based laccase electrodes. We

3. RESULTS AND DISCUSSION In this work, the PTMC and AAMD methods were combined to investigate the orientations of TvL on charged surfaces. The preliminary orientations of TvL on charged surfaces were determined by PTMC. The distance between T1 copper site and charged surfaces, which is the key factor for DET occurring, was analyzed. Furthermore, the RMSD, superimposed structures, gyration radius, eccentricity, and dipole moment of TvL were calculated to study the conformational changes of TvL during the adsorption. Results were obtained by averaging over the last 40 ns AAMD trajectories. Simulation results are summarized in Tables 1−4 and Figures 2−5. Table 1. Interaction Energies of TvL Adsorbed on Charged SAMs by AAMD NH2−SAM COOH−SAM

Utot (kJ·mol−1)

Uele (kJ·mol−1)

Uvdw (kJ·mol−1)

−466.7 ± 51.8 −297.0 ± 64.3

−307.9 ± 34.8 −219.4 ± 64.5

−158.8 ± 19.4 −77.5 ± 18.0 10612

DOI: 10.1021/acs.jpcb.7b08738 J. Phys. Chem. B 2017, 121, 10610−10617

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That means a positively charged surface can promote the stability and conductivity of immobilized TvL. 3.3. Binding Site. To better understand the interactions between TvL and charged surfaces, charged residues of TvL in contact with the SAMs (i.e., binding sites) in the final configuration of AAMD simulations were picked out and are shown in Figure 4 and Table 2. Residues containing atoms

deduce that the weak electrocatalytic current value of laccase on the negatively charged surface is mainly caused by the orientation of T1 copper site, which is almost oriented toward the solution (shown in Figure 2). 3.2. Orientation Distribution and Interaction Energy. Orientation distribution and the interaction energy between proteins and surfaces can be used to quantitatively describe the stability of proteins adsorbed on surfaces. The interaction energies are averaged over the last 40 ns of the MD simulation by block averaging (as shown in Table 1). The adsorption behaviors of TvL on both charged surfaces are induced by the synergy of electrostatic and vdW interactions, while the electrostatic interactions play a more dominant role in the adsorption process. In our previous works, we also concluded that the electrostatic interactions usually dominated the adsorption behavior of charged proteins on charged surfaces.49,51,54,55,65 Due to the stronger electrostatic interaction energy between laccase and NH2−SAM, more side chains move close to the surface, and the vdW interaction energy enhances accordingly. The orientation distributions of TvL on both charged surfaces are shown in Figure 3. The orientations of TvL

Figure 3. Orientation distributions of TvL adsorbed on oppositely charged surfaces by PTMC and AAMD.

calculated by PTMC are consistent with the global-minimumenergy areas in Figure S1, which means the preferred orientations are found by a well sampling. Compared with the orientation distributions of TvL on both charged surfaces predicted by PTMC, the results from AAMD present slight shifts. In PTMC simulations, we ignore the side chains of protein residues and only consider the locations of α-C. Therefore, the dipole direction is slightly deviated. On the other hand, the protein is kept rigid in PTMC, whereas the flexible side chains are taken into account in AAMD simulations, which also contribute to the slight shift of orientation distributions. Moreover, as PTMC is based on the rigid united-residue protein model, the orientation distribution is narrower than that by AAMD. Nevertheless, it is clear that the trends of the orientation distributions of laccase adsorbed on both charged surfaces are basically the same. It can also be found from Figure 2 that the final orientation configurations of TvL on charged surfaces from PTMC and AAMD are quite similar. As can be seen from Figure 3, the orientation distribution of TvL on NH2−SAM is narrower than that on COOH−SAM. The interaction energy between TvL and NH2−SAM is also stronger. It is mainly caused by the net charge of TvL. As TvL carries net negative charges, it tends to adsorb on NH2−SAM.

Figure 4. Final configurations of TvL on NH2−SAM (a, c, e) and COOH−SAM (b, d, f) by AAMD. (a, b) are electrostatic maps of TvL. (c, d) and (e, f) are the side-view and top-view configurations, respectively. Charged residues less than 3.5 Å from the surface are represented in VDW mode. Positively and negatively charged residues are rendered in blue and red, respectively.

within 0.35 nm of the surface are considered as contacting residues.54 As can be seen from Figure 4 and Table 2, four negatively charged residues of TvL, including GLU496, ASP492, ASP364, and GLN499, strongly interact with the Table 2. Key Binding Sites of TvL on Oppositely Charged Surfaces negatively charged residues TvL on NH2− SAM TvL on COOH− SAM 10613

