Muhammad Alif Mohammad Latifa,
Nuno M. Micaêloc and
Mohd Basyaruddin Abdul Rahman*ab
aDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. E-mail: basya@upm.edu.my; Fax: +60 38943 5380; Tel: +60 38946 6798
bEnzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
cDepartamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. E-mail: micaelo@itqb.unl.pt; Fax: +351 25 3604 382; Tel: +351 25 3604 370
First published on 12th September 2014
In this report, molecular dynamics simulations were applied in order to investigate the effect of Room Temperature Ionic Liquid (RTIL) anions toward the structure and dynamic properties of lipases. Two lipases were studied; Candida antarctica lipase B and Candida rugosa lipase were solvated by five RTILs that contained the same cation, with increasing hydration levels. Several properties were investigated: structural deviations and flexibility of the protein conformation, the behaviour of RTILs at the protein surface, and the interactions between RTILs and water molecules in the systems. Both lipases' conformations showed an increased structural stability in RTILs when compared to an aqueous solution. The lowest structural deviation was observed around 15 to 20 percent of water content (w/w protein). The RTIL with the chloride anion was shown to be the exception however, inducing the least structural stability at low water percentages. The flexibility of both lipases was clearly affected when transferred from aqueous into RTILs. The flexible regions found for both lipases in water were significantly more rigid in RTILs. Around the protein surface, the behaviour of RTIL anions and the water molecules was similar to other conventional organic solvents. The water retention ability for all RTIL anions was consistent for both lipases except for the bis(trifluoromethylsulfonyl)imide anion, which showed distinctive behaviour toward different protein surface properties. The effect of water content was more profound compared to the difference between the RTILs anions studied. However, it was found that the structural and dynamic properties of the lipases were affected by the behaviour of anions toward the hydration layer of the enzymes.
Investigations at the atomic level may further explain how enzymes behave in different types of RTIL anions. By using molecular modelling techniques such as molecular dynamics (MD) simulations, it is possible to provide useful information about the structural and dynamic properties of biomolecules21–24 including lipases in organic media.25,26 Previously, there have been several reports on computational studies of enzyme properties in RTILs.27–36 The earliest MD simulation study on the structure and dynamics of a protein in RTILs was reported by Micaêlo and Soares28 where the stability of cutinase in [BMIM][PF6] and [BMIM][NO3] with varying water content at temperatures of 298 K and at 343 K was investigated. The stability of cutinase in RTILs was progressing in a bell-shaped profile across the water percentages. [BMIM][PF6] was found to render native-like structure while [BMIM][NO3] destabilized the protein conformation. Klähn et al.29 reported a MD simulation study on the stability of CALB in eight different RTILs. The stability of CALB was primarily affected by the types of anions following an order of (increasing stability) [NO3]− ≪ [BF4]− < [PF6]−. The MD simulation work by Burney and Pfaendtner have already compared the properties of enzymes when solvated in between RTILs and conventional organic solvents.30 They found that CRL has the least structural deviation in [BMIM][PF6] while the has the most in water. It was also shown that the interactions on the protein surface were dominated by the RTIL anions. Most of these computational studies involved RTIL anions such as [PF6]−, [BF4]− and [NO3]−. Consistent findings from these reports showed that [NO3]− anion destabilizes enzymes. However, to our knowledge, there has been little effort on the more complex RTIL anions such as [TfO]− and [Tf2N]−. As described previously, these anions were also heavily involved in most of the experimental studies regarding enzyme activity. Therefore, a theoretical study involving these anions is important in order to properly explain the contradiction of results from experimental findings. Previously, we have reported the structural and dynamics properties of an α-chymotrypsin in [BMIM]-based RTILs with different anions such as [PF6]−, [BF4]−, [Cl]−, [TfO]− and [Tf2N]− using MD simulations at different water percentages.31 The [Cl]− anion was chosen for its physical properties while the rest of the anions were chosen because enzymes, as pointed out before, were active in them. Several properties which correlated with experimental evidences were explained. In this report, we attempt to extend our understanding by using the same RTILs towards lipases, namely CALB and CRL. We focus our study on comparing the structural deviations and the flexibility between both lipases, while also monitoring the behaviour of water and RTILs at the enzyme surface. Based on the results from our previous work, the lipases studied could exhibit different properties when simulated in different RTILs at different hydration states.28,31,37–39 As we highlighted previously from experimental reports, a certain order can be established between different anions. We anticipated that the order of anions studied will be affected by the concentration of water in the system. Based on the experimental results, we hypothesized that [TfO]− and [Tf2N]− anions will behave differently towards different enzymes and that this contributes toward the different order of activity when different enzymes were solvated in RTIL with different anions.
