Molecular Dynamics Simulations on the Behaviors of Hydrophilic

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Molecular Dynamics Simulations on the Behaviors of Hydrophilic/ Hydrophobic Cyclic Peptide Nanotubes at the Water/Hexane Interface Huifang Lin, Jianfen Fan, Peipei Weng, Xialan Si, and Xin Zhao J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b02465 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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The Journal of Physical Chemistry

Molecular

Dynamics

Simulations

on

the

Behaviors of Hydrophilic/Hydrophobic Cyclic Peptide

Nanotubes

at

the

Water/Hexane

Interface Huifang Lin, Jianfen Fan,∗ Peipei Weng, Xialan Si, and Xin Zhao College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China

∗ E-mail: [email protected] and [email protected]. Tel: 0086-0512-65883271. 1

ACS Paragon Plus Environment


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ABSTRACT:

In this work, nine kinds of amino acid residues, i.e., alanine (A),

leucine (L), valine (V), isoleucine (I), tryptophan (W), glutamine (Q), threonine (T), serine (S) and cysteine (C) were selected to construct seven cyclic peptide nanotubes (CPNTs) with diverse hydrophilic/hydrophobic external surfaces, which were further separately inserted at the water/hexane interface to investigate their microstructures and interfacial properties. Molecular dynamics (MD) simulations reveal that all the CPNTs except the QT- and VL-CPNTs appear different degrees of tilt, fracture and shedding at the interface. The end-CPs are more susceptible to the effect of the surroundings than the mid-CPs. The interactions of individual CP subunits with the neighbourings disclose the firmness of the mid-CPs and the dissociation of the end-CPs. The results indicate that a hydrophobic CPNT is prone to stay at the interface, while a hydrophilic CPNT easily enters the water phase, resulting in many H-bonds with water. Results in this work enrich the dynamic properties of a hydrophilic/hydrophobic CPNT at a biphase interface at an atomic level.

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1. INTRODUCTION An interface region is usually a major site where a phase transition takes place.1 The research on the structure and property of an interface has attracted a great deal of attention, especially in the field of MD simulation, which can achieve profound information and original understanding towards the mechanism and essence of an interfacial behavior at an atomic level.2,3 The first MD investigation of a lipopeptide surfactant with different interfacial concentrations at the water/hexane interface indicated that the surfactin at the interface adopts a tetrahedral conformation and its peptidic backbone exhibits great flexibility strongly depending on its interfacial concentration.4 The peptide ring of a protonated surfactin may slightly tilt at the decane/water interface and the aliphatic chain extends to the decane phase.5 The strong polar interaction between surfactin and water results in the stable adsorption of the surfactin at the interface.5 The backbone of a cyclo-hexapeptide (CHP) may take "horse-saddle" conformation at the water/cyclohexane interface and the structures of amino acid residues on the side chains determine the interfacial property of a CHP.6 For example, the backbone of a CHP with hydrophilic side chains may tilt toward the water phase, which largely weakens the lateral diffusion and rotational motion of the CHP at the interface.6 A CPNT formed from the self-assembly of amino acids, possesses a hollow tubular structure,7,8 favorable biocompatibility9-13 and exhibits distinctive characteristic of effectively transporting molecules and ions, i.e., water,14,15 O2, 3



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CO216,17 and K+, Na+, Ca2+.18-20 Amino acids with different residues endow the external surface of a CPNT with diverse hydrophilicity/hydrophobicity.10,11,13 The surface hydrophilicity/hydrophobicity of a nanoparticle may significantly affect its behavior at a water/oil interface.5,21,22 The stability of a CPNT in a solvent is related to the structures of amino acid residues and the kind of the solvent, etc. MD simulation results demonstrated that the CPNTs with different surface polarities, 8 × cyclo-(WL)4 and 8 × cyclo-(QAEA)2 (E: glutamate), are more stable in a nonpolar solvent than in a polar solvent.23 The solvent effect plays a crucial role in the separation of cyclic peptide (CP) subunits from a CPNT. During the dissociation of CPs, the cooperativity of various CP-CP interactions gradually increases from the terminal to the core of a CPNT. The structure and dynamic property of a CPNT in a single solvent phase have been widely studied.23-26 While its behavior at a biphase interface has not been reported. In this work, MD simulations were performed to explore the dynamical behaviors of CPNTs with various hydrophilic/hydrophobic external surfaces at the water/hexane interface, including their structural morphologies, distributions, and interactions with water and hexane phases, and ultimately to reveal the microscopic mechanisms of deformations, inclinations, movements and fractures of these CPNTs at the interface. Results in this work will fill the void in the study of a CPNT at a biphase interface at an atomic level, and in turn, provide theoretical information for the self-assembly of CPs at an interface and the