GLU496, ASP492, ASP364, GLN499 ASP42, ASP138, GLU142

positively charged residues NA LYS482, ARG43, LYS39, LYS40, LYS130, LYS194, ALA1

DOI: 10.1021/acs.jpcb.7b08738 J. Phys. Chem. B 2017, 121, 10610−10617

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The Journal of Physical Chemistry B NH2−SAM surface while no positively charged residue appears near the surface. Meanwhile, for TvL on COOH−SAM, there are three negatively charged residues (i.e., ASP42, ASP138, and GLU142) and seven positively charged residues (i.e., LYS482, ARG43, LYS39, LYS40, LYS130, LYS194, and ALA1) directly adsorbed on the surface. Among these charged residues, the GLN499 and ALA1 are C-terminal and N-terminal residues. Interactions between TvL and charged SAM can be more visible by examining the distribution of charged residues above the TvL surface by electrostatic maps. From Figures 4a and b, we can see that the negatively charged region of TvL is close to the NH2−SAM while the positively charged region of TvL is close to the COOH−SAM. The number of contact residues of TvL on NH2−SAM is less than that on COOH−SAM. In section 3.1, we mentioned that TvL adsorbed on NH2−SAM with the “end-on” orientation, while it adsorbed on COOH− SAM with the “lying” orientation. In other words, TvL adsorbed on negatively charged surface with more contact residues and had more flat contact. However, as discussed above, we indicated that the adsorption of TvL on NH2−SAM was more stable than that on COOH−SAM. The main reason is that, on NH2−SAM, the dipole direction is parallel to the surface normal and the net charge of TvL is −4 e. Thus, TvL can stably adsorb on NH2−SAM with a narrower orientation distribution and stronger interaction energy. On COOH−SAM, the dipole direction is not antiparallel to the surface normal, as mentioned above. From Figure 4, it can be found that the adsorption of TvL on COOH−SAM is induced by the cooperation of electrostatic interactions and the geometric feature. In our previous work, we also found that the geometric feature and electric dipole of a protein play an important role in determination of orientation distribution.55 3.4. Conformational Changes. The conformational changes of TvL adsorbed on charged surfaces were analyzed by RMSD, superimposed structures, dipole moment, gyration radius, and eccentricity. 3.4.1. RMSD and Superimposed Structures. RMSD and superimposed structures are used to qualitatively analyze the conformational changes of TvL adsorbed on charged surfaces compared with that in bulk solution and crystal state. RMSD values calculated in this work are based on structures of simulated and crystal TvL; only backbone structures are considered as well. The RMSDs of the whole protein and three domains are calculated and shown in Table 3. The three

slightly smaller than that in bulk. In some previous simulation works, researchers also found the same phenomenon that the charged surface can preserve the native conformation of proteins.55,67,68 This is mainly due to the hydrophilic charged surface which limits the free movement of protein’s side chain. For three domains, the conformational change of D3 is relatively larger than those of the other two domains, whether TvL is adsorbed on charged surfaces or dissolved in bulk. Piontek et al.2 mentioned that the D3 domain had more helical contents than the other two domains. It is known that the hydrogen bonds in helix, coil, and turn structures are relatively less than those in sheet structures.55 Therefore, the secondary structure of D3 is more flexible than those of D1 and D2. Yet, this does not mean that TvL will lose its native conformation. The conformation of a protein in the bulk solution can represent the native structures.47,55 RMSDs of TvL on charged surfaces are similar to that in bulk. Thus, the native structure of TvL is well preserved when it is adsorbed on the studied charged surfaces. Furthermore, to provide a visual assessment of TvL structural changes after AAMD, we analyzed the superimposed structures of three domains of the adsorbed TvL on its crystal structure (shown in Figure 5). It reveals that the structural deviation

Figure 5. Simulated structures (red) of three domains (D1, D2, and D3) of TvL superimposed on its crystal structure (cyan).

mainly occurs in domain 3. Structural changes take place in the coil, turn, and helix structures. From Figure 5, it can also be seen that most secondary structures are preserved, which means the changes of simulated structure, with respect to the crystal structure, do not affect the biological activity of TvL. 3.4.2. Dipole Moment, Gyration Radius, and Eccentricity. The conformational changes of TvL adsorbed on charged surfaces are also quantitatively analyzed by dipole moment, gyration radius, and eccentricity. The dipole moment is usually used to represent the distribution of protein charges. From Table 4, it can be seen that the dipole moments of TvL on charged surfaces, 681 D for COOH−SAM and 639 D for NH2−SAM, are relatively larger than that of TvL in bulk (623D). This is mainly caused by the charged surface which can

Table 3. RMSD of TvL and Its Three Domains in the Bulk Solution and Adsorbed on SAM Surfaces by AAMD

in bulk on COOH− SAM on NH2− SAM

RMSD_total (nm)

RMSD_D1 (nm)

RMSD_D2 (nm)

RMSD_D3 (nm)

