Molecular Simulation Study of Feruloyl Esterase Adsorption on

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Molecular Simulation Study of Feruloyl Esterase Adsorption on Charged Surfaces: Effects of Surface Charge Density and Ionic Strength Jie Liu, 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, PR China ABSTRACT: The surrounding conditions, such as surface charge density and ionic strength, play an important role in enzyme adsorption. The adsorption of a nonmodular type-A feruloyl esterase from Aspergillus niger (AnFaeA) on charged surfaces was investigated by parallel tempering Monte Carlo (PTMC) and all-atom molecular dynamics (AAMD) simulations at different surface charge densities (±0.05 and ±0.16 C·m−2) and ionic strengths (0.007 and 0.154 M). The adsorption energy, orientation, and conformational changes were analyzed. Simulation results show that whether AnFaeA can adsorb onto a charged surface is mainly controlled by electrostatic interactions between AnFaeA and the charged surface. The electrostatic interactions between AnFaeA and charged surfaces are weakened when the ionic strength increases. The positively charged surface at low surface charge density and high ionic strength conditions can maximize the utilization of the immobilized AnFaeA. The counterion layer plays a key role in the adsorption of AnFaeA on the negatively charged COOH-SAM. The native conformation of AnFaeA is well preserved under all of these conditions. The results of this work can be used for the controlled immobilization of AnFaeA.

1. INTRODUCTION The physical adsorption stability of a protein can be affected by many factors, such as the hydrophobicity,1,2 crystal faces,3 and surface charge.4 Previous studies indicate that the orientation of charged proteins can be well controlled by charged surfaces.5,6 Zhou et al.5,7−13 proposed that the protein dipole played an important role in determining the orientation of a protein adsorbed on a charged surface when electrostatic interactions dominate. Kubiak-Ossowska and Mulheran14 also found that the adsorption of lysozyme on a charged surface was driven by electrostatic interactions, where the orientation of the protein dipole moment defines the direction of protein movement. Ravichandran et al.15 found that the distribution of charged amino acid residues on the protein surface was one of the key factors in determining electrostatic interactions between the protein and the surface. Besides the charge character of the surface, the surface charge density (SCD) is another important factor. Many researchers10,11,16 found that SCD could regulate electrostatic interactions between proteins and surfaces. Wang et al.16 found that SCD determined the protein adsorption kinetics, while the surface charge character determined the conformation and orientation of the assembled proteins. Researches also have provided some insights into the effect of ionic strength (IS) on the electrostatic interactions between proteins and charged surfaces.17−19 Schlapak et al.20 found that surface charges were screened at high IS and the electrostatic attraction diminished. Xie et al.9 performed parallel tempering © XXXX American Chemical Society

Monte Carlo (PTMC) to investigate the effect of IS on the orientation of lysozyme on charged surfaces. They found that IS could induce the reorientation of the adsorbed protein. Consequently, it is necessary to investigate the effects of surface charge and IS in solution on the adsorption and orientation of proteins. Feruloyl esterases, a diverse group of hydrolases which can naturally catalyze the hydrolyzation of ester bonds between hydroxycinnamic acids and plant cell wall hemicellulose, play a significant role in biotechnological processes for many industrial and medicinal applications.21 In biocatalysis processes, feruloyl esterases are usually used for the refinement of hydroxycinnamic acids. Hydroxycinnamic acids have been known by their antioxidant and antimicrobial properties, and it is necessary to modify the solubility of acids by esterification and transesterification for different commercial products.22−24 It is beneficial to the reaction of biocatalytic esterification or transesterification while the hydrolytic reaction can be reversed when increasing the concentration of organic solvents. To enhance the stability of enzymes, it is of utmost importance to immobilize enzymes on solid support materials.25,26 Thorn et al.27 immobilized feruloyl esterase into mesoporous silica materials. They found that feruloyl esterase adsorption into Received: April 25, 2015 Revised: September 16, 2015