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separation of CPNTs with diverse hydrophilicity/hydrophobicity in a water/oil biphase system.

2. MATERIALS AND METHODS 2.1. Modeling Systems and Simulation Details. In this work, nine kinds of amino acid residues, i.e., Ala (A), Leu (L), Val (V), Ile (I), Trp (W), Gln (Q), Thr (T), Ser (S), and Cys (C), with diverse hydrophilic/hydrophobic characteristics, were used to construct seven kinds of cyclic-octa-peptide subunits, shown in Figure 1. The process of constructing a CPNT is as follows. Firstly, two different kinds of amino acid residues were alternately arranged using our self-written Tcl script8,15 to construct the first flat CP subunit, named as P1. Secondly, the P1 was successively translated and flipped using the VMD 1.9.1 software27 to obtain the structures of the remaining seven CP subunits. Thirdly, eight flat CP subunits self-assembly to form the initial configuration of a CPNT by stacking through an antiparallel β-sheetlike H-bonding network.7,13,15,18 Each CPNT underwent energy minimization of 10,000 steps, 10-ns simulated annealing from 400 K to 200 K at a step of 0.1 K per 5 ps in an NPT ensemble, and further 5-ns equilibration simulation at 310 K to obtain the equilibrium structure. The simulated annealing and equilibration process of each CPNT were repeated 10 times, respectively. The conformation of a CPNT with the lowest total energy among these parallel simulations was selected as the equilibrium structure of the CPNT. The symbols of seven CPNTs and their corresponding short forms, and the outer-wall features are

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listed in Table 1. The tube length of each equilibrated CPNT was measured as the average distance between the centers of mass (COMs) of the first and last CP rings during the last 2-ns equilibration simulation. The region between two adjacent CP subunits is defined as a gap. As examples, Figure S1 in the Supporting Information illustrates the time evolutions of the intersubunit distances (in individual gaps) between the COMs of two adjacent CP subunits of the IL- and QT-CPNTs along the z-direction during the 5-ns equilibration simulations. It can be seen that the seven intersubunit distances of the two CPNTs all reach equilibrium after 2.5 ns. The average intersubunit distance of a CPNT refers to the average value of intersubunit distances in seven gaps during the last 2-ns equilibrium simulation. It basically equals to the quotient of the average tube length by 7. The intersubunit distances between 4.7∼5.0 Å of seven CPNTs were basically consistent with the experimental and simulation results (4.8 Å) for similar CPNTs.28-30 These equilibrated CPNTs were used for subsequent constructions of individual simulation systems. Two solvent boxes with fixed x and y dimensions (i.e., 50 Å × 50 Å), containing 4250 water and 576 hexane molecules, were set up, and equilibrated for 10 ns in NPT ensembles, respectively. The final dimensions of the water and hexane boxes are 50 Å × 50 Å × 52.23 Å and 50 Å × 50 Å × 52.65 Å, with the densities of 0.97 g/cm-3 and 0.63 g/cm-3, respectively, which are in good agreement with their respective theoretical densities at 310 K.31 An water/oil interface was constructed by jointing the water and hexane boxes along the z-direction, with a narrow slit of 6