Enzyme | %Water (w/w protein) | ||||
---|---|---|---|---|---|
5 | 10 | 15 | 20 | 50 | |
CALB | 92 | 184 | 275 | 367 | 917 |
CRL | 158 | 317 | 475 | 634 | 1584 |
RTIL | %Water (w/w protein) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
5 | 10 | 15 | 20 | 50 | ||||||
Ncat | Nan | Ncat | Nan | Ncat | Nan | Ncat | Nan | Ncat | Nan | |
(a) CALB | ||||||||||
[BMIM][PF6] | 1410 | 1409 | 1404 | 1403 | 1398 | 1397 | 1393 | 1392 | 1358 | 1357 |
[BMIM][BF4] | 1538 | 1537 | 1530 | 1529 | 1523 | 1522 | 1516 | 1515 | 1472 | 1471 |
[BMIM][Cl] | 1990 | 1989 | 1980 | 1979 | 1971 | 1970 | 1961 | 1960 | 1904 | 1903 |
[BMIM][TfO] | 1395 | 1394 | 1389 | 1388 | 1383 | 1382 | 1378 | 1377 | 1343 | 1342 |
[BMIM][Tf2N] | 1089 | 1088 | 1085 | 1084 | 1082 | 1081 | 1078 | 1077 | 1054 | 1053 |
(b) CRL | ||||||||||
[BMIM][PF6] | 2577 | 2560 | 2567 | 2550 | 2557 | 2540 | 2547 | 2530 | 2487 | 2470 |
[BMIM][BF4] | 2812 | 2795 | 2799 | 2782 | 2787 | 2770 | 2774 | 2757 | 2698 | 2681 |
[BMIM][Cl] | 3277 | 3260 | 3261 | 3244 | 3244 | 3227 | 3228 | 3211 | 3130 | 3113 |
[BMIM][TfO] | 2550 | 2533 | 2540 | 2523 | 2530 | 2513 | 2520 | 2503 | 2461 | 2444 |
[BMIM][Tf2N] | 1991 | 1974 | 1984 | 1967 | 1978 | 1961 | 1971 | 1954 | 1930 | 1913 |
RTIL molecules were introduced into the system as the outermost layer of the system, filling up the simulation box. Packing of molecules was accomplished using the Packmol program.47 The net charge in the pure aqueous system was kept neutral by replacing water molecules with sodium or chloride counter ions. For systems in RTILs, the net charge was neutralized by varying the number of cation and anion in the starting configuration. The protonation states for titratable amino acid residues were taken at pH 7.0 for aqueous MD simulations and were unchanged during transfer to RTILs systems.48
In an effort to overcome the “slow dynamics” of RTIL molecules and to achieve faster convergence, all systems were simulated using positional-restraints with a force constant of 1 × 106 kJ mol−1 nm2.28 Several steps of energy minimizations were employed for all systems. Firstly, 5000 steps of steepest-descents energy minimization were performed on the system where all heavy atoms were position-restrained. The RTIL cations and anions, water molecules, protein heavy atoms and protein Cα atoms were released from positional-restraints consecutively in further energy minimization steps. The final step of energy minimization was another 5000 steps of steepest-descents without any position-restraints condition applied. Similar steps were applied for canonical ensemble (NVT), position-restrained MD simulations, each for 500 ps (100 ps for aqueous). When necessary, extra simulations were carried out to ensure the convergence of temperature and pressure for each step of the canonical ensemble simulations. For analysis purposes, all systems were further simulated for 10 ns (6 ns for aqueous) in an isothermal–isobaric condition (NPT). All trajectory analysis on the protein structure and dynamics were performed over the last 2 ns of MD simulations. For statistical accuracy, all systems were simulated in triplicates using different initial velocities for the first NVT simulations.