A

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calculated by using PDB2PQR and APBS algorithms39,40 with the CHARMM force field. Each amino acid was reduced to a sphere centered on the α-carbon atom of the residue, which is called the united-residue model.5 The charged SAM was treated as a flat surface. The van der Waals (vdW, UvdW) and electrostatic interactions (Uele)5 between the protein and the charged surface were calculated. MC cycles (40 000 000) were performed, in which the first 20 000 000 cycles were for preequilibration and the last 20 000, 000 cycles were for production. Six replicas at temperatures of 310, 350, 400, 500, 700, and 1000 K were used to ensure sufficient energy overlap between neighboring replicas. As PTMC is based on a united-residue model and implicit solvent model, the dynamic behaviors of AnFaeA on charged surfaces and the atomisticlevel interactions between AnFaeA and the charged surface under the explicit solvent model are absent. Thus, it is necessary to perform AAMD simulations to further investigate the detailed atomistic-level information on AnFaeA adsorption. 2.2. AAMD. The initial orientations of AnFaeA adsorbed on different charged SAMs were gained from PTMC simulations. The orientations of AnFaeA were obtained by fitting the α-C of the crystal structure with the coarse-grained structure (i.e., residue model) determined by PTMC.6−8,37 Hydrogen atoms within AnFaeA were added by GROMACS 4.5.4.41 The protonation and deprotonation states of charged residues and histidines were the same as that in PTMC simulations. The net charge of AnFaeA was −20 e. The CHARMM27 force field42 was used. For charged SAMs, the HS(CH2)10COOH SAM and HS(CH2)10NH2 SAM on Au(111) with 31/2 × 31/2 structures, in which the packing density was 2.16 nm2 per molecular chain, were simulated as surfaces. In total, there are 224 thiol chains and 2016 gold atoms. In a previous work,10 Zhou et al. mentioned that a strongly charged surface (50% dissociation of COOH-SAM) can induce distinct conformational changes in a protein. Thus, in this study, we chose charged SAMs with degrees of dissociation of 5 and 25% to investigate the orientation of AnFaeA with a retained native conformation. Thus, 15 or 56 chains were deprotonated/protonated, representing an SCD degree of ±0.05 or ±0.16 C·m−2 (i.e., a dissociation of 5 or 25%) for charged SAMs, respectively. During the whole AAMD simulations, the sulfur atoms within thiol chains and all gold atoms were kept fixed. LJ parameters σAu−Au and εAu−Au for a gold atom are 3.20 Å and 0.48 kJ·mol−1, respectively. At the start of AAMD, AnFaeA was put above the surface with the closest distance of 0.5 nm. Water molecules were added to the box with the dimension of 6.99 × 6.92 × 6.00 nm3. The water molecules were described by the TIP3P model.43 To keep the systems neutral, counterions (Na+ and Cl−) were added. For the ionic strength, 0.007 M indicated a distinctly low concentration and 0.154 M indicates the physiological concentration of the human body. Thus, these two salt concentrations were considered during MD simulations. The height of the SAM was 2.8 nm, and the whole simulation box dimension was 6.99 × 6.92 × 8.8 nm3. As the objective was to investigate the interfacial region between the protein and surfaces, periodic boundary conditions were applied only in the x and y directions.44 There were a total of twelve interface systems and two reference bulk (i.e., solution) systems studied in this work. In order to eliminate the steric overlap or inappropriate geometry in the initial configurations, each system was optimized with the

mesoporous silica changed the product specificity of the enzyme to favor transesterification and decreased the rate of hydrolysis when compared to that of free enzymes. Additionally, the immobilized enzymes showed excellent operational stability and reusability. Experimental studies can provide the catalytic efficiency of the immobilized feruloyl esterases; however, detailed information on the changes in interactions between feruloyl esterases and charged surfaces influenced by the surface charge character, SCD and IS, can hardly be obtained from experiments. Molecular simulations could complement this. Molecular dynamics (MD) simulations have been used to study the orientation and conformation changes of proteins adsorbed on surfaces in past decades.5,6,9,11,28−36 In our previous works,6,8,37 we found that the combination of PTMC9 and MD simulations is an effective approach to studying the adsorption behavior of proteins on solid surfaces. In this work, we combine PTMC and all-atom molecular dynamics (AAMD) simulations to study the adsorption of feruloyl esterases on oppositely charged self-assembled monolayers (SAMs) at different SCDs (±0.05 and ±0.16 C· m−2) and ISs (0.007 and 0.154 M). Meanwhile, the feruloyl esterases solved in bulk solution at different ISs were also simulated as reference systems. By comparing the adsorption behavior of enzyme adsorption at oppositely charged SAMs (i.e., COOH-SAM and NH2-SAM) under different solution conditions, an experimental strategy to modulate feruloyl esterase adsorption was suggested.

2. SIMULATION DETAILS The nonmodular type-A feruloyl esterase from Aspergillus niger (AnFaeA) (PDB ID 1USW)38 was used to investigate interactions between feruloyl esterase and charged surfaces. AnFaeA has 260 amino acid residues. Its protein scaffold is based on an α/β hydrolase fold. It consists of a major ninestranded mixed β-sheet, two minor two-stranded β-sheet arrangements, and seven helices (Figure 1).38 The active site of AnFaeA contains catalytic triad residues (SER133, ASP194, and HIS247).38

Figure 1. Secondary structure of AnFaeA. Green beads represent the active site of AnFaeA.

2.1. PTMC. PTMC9 simulations were performed here to obtain the preferred orientations of AnFaeA on charged SAMs, which can be used as the initial configurations in MD studies. It can obtain the global minimum-energy state through a single simulation.9 The electric potentials of charged amino acids and histidine within AnFaeA under physiological conditions were B

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Langmuir Table 1. Interaction Energies between AnFaeA and Surfaces COOH-SAM O1

−2

−0.05 C·m

−0.16 C·m−2 COOH-SAM O2

−0.05 C·m−2 −0.16 C·m−2

NH2-SAM

0.05 C·m−2 0.16 C·m−2

0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154

M M M M M M M M M M M M

Utotal (kJ·mol−1)

Uele (kJ·mol−1)

UvdW (kJ·mol−1)

−346.6 −64.1 NA NA −365.5 −83.3 −286.9 −152.6 −488.0 −444.8 −709.7 −597.8

−226.6 −24.3 NA NA −255.7 −56.6 −223.5 −21.4 −409.0 −327.4 −621.0 −531.6

−80.1 −39.8 NA NA −109.8 −26.7 −63.4 −131.3 −79.0 −117.4 −88.7 −66.3

± 72.3 ± 83.2

± ± ± ± ± ± ± ±

106.4 59.6 73.3 94.5 79.4 105.2 96.7 99.3

± 76.2 ± 83.2

± ± ± ± ± ± ± ±

90.6 59.6 75.6 91.2 79.8 90.8 101.2 97.0

± 19.6 ± 23.1

± ± ± ± ± ± ± ±

36.7 12.8 16.4 21.7 29.2 43.7 23.1 17.9

steepest descent method and conjugate gradient method until the maximum force between atoms was lower than 10 kJ·mol−1· nm−1. After that, NVT-MD simulations were performed with the integrating time step of 2 fs. A Nose-Hoover thermostat45,46 was used to control the temperature to be 310 K with a time constant of 0.5 ps. Bonds containing hydrogen atoms were constrained by the LINCS algorithm.47 The cutoff distance was 1.0 nm for the nonbonded interactions. The Ewald summation for systems with slab geometry44 was used to calculate the electrostatic interactions. Each system was simulated for 50 ns. AnFaeA in the bulk at 0.007 and 0.154 M was also simulated as a reference for comparison. The periodic boundary conditions were applied in 3D directions for AnFaeA solvated in the bulk. Other simulation parameters were the same as that in slab systems. The visual molecular dynamics (VMD) program48 was used for structure visualization.