0.45 ± 0.02 0.40 ± 0.02

0.11 ± 0.01 0.12 ± 0.01

0.13 ± 0.02 0.11 ± 0.01

0.41 ± 0.02 0.32 ± 0.01

0.43 ± 0.04

0.18 ± 0.01

0.12 ± 0.01

0.27 ± 0.02

Table 4. Averaged Properties of TvL in Bulk Solution and Adsorbed on Charged SAMs by AAMD

domains (i.e., domain 1, domain 2, and domain 3) are named as D1, D2, and D3, respectively. When TvL adsorbs on positively and negatively charged surfaces, the RMSD values are 0.43 and 0.40 nm, respectively. The RMSD is 0.45 nm when TvL solved in the bulk solution. It indicates that the charged surfaces do not induce significant conformational change of TvL. Indeed, the conformational changes of TvL on charged surfaces are

crystal in bulk on COOH−SAM on NH2−SAM 10614

orientation

dipole (Debye)

Rg (nm)

eccentricity

− − −0.36 0.99

621 623 ± 86 681 ± 47 639 ± 40

2.15 2.32 ± 0.01 2.31 ± 0.01 2.33 ± 0.02

0.15 0.19 0.19 .

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charged surfaces. The conformational changes in domain 3 are relatively more distinct than in the other two domains. But this does not matter in the inactivation of TvL, as the overall conformations of TvL on charged surfaces are similar to that in bulk. This work shed lights on the orientation control of TvL on charged surfaces and provides the mechanism of laccase adsorption on charged surfaces by molecular simulations. The findings are helpful for the design and development of laccasebased electrodes.

induce side chains of residues bearing the same sign of charge to orient away from the surface. It can also be found in Table 4 that the dipole moment of TvL on COOH−SAM is larger than that on NH2−SAM. When adsorbed on NH2−SAM with the “end-on” orientation, the principal axis of TvL is nearly perpendicular to the SAM; the effect of NH2−SAM on the electric dipole moment of TvL is weaker than that of the COOH−SAM, which contributes to TvL adsorption with the “lying” orientation. Moreover, TvL carries a net charge of −4 e; the number of negatively charged residues on the protein surface is more than that of positively charged residues. Thus, when it adsorbs on COOH−SAM, the negatively charged residues on the protein surface tend to move away from the surface. That is another reason why the dipole moment of TvL on COOH−SAM is larger than that on NH2−SAM. In our previous work, we also found the same interesting phenomenon that the dipole moment of ribonuclease A, a positively charged protein, adsorbed on NH2−SAM is larger than that on COOH−SAM.55 It is worth to mention that the dipole moment changes are induced by the movement of side chains, because the backbone structure of the protein is well preserved as shown in RMSD results. Gyration radius and eccentricity are usually used together to analyze the overall morphology of a protein. The gyration radius, defined as the mass-weighted root-mean-square average distance of all atoms in a protein from its center of mass,47 demonstrates the overall size of a protein. The eccentricity represents the value of 1 − Iave/Imax, in which Imax and Iave are the maximal principal moment of inertia and average of three principal moments of inertia, respectively.49 It reveals the overall shape or ellipsoidal degree of a protein. From Table 4, we can see that the gyration radii of TvL on charged surfaces are nearly the same as those in bulk. The same result can also be found in the eccentricity analysis. This further demonstrates that the charged surface does not influence the overall morphology of TvL.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b08738. Potential energy surface and orientations in PTMC simulation and RMSD of TvL during MD simulation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 20 87114069. Tel.: +86 20 87114069. ORCID

Jian Zhou: 0000-0002-3033-7785 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support from National Natural Science Foundation of China (Nos. 21376089, 21706197, 21606053, 21706045, 21776093, and 91334202), the National Key Basic Research Program of China (No. 2013CB733500), Guangdong Science Foundation (No. 2014A030312007), and the Fundamental Research Funds for the Central Universities (SCUT-2015ZP033) is gratefully acknowledged. An allocation time from the SCUTGrid at South China University of Technology is gratefully acknowledged.

4. CONCLUSION In this work, the orientation and the effective DET of TvL on charged surfaces (i.e., COOH−SAM and NH2−SAM) are studied by the combination of PTMC and AAMD methods. The results are consistent with previous experiment investigations which are summarized below. The DET can be achieved by orienting the T1 copper site within TvL toward the surface. Our results show that the NH2− SAM can make the T1 copper site closer to the surface. Furthermore, from the analysis of orientation distribution and interaction energy, it can be found that TvL can adsorb stably on the positively charged surface. It means that the positively charged electrode is beneficial for the DET between TvL and electrode. The adsorptions of TvL on both charged surfaces are induced by the synergy of electrostatic and vdW interactions, and the electrostatic interactions play the dominant role. The orientation of TvL on the positively charged surface (i.e., “endon” orientation) is controlled by the electric dipole of the protein. The orientation of TvL on the negatively charged surface (i.e., “lying” orientation) is caused by the combination of electrostatic interaction and the geometric feature. The conformational changes of TvL on charged surfaces are analyzed by RMSD, superimposed structures, dipole moment, gyration radius, and eccentricity. Results show that the native conformations of TvL are well preserved when it adsorbs on

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