The average RMSD for both lipases in RTILs at different solvation conditions is shown in Fig. 1. The first striking observation is that the lipases RMSD in all these conditions were lower than that in aqueous condition, which was 0.15 ± 0.004 nm for CALB and 0.18 ± 0.009 nm for CRL. It was clear that these enzymes were closer to their respective crystal structures in RTILs than in aqueous solution. Considering the data from aqueous simulation, the RMSD profiles for both lipases showed a bell-shaped progression across the water percentages, with an observed minimum around 10 to 20% of water. [BMIM][TfO] showed the lowest average RMSD at this percentage content, closely similar to [BMIM][PF6] and followed by [BMIM][Tf2N], [BMIM][BF4] and [BMIM][Cl]. This behaviour was similar to that found in other RTIL-solvated systems and conventional organic solvents that have been previously reported.28,31,38,39,55,56 At 15% of water, both lipases showed an order of RTIL anions (in increasing order of average RMSD) of [TfO]− ∼ [PF6]− > [Tf2N]− > [BF4]− > [Cl]−. This order agreed with the order of stability as reported by Klähn et al.29 As pointed in Fig. 1, [BMIM][Cl] demonstrated highest RMSD profiles for both enzymes when compared with other RTILs. However, the difference of average RMSD between anions, especially [Cl]− was reduced at high water percentage (50%). Similarly to what was observed in our previous study,31 the [Cl]− anions have a tendency to penetrate into the protein-core, and the presence of ions inside the protein-core have been reported to destabilize enzymes.29
This effect, however, was reduced at higher water content due to the thicker layer of water molecules surrounding the protein conformation. Other than [BMIM][Cl], average RMSD for both lipases in RTILs did not show a significant gap across the water percentages, suggesting that the structural deviations of the proteins studied here was not greatly affected by these four types of anions, but rather by the water concentration in the system. These findings also support the observation that partitioning of the hydration level between the enzyme and the bulk solvent was a determinant factor that differentiates molecular-level details in different organic media.57
From the bell-shaped RMSD profiles, it can be deduced that instead of RTIL anions, the water content (which is related to the water activity) played a major role in determining the lipases' stability in RTILs. The dependency of lipase activity on the water content have been observed experimentally, with CALB producing better initial rates when compared with CRL.17 The same report also stated that when [BMIM]+ cation was used, the initial reaction rate was hugely dependent on the nature of the anions, which is in line with the observations in this work at low water content. Schőfer et al.58 also revealed that using CALB resulted in better activity than CRL for the kinetic resolution of 1-pheenylethanol in RTILs. From these reports, CALB was shown better in RTILs when compared to CRL. However, according to Kaar et al.,59 the catalytic activity of CRL in RTILs was still better than organic solvents. They found that in [BMIM][PF6], CRL can increase the initial reaction rate up to 1.5 times faster than in hexane. In relation to the simulation results, the structural deviations of both lipases were almost identical, with only small differences found in the range of the RMSD values for each RTIL. A non-distinguished order of stability between the types of RTIL anions for these lipases was also an indication of why no definite order could be established from various experimental evidences. Therefore, it can be suggested that the difference between the lipases' activity in RTILs were not directly related to their structural stability when solvated by different RTIL anions.
Fig. 2 RMSF of CALB (a) and CRL (b) main chain in RTILs at different water percentages. Values are averaged from the last 2 ns of triplicate MD simulations. |
Fig. 3 Theoretical b-factors of CALB (a) and CRL (b), calculated as per residue in aqueous and RTILs. |
On the other hand, CRL exhibited significantly higher flexibility in aqueous, notably from residue 69 through 91 (Tyr69, Glu70, Glu71, Asn72, Leu73, Pro74, Lys75, Ala76, Ala77, Leu78, Asp79, Leu80, Val81, Met82, Gln83, Ser84, Lys85, Val86, Phe87, Glu88, Ala89, Val90, and Ser91) which is an α-helix connected by loops at each end (Fig. 3b). These residues made up a ‘lid’ for the enzyme conformation. Tejo et al.25 also observed the lid flexibility in water in their simulation studies. They compared the flexibility of the lid in the open conformation which was used in this study (PDB ID: 1CRL) and the closed conformation (PDB ID: 1TRH). Results from their MD simulations also showed the same phenomenon where the lid of CRL for both opened and closed conformation lost its flexibility in organic solvent used (CCl4). Although enzymes were observed to be highly stable in anhydrous media, they also can produce lower activities.60 The presence of water around the surface and inside the enzyme structure is therefore important for biocatalysis as it provides flexibility toward the conformation61 and increases the enzyme-substrate interactions. The flexibility for both lipases at higher water content was clearly affected by the types of the anions. Water-immiscible anions such as [PF6]− and [Tf2N]− promoted higher flexibility while the other anions maintained a low flexibility profile across the water percentages. The decrement in flexibility caused by the presence of RTILs on both lipases studied could restrict the ability of both enzymes to catalyze substrates that are not naturally tailored to their conformations. Furthermore, the interactions of the protein surface with water and RTIL anions were also a contributing factor toward the lipases' flexibility in RTILs, which will be discussed in the following section. Meanwhile, based on our previous observation in Fig. 2, it is possible to ‘tune’ the flexibility of enzymes in RTILs by varying the water content in the system. For example, in reactions involving hydrophobic anions such as [PF6]− and [Tf2N]−, increasing the water content can contribute toward higher enzyme flexibility while not in the water-miscible anions such as [BF4]− and [TfO]−.