3. RESULTS AND DISCUSSION The interactions between AnFaeA and charged surfaces (COOH-SAM and NH2-SAM) at different SCDs (±0.05 and ±0.16 C·m−2) and different ISs (0.007 and 0.154 M) are investigated by a combined PTMC and AAMD simulation approach. For reference, AnFaeA in bulk solutions at 0.007 and 0.154 M is also studied. The preliminary orientations of AnFaeA on charged surfaces are determined by PTMC simulations. The interaction energies and minimal distance (min-distance) between AnFaeA and charged surfaces and orientation and conformational changes are analyzed from MD simulations. Results are summarized in Tables 1−4 and Figures 2−10. 3.1. Orientation of AnFaeA on Charged Surfaces. The results of PTMC simulation are shown in Figure 2. There is only one dominant orientation when AnFaeA adsorbs on NH2SAM, while AnFaeA adsorbs on COOH-SAM with two preferred orientations (named O1 and O2, respectively). The orientations calculated by PTMC are used as initial orientations in AAMD. The detailed interactions between AnFaeA and charged SAMs on the atomic level are analyzed below. The orientation of proteins adsorbed on charged surfaces is quantitatively characterized by the orientation angle. It is defined as the angle between the unit vector normal to the surface (n) and the unit vector along the protein dipole (m).3,5−9,11−13,37,49,50 The orientational distribution is the frequency counts of the cosine value of the orientation angle during the last 20 ns. Figure 3 shows the orientational distribution of AnFaeA on charged surfaces under different conditions.

Figure 2. Orientational distributions of AnFaeA on charged surfaces calculated by PTMC.

For the system of AnFaeA adsorbed on COOH-SAM with an O1 orientation (COOH-SAM (O1)), when the SCD is high, AnFaeA is not adsorbed. It is consistent with the results of the interaction energy and min-distance, which will be discussed in section 3.2. For COOH-SAM (O1) at the low SCD, AnFaeA adsorbed with a preferred orientation (as shown in Figure 3). When IS increases, the orientational distribution becomes wider. In other words, a greater IS affects the orientational distribution of AnFaeA on negatively charged surfaces. For AnFaeA adsorbed on COOH-SAM with an O2 orientation (COOH-SAM (O2)), AnFaeA can adsorb on COOH-SAM under all conditions with an opposite orientation compared to that on NH2-SAM (Figure 3). On the low SCD surface, the orientational distribution is influenced distinctly by IS, and the orientational distribution of COOH-SAM (O2) is steadier at low IS than that at high IS. Figure 4 shows the number density distribution profiles of ions (i.e., Na+ and Cl−) above charged SAMs. For COOH-SAM (O2) on a low SCD C

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Figure 3. Orientational distribution of AnFaeA on charged surfaces. The maximum values of the y axis in black, blue, and red are present for 5, 8, and 20, respectively. Panels without peaks denote no adsorption.

surface, a Cl− layer appears when the IS increases. Thus, when the SCD is low, the orientational distribution of COOH-SAM (O2) is wider at high IS than that at low IS due to the effect of the Cl− layer. For the high SCD surface, the orientational distributions of AnFaeA are similar under both ISs. It can also be seen from Figure 4 that the variation in the Cl− layer above the high SCD surface can be ignored as the distinct Na+ layer appears at the interface with a high number density. For the NH2-SAM surface, AnFaeA shows a distinct orientational distribution under all conditions. From Figure 3, on the low SCD surface, it can be seen that the orientational distribution of AnFaeA is wider at the high IS than that at the low IS. On the low SCD surface, the orientational stability is slightly weakened by the increase in IS. Figure 4 shows the number density of ions (i.e., Na+ and Cl−) above the charged surface along the z direction. It can be seen that, at low SCD, the first ion layer above the NH2-SAM is more distinct at the high IS than that at the low IS. That means that, with the increase in IS, the ionic concentration near NH2-SAM increases. Thus, at the low SCD, the orientational distribution becomes wider at the higher IS, yet this slight change does not affect the overall adsorption state of AnFaeA. As is shown in Table 1, the interaction energy between AnFaeA and a positively charged surface at low SCD and high IS is strong enough to ensure stable adsorption. The same result was also observed in our previous work that high IS can make the orientational distribution wider than low IS.9 At the high SCD, the orientational distribution of AnFaeA presents the same trend at both low and high IS. This indicates that electrostatic interactions between AnFaeA and NH2-SAM at the high SCD are strong enough that IS cannot affect the orientational stability. The interaction energies between AnFaeA and NH2SAM at the high SCD also support this view (shown in Table 1). As is shown in Figure 4, at the high SCD, the relative