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about 2 Å to avoid poor van der Waals interactions between two phases. A 10-ns equilibration was then carried out to obtain a steady two-phase (water/hexane) system with the dimensions of 50 Å × 50 Å ×106.64 Å. The density distribution profiles in Figure 2 show that the water phase occupies the z < 6.3 Å region and hexane the z > -4.5 Å region of the simulation box, and the water/hexane interface locates at z = 2.6 Å. Each CPNT was inserted at the equilibrated water/hexane interface with its tube axis parallel to the interface. All the overlapped solvent molecules within 4 Å of the CPNT were deleted to avoid poor van der Waals interactions. Figure 3 illustrates the incorporation mode of the WL-CPNT at the water/hexane interface as an example. Each system first underwent 10-ns equilibrium in an NPT ensemble by fixing the position of the CPNT, and then experienced energy minimization of 10,000 steps using the steepest descent method,32 further followed 18-ns equilibrium without any constraints. The 18-ns equilibrium process of each system was repeated 3 times. The conformation of each system with the lowest total energy was chosen for subsequent analysis. Figure S2 in the Supporting Information illustrates the average pressures and corresponding standard deviations of the seven systems, indicating the average data of the system pressures are about 1.09∼1.30 bar. The modeling systems are too small to cause the thermodynamics including pressures to fluctuate too much. As can be seen from the time evolutions of the total energies of individual “water/CPNT/hexane” systems shown in Figure S3, all seven systems reach 7



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equilibrium after 8 ns. Thus, the last 10-ns trajectories can be used for the analyses of individual properties. All the energy minimizations and MD simulations were performed using the NAMD 2.9 program33 with the all-atom CHARMM 27 force field34 and TIP3P water model.35 The Nosé−Hoover Langevin piston method (with 100 fs Langevin piston period and 50 fs decay time)36 and Langevin dynamics (with 5/ps damping coefficient)37 were applied to maintain the pressure of a system at 1.01325 bar and the temperature at 310 K, respectively. Periodic boundary conditions were applied in all three directions, and the x and y dimensions of a simulated system remain unchanged, while the z dimension is allowed changeable. Particle-mesh Ewald (PME)38 summation approach was adopted to describe full electrostatic interactions. The non-bonded interactions including long-range electrostatic and van der Waals terms were computed using a cutoff radius of 12.0 Å, with a smoothing function switched on from 10.0 Å. The bond lengths between H and heavy atoms were constrained to the equilibrium values using the SHAKE algorithm.39 A time step of 1.0 fs was used during all simulation runs. The analyses and visualizations of all systems were completed using the molecular graphics program VMD 1.9.1.27

2.2. Aromatic π-π π Stacking Interactions. It is considered that a special hydrophobic interaction—π-π stacking interaction may exist between two aromatic ring planes,40 which may affect the crystal structures of aromatic molecules and the stabilities of biological systems.41-43 According to McGaughey 8


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et al.′s suggestion,44 the intensity of such an interaction is closely related to the relative orientation of two aromatic ring planes, which can be collectively described by the distance Rcen between the two plane centers and the angle θ ur

between the vector R (from one plane center to another plane center) and the r surface normal vector n of one plane, shown in Figure S4 of the Supporting

Information. A π-π interaction exists only when Rcen ≤ 7.5 Å and is the strongest when

10° ≤ θ ≤ 30°.44

In this work, tryptophan (W) participates in forming cyclic-octa-peptide subunits which further self-assemble into the WL-CPNT. The π-π stacking interactions between neighbouring indole rings of Trp (W) side chains were speculated to have a non-ignorable effect on the structure and stability of the CPNT.

3. RESULTS AND DISCUSSION 3.1. Interfacial Behaviors. 3.1.1. Structural Changes. The backbone structures of seven CPNTs obtained from the last frames of 18-ns MD simulations are illustrated as the thick-lined graphs in Figure 4. Individual CP subunits in each CPNT, are sequentially marked as P1 to P8 along the tube axis, shown at the right bottom of Figure 4. The results indicate that all the CPNTs except the VL- and QT-CPNTs exhibit different degrees of fractures at the water/hexane interface. Figure 4 presents a particularly eye-catching phenomenon that the first or last CP ring of four hydrophobic CPNTs (i.e., P8/AL, P1/VL, P1/IL, P8/WL) has a