In the case of [BMIM][PF6] and [BMIM][Tf2N], the anions and water molecules were highly mobile at higher water percentage, which was clear at 50%. This finding explained why the flexibility of enzyme in these RTILs increased faster at higher water content (Fig. 2). Water localization at the surface was least in [BMIM][Cl]. Due to its significantly smaller size and its high solubility in water,62 there was smaller coverage of anions around the protein surface. From this observation, it is clear that the localization of water was greatly influenced by the RTIL anions, which is further supported by the fact that localization of water molecules was favourable on the surface where the anions were covering. The interactions between RTILs and water molecules are reported to be strongly dependent on the type of anions.63,64 In this study, the behaviour of different RTIL anions and water were observed by monitoring the average percentage of water at the protein surface across the water percentages (Fig. 6). The percentage of water on the surface was determined based on the number of water located within 0.5 nm from protein surface in the starting configuration and the average number of water in the same region from the last 2 ns of MD simulations. We found that, as conventional organic solvents, RTILs stripped water molecules away from protein surface.37,64 The percentage of water at the surface for four types of anions ([PF6]−, [TfO]−, [BF4]− and [Cl]−) were consistent for both of the lipases studied. The amount of water that was stripped from the surface was increased as the water concentration increased. However, the number of water in the calculated region was actually increasing despite the strong, water-stripping capability that was exhibited by the RTILs. We estimated that the water-stripping capability of different anions was related to their tendency to hydrogen-bond with water molecules. RTILs such as [BMIM][PF6] and [BMIM][TfO] exhibited lower stripping strength due to less hydrogen bond interactions with water molecules. As for [BF4]− and [Cl]− anions, which are highly hydrophilic and therefore water-miscible,65 more water molecules were stripped away from protein surface. Previously, [BMIM][TfO] was found to reduce the enzyme flexibility but this RTIL stripped less water from the protein surface. It can be suggested that the [TfO]− anions exhibited the capability to hydrogen-bond with water through the oxygen atoms. These anions mostly interact with water at the surface as compared to the protein surface residues themselves. Hence, water could remain near the protein surface but lose their effect towards the protein flexibility.
As observed in Fig. 6a, CALB was found to maintain a certain level of hydration in [BMIM][Tf2N] despite the increasing water concentration in the system. This was also observed in our previous work.31 However, Fig. 6b showed that for CRL, the strength of water-stripping exhibited by different anions was in increasing order of [Tf2N]− < [PF6]− ∼ [TfO]− < [BF4]− ∼ [Cl]−. The variation of [BMIM][Tf2N] behaviour toward the two lipases studied can be related to the difference of the enzyme surface properties. [Tf2N]− anion is weakly-coordinated, possesses low capacity for ion-ion interactions, has poor water-solubility properties, and is most hydrophobic compared to other anions studied.66 However, the amide group in the [Tf2N]− anion is able to form strong hydrogen bond interactions with water molecules that can pull them away from the protein surface. For enzymes such as CALB and previously-studied α-Chymotrypsin,31 [BMIM][Tf2N] stripped away the most number of water molecules consistently at each water percentage studied as compared with other RTILs. However, CRL possesses more than twice the number of Asparagine residues at the surface compared to CALB (Fig. 7).
Fig. 7 Amino acid residues that were found exposed on the surface of CALB and CRL. The amino acid residues were probed on the surface of the crystal structures for the respective enzymes. |
Asparagine has a high propensity to hydrogen bond, since it is possible for the amide group to accept two and donate two hydrogen bonds. The competition from these residues at the surface to hydrogen-bond with water molecules resulted in fewer [Tf2N]–water interactions. Thus, a higher percentage of water molecules was found on the surface. This finding coincided with the rapid increment of the average number of hydrogen bonds between the water and CRL in [BMIM][Tf2N] when compared to the ones in [BMIM][PF6] (Table 3). However, as the number of water found on the surface was increasing with every water percentage, the differences between the amounts of water on the surface between the RTILs did not greatly affect the overall protein–water interactions. The data from Table 3 shows that the average hydrogen-bonding between CALB in [BMIM][Tf2N] was slightly higher than [BMIM][PF6] at each water percentage. This can explain why [BMIM][Tf2N] produced a closely similar effect toward the structural deviations and flexibility of CALB, even with less water molecules on the surface when compared with other RTILs, which was consistent with our previous study.31
%Water (w/w) | CALB | CRL | ||
---|---|---|---|---|
[BMIM][PF6] | [BMIM][Tf2N] | [BMIM][PF6] | [BMIM][Tf2N] | |
5 | 130.27 | 136.76 | 228.52 | 256.01 |
10 | 207.72 | 215.43 | 408.30 | 454.57 |
15 | 254.17 | 263.09 | 494.96 | 567.23 |
20 | 247.80 | 257.42 | 533.35 | 643.67 |
50 | 318.88 | 352.45 | 647.61 | 809.92 |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07460b |
This journal is © The Royal Society of Chemistry 2014 |