change of ion distribution above the NH2-SAM is not as distinct as that at the low SCD. It also indicates that the orientational distribution of AnFaeA on NH2-SAM at the high SCD is not affected by IS. Comparing the orientational distributions of AnFaeA on oppositely charged surfaces, it can be seen that the dipole direction of AnFaeA is oriented toward the surface when AnFaeA adsorbs on COOH-SAM with an O2 orientation, whereas the dipole direction of AnFaeA is oriented toward the solution when AnFaeA adsorbs on NH2-SAM (Figure 5). In a previous work, we also found that the mutated protein G B1 domain adsorbed on oppositely charged surfaces with imperfect opposite orientations.6 Previous studies also indicated that the orientation of a protein on charged surfaces was determined by the direction of the dipole of the protein when electrostatic interactions dominate.9−11 However, in this study, another orientation (i.e., O1 orientation) with the dipole direction pointing toward the solution appears when AnFaeA adsorbs on COOH-SAMs. We deduce that this is mainly caused by the counterion layer above COOH-SAM surfaces. For COOHSAMs, there is an obvious Na+ layer above the surface at either low or high IS, as shown in Figure 4. As AnFaeA bears the same charge character with the COOH-SAM, Na+ ions in solution can easily adsorb on the surface and form a counterion layer (i.e., a Na+ layer). Thus, the adsorption behaviors of AnFaeA on COOH-SAMs at low SCD with a O1 orientation are induced by the synergy of electrostatic interactions between positively charged residues and COOH-SAM as well as the electrostatic interactions between negatively charged residues and the Na+ layer, whereas the O2 orientation is induced by the dipole direction. It can also be found from Figures 3 and 5 that AnFaeA cannot be adsorbed on COOH-SAM with a O1 orientation when the SCD increases, whereas AnFaeA can adsorb on COOH-SAM with a O2 orientation under all D

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Figure 4. Number density distribution profiles of ions (i.e., Na+ and Cl−) above charged surfaces along the z direction. The number density distributions of Na+ and Cl− are presented in black and red, respectively.

AAMD are summarized in Table 1. The electrostatic interaction energy (Uele) and vdW interaction energy (UvdW) are calculated separately, thus the dominant interaction which leads to protein adsorption can be distinguished. For COOH-SAM (O1), AnFaeA can only be adsorbed at a low SCD of −0.05 C·m−2. At a high SCD of −0.16 C·m−2, AnFaeA cannot be adsorbed with O1 orientation due to the electrostatic repulsion between the protein and the surface. As shown in Table 1, when AnFaeA adsorbs on COOH-SAM with O1 orientation, the interaction energies between AnFaeA and COOH-SAMs at high SCD are not available (NA). The time evolution of min-distances of AnFaeA adsorbed on charged surfaces at different SCDs and ISs is shown in Figure 6. From Figure 6, it can also be seen that AnFaeA, adsorbed with O1

conditions. Further analysis of the role of the key residues will be discussed in section 3.3. The final configuration of AnFaeA on surfaces is shown in Figure 5, and the active sites within AnFaeA are presented in green beads. For the adsorbed systems, the active sites are not blocked during the adsorption, which means both kinds of surfaces can preserve the activity of the adsorbed AnFaeA. For NH2-SAM at low SCD and high IS, the active site of AnFaeA is totally exposed to the solution. Thus, the positively charged surface under low SCD and high IS conditions can maximize the utilization of the immobilized AnFaeA. 3.2. AnFaeA−Surface Interaction Energy and MinDistance. The interaction energies between AnFaeA and charged surfaces at different SCDs and ISs calculated from E

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Figure 5. Final configurations of AnFaeA orientations on COOH-SAM (above) and NH2-SAM (below) from MD simulations. The surface with the top red groups is for COOH-SAM, and that with the top blue groups is for NH2-SAM. The active sites are represented in green beads. The direction of the dipole is denoted by a red arrow.

Figure 6. Time evolution of the min-distance between AnFaeA and charged surfaces. (a) AnFaeA on COOH-SAM with the O1 orientation. (b) AnFaeA on COOH-SAM with the O2 orientation. (c) AnFaeA on NH2-SAM.

orientation, moves away from the COOH-SAM at the high SCD. At the low SCD, the adsorption of AnFaeA on COOHSAM with an O1 orientation is relatively stable under the low IS condition and is dominated by the electrostatic interactions (shown in Table 1). The adsorption is induced by positively charged residues, which will be illustrated in detail in section 3.3. When IS increases, the protein−surface interactions are slightly screened. Therefore, the interactions between AnFaeA and the surface under the high IS condition is weaker than that under the low IS condition. The same phenomenon can also be found in Figure 6(a); under the low SCD and high IS

conditions, the min-distance of AnFaeA to the surface changes frequently during the whole adsorption process. This indicates that AnFaeA adsorption on COOH-SAM with a O1 orientation at the low SCD and the high IS is not stable. As mentioned above, AnFaeA can adsorb on COOH-SAM with a O2 orientation under all conditions, which is consistent with the interaction energy and the min-distance between AnFaeA and the charged surfaces (Table 1 and Figure 6). It is shown in Table 1 that the electrostatic interaction energy between AnFaeA and COOH-SAM with an O2 orientation is also affected by the variation of IS. When IS increases, the F

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Figure 7. Charged residues close to surfaces during AnFaeA adsorption. The positively charged and negatively charged residues near the surface are presented in blue and red, respectively. The surface with top red groups is for COOH-SAM whereas that with top blue groups is for NH2-SAM. COOH-SAM (O1) under high SCD is not shown here since it does not adsorb on the surface.