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considerable displacement. This is due to the application of periodic boundary conditions during the simulations. Thus, the VL-CPNT keeps a tubular structure, but with slight deformations of the first and last CP subunits (P1 and P8). As for the QT-CPNT, it almost maintains the original tubular structure, but seriously tilts at the interface. The sub-weak deformed peptide tubes include the AL- and QC-CPNTs. Their first CP rings (P1) present slightly deformations and sheddings. Nevertheless, the remaining three CPNTs (WL, IL and QS) exhibit distinct sheddings of two, three and four CP subunits, respectively, and the exfoliated CP subunits have undergone significant deformations. Theoretically, a CP subunit displays a circular structure in vacuum. It may deform in an actual environment. Such deformation can be described by the quantity of ellipticity, which is defined as the ratio of the largest (Dmax) and smallest diameters (Dmin) of a CP subunit,

Ellipticity = Dmax Dmin

( Ellipticity ≥ 1)

(1)

Here, the diameter of a CP subunit refers to the distance between two opposite atoms on the ring. As there are 24 atoms on the backbone ring of a cyclic octa-peptide, 12 values of the diameters can be obtained. Dmax and Dmin are the largest and smallest diameters among the 12 values. The deviation of the ellipticity from 1.0 indicates the degree of the deformation of a CP subunit from a circular structure. MD simulation trajectories reveal that a CP subunit has only minor deformation when its ellipticity is no more than 1.4.

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The average ellipticities of individual CP subunits of seven CPNTs at the water/hexane interface during the last 10-ns trajectories are illustrated in Figure 5. It is clear that the four middle CP (mid-CP) subunits (P3, P4, P5 and P6), no matter of a hydrophobic CPNT or of a hydrophilic CPNT, maintain the ellipticities of 1.2∼1.3, while the terminal CPs at both ends (end-CPs) go through severer deformations, which is consistent with the phenomena shown in Figure 4 and the result of Subramanian et al.28 Following points can be found by careful observation. (1) The ellipticities of individual CP subunits of the QT-CPNT are all less than 1.4, which is consistent with the complete structure presented in Figure 4. Although the first CP rings (P1) of the AL- and QC-CPNTs are detached, as far as the ellipticity is merely concerned, the variation amplitudes in these two CPNTs are not obviously different from that of the QT-CPNT. (2) The ellipticities of the seven CP rings of the WL-CPNT are all below 1.4, except that (1.6) of the last CP ring (P8), meaning that only the last CP subunit has a slightly larger deformation. (3) The VL- and IL-CPNTs have the similar results that the ellipticities of the six mid-CPs are all less than 1.4, while the end-CPs (P1 and P8) show prominent deformations with ellipticities of 1.7∼2.0. (4) Special phenomenon occurs in the QS-CPNT, that is the ellipticities of P2, P7 and P8 subunits are much outstanding, as high as 1.7∼2.7 compared to those (1.40∼1.45) of the remaining five CP subunits. In fact, the QS-CPNT exhibits the worst integrity at the interface, shown in Figure 4.

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Theoretically, up to eight H-bonds can exist between the backbones of two adjacent cyclic-octa-peptide subunits, and the presence of the H-bonds between their side chains depends on the structures of the amino acid residues. The deformation or detachment of a CP subunit is closely related to the weakening of the H-bonded interactions with its neighbouring CP subunits.23 Usually, whether a H-bond exists between two molecules can be determined by energy criterion45 or conformation judgement46. In this work, the default plugin of conformation judgement in the VMD software was applied. That is, if the distance between a H donor and acceptor is less than or equal to 3.0 Å, and the supplementary angle formed by a H donor, H and a H acceptor is no more than 20°, the presence of a H-bond can be determined. Figure 6 shows the probability distributions of the total numbers of backbone and side chain H-bonds in seven CPNTs during the last 10-ns trajectories. Several features can be found from Figure 6. First, in the middle gaps (mid-gaps) of all seven CPNTs, the fluctuations of the H-bond numbers all exhibit peaks at 2∼5 H-bonds, while those in the terminal gaps (end-gaps) even display peaks at zero H-bonds, implying thorough fractures. Second, the end-gaps of four hydrophobic CPNTs seem more likely to fracture, compared to those of the hydrophilic CPNTs except the QS-CPNT. The AL-, VL-, ILand WL-CPNTs in turn have 3, 2, 3 and 3 gaps with probability peaks at zero H-bonds. Among three hydrophilic CPNTs, the QT-CPNT seems the most stable with no occurrence of the distribution peak at zero H-bond. Nevertheless, the distribution peaks in one or two end-gaps of the QC- and QS-CPNTs appear at 12


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zero H-bonds, indicating that the broken phenomena of these two CPNTs would appear at the ends, which is in line with the results shown in Figure 4.