Table 2. Averaged Properties of AnFaeA in the Bulk and on Different Charged Surfaces under Different ISs by MD Simulations orientation in bulk COOH-SAM (O1)

−0.05 C·m−2 −0.16 C·m−2

COOH-SAM (O2)

−0.05 C·m−2 −0.16 C·m−2

NH2-SAM

0.05 C·m−2 0.16 C·m−2

0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154 0.007 0.154

M M M M M M M M M M M M M M

0.53 0.78 NA NA −0.88 −0.36 −0.55 −0.62 0.96 0.31 0.94 0.95

electrostatic interactions between AnFaeA and COOH-SAM decrease. As the O2 orientation is determined by the dipole within AnFaeA and there are more positively charged residues which are close to COOH-SAM rather than the O1 orientation (shown in Figure 7), the increase in SCD does not induce the desorption of AnFaeA from the COOH-SAM when it adsorbs

± 0.07 ± 0.11

± ± ± ± ± ± ± ±

0.09 0.19 0.08 0.06 0.02 0.07 0.03 0.02

RMSD (nm) 0.14 0.13 0.13 0.13 NA NA 0.14 0.15 0.13 0.15 0.19 0.13 0.17 0.11

± 0.01 ± 0.01

± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

Rg (nm)

eccentricity

1.71 1.70 1.72 1.72 NA NA 1.72 1.72 1.72 1.72 1.73 1.72 1.72 1.71

0.10 0.10 0.11 0.09 NA NA 0.09 0.10 0.10 0.10 0.12 0.11 0.09 0.10

with the O2 orientation. In other words, it also indicates that the orientation determined by the dipole direction is steadier than that determined by the partially charged residues or electrostatic interactions between AnFaeA and counterions. For the positively charged surface (NH2-SAM), it can be seen from Figure 6 that AnFaeA can be adsorbed under all G

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energy is mainly caused by the electrostatic attraction between the protein and the surface. Because AnFaeA bears a net charge of −20 e, increasing the SCD of a positively charged surface leads to an enhancement of the electrostatic attraction between the protein and the surface. Either for COOH-SAM or NH2-SAM, the effects of IS are the same. High IS could screen the electrostatic attraction/ repulsion between the protein and the charged surface. This phenomenon has also been observed in previous studies. Zhou et al.11 found that, with the increase in surface charge density, antibody−surface interactions became stronger. They also indicated that the electrostatic interactions were screened when IS was high. Wang et al.16 found that with the increase in KCl concentration the electrostatic interactions between cytochrome c and negatively charged SAMs decreased gradually. Schlapak et al.20 indicated that at high IS the electrostatic attraction diminished, and the adsorption of DNA was reduced. All of these previous studies are consistent with our results. We also find that AnFaeA cannot adsorb on COOH-SAM at the high SCD with an O1 orientation, which means that the vdW interactions between AnFaeA and COOHSAM are not strong enough to overcome the electrostatic repulsion between AnFaeA and COOH-SAM. Thus, the key factor which controls protein adsorption onto charged surfaces is SCD and the electrostatic interactions between AnFaeA and charged surfaces, while IS can affect only the electrostatic interactions between AnFaeA and positively charged surfaces. Furthermore, the O2 orientation is steadier than the O1 orientation, which means that the orientation determined by the dipole direction is the dominant orientation. 3.3. Binding Sites. The charged residues near the surfaces within 0.3 nm are further analyzed, as shown in Figure 7. For COOH-SAM (O1), the adsorption is dependent on the positively charged ALA1 (i.e., N-terminus), which directly contacts the surface, and other negatively charged residues, which indirectly contact the counterion layer above the surface (shown in Figure 7). At low IS, the negatively charged residues close to the surface are GLU8, ASP9, GLU182, GLU231, and ASP230, whereas they are ASP174, GLU182, ASP230, and GLU231 under the high IS condition. This is consistent with the results which discussed in section 3.2. The adsorption behavior of AnFaeA on COOH-SAMs with the O1 orientation is induced by the synergy of the charged surface and the counterion layer. The electrostatic interactions between AnFaeA and COOH-SAM with a O1 orientation are mainly caused by the positively charged ALA1 (i.e., N-terminus), while the Na+ layer contributes to the adsorption of several negatively charged residues (five negatively charged residues at the low IS and four negatively charged residues at the high IS). Thus, the O1 orientation is mainly caused by the counterion layer above the surface. The important role of the counterion layer in protein adsorption has also been found in Yu et al.’s work.12 They determined that the key residues are positively charged residues of lysozyme (net charge +8 e) when adsorbed on a positively charged surface, due to the interactions between lysozyme and the counterion layer. For COOH-SAM (O2), when SCD and IS are low, the adsorption behavior of AnFaeA on COOH-SAM is induced by three positively charged residues (i.e., LYS37, LYS40, and ARG66) directly interacting with the COOH-SAM and four negatively charged residues (i.e., ASP27, GLU39, ASP47, and ASP71) indirectly interacting with the counterionic layer. Obviously, the number of positively charged residues which

Figure 8. Time evolution of secondary structures of AnFaeA solved in the bulk and adsorbed on charged surfaces at different SCDs and ISs. AnFaeA on COOH-SAM with high SCD is not shown here since it does not adsorb on the surface.

conditions. That is mainly caused by the electrostatic attraction between the protein and the surface, as the net charge of AnFaeA is −20 e. From Table 1, it is obvious that the adsorption behavior of AnFaeA on NH2-SAM is induced by the synergy of electrostatic and vdW interactions, while the electrostatic interactions dominate the adsorption behavior under all conditions. The increase in the solution IS results in a decrease in electrostatic interactions between the protein and the surface. With the increase in SCD, the interaction energy between the protein and surface also increases. The increase in H

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Figure 9. RMSF of AnFaeA adsorbed on charged surfaces at different SCDs and ISs. AnFaeA on COOH-SAM with high SCD is not shown here since it does not adsorb on the surface.

carry oppositely charge, referring to the COOH-SAM, is distinctly larger than the O1 orientation with only one positively charged residue (i.e., ALA1) close to the COOHSAM. It also indicates that the O2 orientation is steadier than the O1 orientation. At low SCD, with the increase in IS, the charged residues move away from the surface due to the appearance of the Cl− layer, and the adsorption behavior is mainly maintained by the indirect interaction between GLU8 and the NA+ layer. Thus, the adsorption behavior of AnFaeA on COOH-SAM with O2 orientation at low SCD and high IS is less stable than that under other conditions, which is also consistent with the orientational distribution results (shown in Figure 3). With the increase in SCD, the number of charged residues above the COOH-SAM is decreased due to the increase in repulsive electrostatic interactions. But it still has four charged residues close to the COOH-SAM which induce

the adsorption behavior of AnFaeA. For high SCD, the binding sites of COOH-SAM (O2) under low IS are GLU39, LYS40, ASP109, and LYS116; the binding sites of COOH-SAM (O2) under high IS are LYS37, GLU39, LYS40, and ASP109. For NH2-SAM, the adsorption is induced by the direct interactions between negatively charged residues and the positively charged surface (shown in Figure 7). It can be seen from Figure 7 that there are no ions between the binding site and the surface. Except for AnFaeA adsorbed on NH2-SAM at low SCD and high IS, the key binding sites of AnFaeA adsorption on the positively charged surface are ASP93, GLU95, ASP203, GLU204, ASP230, and GLU231. The same result can also be found from Figure 3 as the trends in the orientational distributions under the other three conditions are the same. For AnFaeA adsorption on the NH2-SAM at low I

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SCD and high IS, the key binding sites are ASP93, ASP174, GLU182, ASP151, and ASP124. 3.4. Conformation Changes. The conformational changes in AnFaeA can be characterized by the root-mean-square deviation (RMSD), the definition of the secondary structure of protein (DSSP), the root-mean-square fluctuation (RMSF), superimposed structures, the radius of gyration, eccentricity, and hydrogen bonds. The results present in Tables 2−4 are obtained by statistical analysis of the last 25 ns MD trajectories. The conformational changes in AnFaeA on COOH-SAM with O1 orientation at high SCD are not presented in Table 2 since they are not adsorbed. 3.4.1. RMSD, DSSP, RMSF, and Superimposed Structures. The RMSD value is calculated by comparing the backbone structure between the crystal structure and the simulated structure. The RMSD values of AnFaeA in the bulk at different ISs are also calculated as references. In Table 2, it can be seen that the RMSD values of AnFaeA on COOH-SAMs with both orientations are similar to those in the bulk, which means that the COOH-SAM does not affect the backbone structure of AnFaeA. As shown in Table 1, the absolute value of the interaction energy between AnFaeA and COOH-SAM is relatively low. Therefore, the conformational change of AnFaeA on COOH-SAM is close to that in the bulk. For NH2-SAMs, the RMSD values of AnFaeA at the low IS are higher than those in bulk whether the SCD is low or high. It is indicated that the positively charged surface at low IS can slightly induce the conformation changes in AnFaeA. At low IS, the RMSD values of AnFaeA on NH2-SAMs of low SCD and high SCD are 0.19 and 0.17 nm, respectively. It is worth emphasizing that the values are in the rational ranges in which the conformational changes do not affect the natural functions of AnFaeA. When IS increases, the RMSD values of AnFaeA are close to the values in the bulk. It can be seen from Figure 4 that the number density of Na+ above the NH2-SAM increases at the high IS. As mentioned above, high IS could decrease the electrostatic interactions between AnFaeA and charged surfaces (shown in Table 1). Thus, for NH2-SAM, with the increase in IS, the conformation of AnFaeA can be well preserved. The DSSP (shown in Figure 8), RMSF (shown in Figure 9), and superimposed structures (shown in Figure 10) are further analyzed to understand the conformational changes in more detail. From Figures 8 and 10, it is obvious that the conformational changes mainly occur in the random coils and turns, whether AnFaeA is solved in the bulk or adsorbed on the charged surface. The binding sites of AnFaeA are shown in Figure 7, the secondary structures containing residues labeled in Figure 7 are suffering similar conformation changes whether in the bulk or on surfaces. In a previous work, we also found that the conformation changes of adsorbed protein usually occurred in the coil and turn structures which are relatively flexible secondary structures7 because the number of hydrogen bonds within the coil and turn structure is relatively less than that within the α-helix and β-sheet structures. The deviation between the residues and crystal structures during the whole

Figure 10. Simulated structures of AnFaeA (in red) superimposed on the crystal structure of AnFaeA (in cyan). AnFaeA on COOH-SAM with a high SCD is not shown here since it does not adsorb on the surface.

Table 3. RMSD Values of Catalytic Sites of AnFaeA in Bulk Solution and on Charged Surfaces COOH-SAM (O1) (nm) 0.007 M 0.154 M

−2

in bulk (nm)

−0.05 C·m

0.06 ± 0.01 0.05 ± 0.01

0.07 ± 0.01 0.09 ± 0.01

COOH-SAM (O2) (nm) −0.05 C·m

−2

0.13 ± 0.03 0.06 ± 0.01 J

NH2-SAM (nm) −2

−0.16 C·m

0.05 C·m−2

0.16 C·m−2

0.07 ± 0.01 0.09 ± 0.02

0.06 ± 0.01 0.08 ± 0.01

0.07 ± 0.01 0.06 ± 0.01

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Langmuir Table 4. Number of Hydrogen Bonds in AnFaeA as Well as between Protein and Water Molecules COOH-SAM (O1) in bulk 0.007 M 0.154 M

in protein between protein and water in protein between protein and water

192 504 192 487

± ± ± ±

6 12 6 13

−0.05 C·m−2 190 489 189 494

± ± ± ±

7 14 7 14

COOH-SAM (O2) −0.05 C·m−2 189 502 187 491

± ± ± ±

8 14 6 13

NH2-SAM

−0.16 C·m−2 192 485 192 478

± ± ± ±

6 15 6 16

0.05 C·m−2 191 511 192 487

± ± ± ±

6 13 6 13

0.16 C·m−2 187 512 193 479

± ± ± ±

6 12 6 13

surfaces, the numbers of hydrogen bonds inside a protein are similar, which means that most secondary structures of AnFaeA on charged surfaces are well preserved. For AnFaeA in the bulk and on NH2-SAM surfaces, the number of hydrogen bonds between AnFaeA and water molecules in low IS is slightly smaller than that in high IS. The main reason is that some ions replace a few water molecules around AnFaeA when the IS is high. The difference in hydrogen bonds between AnFaeA and water molecules is quite small under different conditions, and thus it does not affect the native property of AnFaeA. In other words, the native conformation of AnFaeA is well preserved when it adsorbs on charged surfaces, which in consistent with the results we have mentioned above.

adsorption simulation was analyzed by RMSF (shown in Figure 9). It can be seen from Figure 9 that, except for residues in coil and turn structures (the relative secondary structure of residue refers to DSSP analysis), the terminal residues, including Nterminus and C-terminus, also incur obvious structural changes, as the structures of terminal residues can be influenced by solvent molecules more easily than can other residue structures. For superimposed structure analysis (shown in Figure 10), the crystal structure, which is more comparable than the protein structure in the bulk, is used as a reference structure to analyze the conformation changes in AnFaeA on surfaces and in the bulk. From the superimposed structures, it can also been seen that the overall structures of AnFaeA are preserved during the adsorption, which is consistent with the RMSD result. Thus, during the adsorption process, AnFaeA could preserve its native structure. The conformation changes in catalytic sites within AnFaeA are also an important factor for AnFaeA activity. The catalytic sites of AnFaeA contain catalytic triad residues (SER133, ASP194, and HIS247). From Figure 8, it can be seen that the conformations of SER133 and ASP194 are stable during the whole simulations under all conditions. For HIS247, as it is located in a loop structure, the secondary structure is more flexible than others. Thus, the conformation was not as stable as the other two catalytic sites. However, it does not influence the catalytic activity of AnFaeA because the overall structure of AnFaeA adsorbed on a charged surface is similar to that in the bulk, as shown in DSSP, RMSD, and superimposed structure analysis. Furthermore, the RMSD values of the simulated catalytic triad residues compared to the crystal structure are analyzed (Table 3). As is shown in Table 3, the RMSD values of catalytic sites of AnFaeA are quite small whether in the bulk or on surfaces, which means that the adsorption behavior of AnFaeA on charged surfaces does not influence the conformation of catalytic sites. 3.4.2. Radius of Gyration and Eccentricity. The radius of gyration (Rg) and eccentricity of AnFaeA are presented in Table 2. The radius of gyration of a protein refers to a massweighted root-mean-square average distance of all atoms in a protein from its center of mass.7 The eccentricity is defined as 1 − (Iave/Imax), in which Imax is the maximal principal moment of inertia and Iave is the average of three principal moments of inertia.6,10 The overall shape change can be characterized by the combination of the radius of gyration and eccentricity. It can be seen from Table 2 that the values of the radius of gyration and the eccentricity of AnFaeA on the charged surface are close to those in the bulk. This indicates that the charged surface at different ISs and SCDs do not affect the overall shapes of AnFaeA, which is consistent with the results in section 3.4.1. 3.4.3. Hydrogen Bonds. The hydrogen bond is an important parameter which could be used to assess the protein−surface interactions. The numbers of hydrogen bonds in AnFaeA as well as between AnFaeA and water molecules are shown in Table 4. It can be seen that, whether in bulk or on charged

4. CONCLUSIONS In this work, the effects of surface charge density and ionic strength on the AnFaeA adsorption orientation and conformational changes on oppositely charged surfaces are simulated by PTMC and AAMD simulations. The results reveal that the adsorption behavior is controlled by the synergy of vdW interactions and electrostatic interactions. Whether AnFaeA can adsorb onto the charged surface is mainly controlled by the electrostatic interactions between AnFaeA and surfaces. The ionic strength could adjust the stability of the protein adsorption. The electrostatic interactions between AnFaeA and charged surfaces are weakened when the solution ionic strength increases. The active site of AnFaeA is totally exposed to the solution when AnFaeA adsorbs on NH2-SAM at low surface charge density and high ionic strength. Thus, the positively charged surface at low surface charge density in a high ionic strength solution can maximize the utilization of the immobilized AnFaeA. For COOH-SAM, the adsorption of AnFaeA is induced by the direct interactions between positively charged residues and the surface as well as the interactions between negatively charged residues and the counterion layer above the surface. The orientation determined by the dipole direction (i.e., O2 orientation of AnFaeA on COOH-SAM) is steadier than that determined by the partially charged residue distribution (i.e., O1 orientation of AnFaeA on COOH-SAM). For NH2-SAM, the adsorption is induced by the direct interactions between negatively charged residues and the positively charged surface. The orientation of AnFaeA on NH2-SAM is directly induced by the positively charged surface. When AnFaeA adsorbs on NH2-SAM at low ionic strength, the backbone structure of AnFaeA changes slightly relative to that in the bulk. That is mainly caused by the strong electrostatic interactions between AnFaeA and the positively charged surface. When the ionic strength increases, the conformation of AnFaeA can be better preserved. The native functions of AnFaeA are well preserved under all studied conditions. The results of this work might provide some K

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(16) Wang, G. X.; Wang, M.; Wu, Z. Q.; Bao, W. J.; Zhou, Y.; Xia, X. H. Dependence of the direct electron transfer activity and adsorption kinetics of cytochrome c on interfacial charge properties. Analyst 2013, 138, 5777−5782. (17) Petrovic, J.; Clark, R. A.; Yue, H. J.; Waldeck, D. H.; Bowden, E. F. Impact of surface immobilization and solution ionic strength on the formal potential of immobilized cytochrome c. Langmuir 2005, 21, 6308−6316. (18) Pasche, S.; Voros, J.; Griesser, H. J.; Spencer, N. D.; Textor, M. Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces. J. Phys. Chem. B 2005, 109, 17545−17552. (19) Biesheuvel, P. M.; Stroeve, P.; Barneveld, P. A. Effect of protein adsorption and ionic strength on the equilibrium partition coefficient of ionizable macromolecules in charged nanopores. J. Phys. Chem. B 2004, 108, 17660−17665. (20) Schlapak, R.; Armitage, D.; Saucedo-Zeni, N.; Chrzanowski, W.; Hohage, M.; Caruana, D.; Howorka, S. Selective protein and DNA adsorption on PLL-PEG films modulated by ionic strength. Soft Matter 2009, 5, 613−621. (21) Fazary, A. E.; Ju, Y. H. Feruloyl esterases as biotechnological tools: Current and future perspectives. Acta Biochim. Biophys. Sin. 2007, 39, 811−828. (22) Topakas, E.; Vafiadi, C.; Christakopoulos, P. Microbial production, characterization and applications of feruloyl esterases. Process Biochem. 2007, 42, 497−509. (23) Otero, J. M.; Papadakis, M. A.; Udatha, D.; Nielsen, J.; Panagiotou, G. Yeast Biological Networks Unfold the Interplay of Antioxidants, Genome and Phenotype, and Reveal a Novel Regulator of the Oxidative Stress Response. PLoS One 2010, 5, e13606. (24) Vafiadi, C.; Topakas, E.; Alderwick, L. J.; Besra, G. S.; Christakopoulos, P. Chemoenzymatic synthesis of feruloyl Darabinose as a potential anti-mycobacterial agent. Biotechnol. Lett. 2007, 29, 1771−1774. (25) Zhou, Z.; Hartmann, M. Recent Progress in Biocatalysis with Enzymes Immobilized on Mesoporous Hosts. Top. Catal. 2012, 55, 1081−1100. (26) Thorn, C.; Udatha, D. B. R. K. G.; Zhou, H.; Christakopoulos, P.; Topakas, E.; Olsson, L. Understanding the pH-dependent immobilization efficacy of feruloyl esterase-C on mesoporous silica and its structure activity changes. J. Mol. Catal. B: Enzym. 2013, 93, 65−72. (27) Thorn, C.; Gustafsson, H.; Olsson, L. Immobilization of feruloyl esterases in mesoporous materials leads to improved transesterification yield. J. Mol. Catal. B: Enzym. 2011, 72, 57−64. (28) Yu, X.; Wang, Q. M.; Lin, Y. A.; Zhao, J.; Zhao, C.; Zheng, J. Structure, orientation, and surface interaction of alzheimer amyloidbeta peptides on the graphite. Langmuir 2012, 28, 6595−6605. (29) Zhang, L.; Zhao, G.; Sun, Y. Molecular insight into protein conformational transition in hydrophobic charge induction chromatography: A molecular dynamics simulation. J. Phys. Chem. B 2009, 113, 6873−6880. (30) Zhang, L.; Zhao, G.; Sun, Y. Molecular dynamics simulation and experimental validation of the effect of pH on protein desorption in hydrophobic charge induction chromatography. Mol. Simul. 2010, 36, 1096−1103. (31) Zhang, L.; Zhao, G.; Sun, Y. Effects of ligand density on hydrophobic charge induction chromatography: molecular dynamics simulation. J. Phys. Chem. B 2010, 114, 2203−2211. (32) Zhang, L.; Bai, S.; Sun, Y. Modification of Martini force field for molecular dynamics simulation of hydrophobic charge induction chromatography of lysozyme. J. Mol. Graphics Modell. 2011, 29, 906− 914. (33) Zheng, J.; Li, L. Y.; Tsao, H. K.; Sheng, Y. J.; Chen, S. F.; Jiang, S. Y. Strong repulsive forces between protein and oligo (ethylene glycol) self-assembled monolayers: A molecular simulation study. Biophys. J. 2005, 89, 158−166. (34) Michael, K. E.; Vernekar, V. N.; Keselowsky, B. G.; Meredith, J. C.; Latour, R. A.; Garcia, A. J. Adsorption-induced conformational

guidelines for the design and control of AnFaeA immobilization.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support from the National Key Basic Research Program of China (no. 2013CB733500), the National Natural Science Foundation of China (nos. 21376089 and 91334202), the Guangdong Science Foundation (nos. S2011010002078 and 2014A030312007), and the Fundamental Research Funds for the Central Universities (SCUT-2015PY) is gratefully acknowledged. An allocation of time from the SCUTGrid at South China University of Technology is gratefully acknowledged.

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