3.1.2. Stabilities. The stability of a CPNT at the water/hexane interface is attributed

to

the

several

factors

including

solvent

effects,

the

backbone–backbone H-bonded interactions, and various interactions between side chains.47-49 The latter involved intermolecular H-bonds, aromatic π-π stacking interactions, van der Waals and electrostatic interactions, etc. Among these different types of interactions, aromatic π-π stacking interactions may exist between neighbouring CP subunits of the WL-CPNT. The stabilities of the backbones and side chains of seven CPNTs were described by the average RMSDs (root mean square deviations) and corresponding standard deviations of the backbones and heavy atoms of the side chains during the last 10-ns simulation, collectively shown in Figure 7. First, among seven CPNTs, the variation trends of the average RMSDs and the corresponding standard deviations of the side chains are basically consistent with those of the backbones, which confirms that the interactions between the side chains are significant for the stability of a CPNT. Second, the average RMSDs and the corresponding standard deviations of the backbones and heavy atoms of the side chains of three hydrophilic CPNTs (i.e., QT, QS and QC) are obviously smaller than those of four hydrophobic CPNTs (i.e., AL, VL, IL and WL). It is the presence of an acylamino group in Gln (Q) moiety (see Figure 1) that promotes the formation of the H-bonds between the side chains of 13



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three hydrophilic CPNTs.47 Nevertheless, there is no possibility forming H-bonded interactions between the side chains of four hydrophobic CPNTs (see the structural formulas shown in Figure 1). As a result, the side chains of three hydrophilic CPNTs are more firmly connected, which further enhances the stabilities of the backbones. Third, the AL-CPNT has significant higher RMSDs both for its backbone and side chains, compared to those of the VL-CPNT. Among four hydrophobic amino acids (A, V, I and W), the Ala (A) has the smallest residue of ̶ CH3 on its side chain. It was reported that the presence of Ala in a CPNT would increase the flexibility of the CPNT, resulting in large RMSDs of the backbone and side chains of the CPNT.47 The presence of small groups (̶ CH3) on the side chains of the AL-CPNT also makes water molecules more accessible to the backbone carbonyl groups,47 forming H-bonded interactions, which is in competition with the original H-bonded interactions between the backbones of individual CP subunits. As a result, the AL-CPNT exhibits distinct instability compared with the VL-CPNT. In addition, the RMSDs of the WL-CPNT are slightly lower than those of the IL-CPNT. The aromatic structure (indole ring) of Trp (W) is made up of a hydrophobic benzene ring and a hydrophilic pyrrole ring (see Figure 1). As mentioned before, an aromatic π-π stacking interaction maybe exists between two neighbouring benzene rings. According to McGaughey et al.’s suggestion,45 the intensity of a π-π stacking interaction can be collectively described by the parameters of Rcen and θ (see the section “Materials and methods”). As four Trp 14


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(W) moieties numbered as resides 1, 3, 5 and 7 alternately exist in each side chain of the WL-CPNT, there are totally 32 Trp moieties in the CPNT. For the sake of conciseness, the π-π stacking interactions between resides 1 of P2 and P3, between resides 5 of P2 and P3 and between resides 5 of P4 and P5 were shown in Figure S5 (a), (b) and (c), respectively. The high probabilities of Rcen ≤ 7.5 Å in these regions, together with the occurrences of θ between 10° and 30° distinctly indicate that the π-π stacking interactions really exist between the side chains of the WL-CPNT. Besides, amphiphilic indole rings may stretch out, destroying the neighbouring water structure, and further resulting in their more preferable configurations.50 In a word, the π-π stacking interactions between the neighbouring side chains of the WL-CPNT distinctly enhance the stability of the CPNT, resulting in smaller RMSDs compared to those of the IL-CPNT. Fourth, among three hydrophilic CPNTs (QS, QT and QC), the QS-CPNT exhibits the highest RMSDs and corresponding deviations. The residue ( ̶ CH2OH) of Ser (S) is the smallest among the amino acid residues of Ser (S), Thr (T) and Cys (C). This case is similar to the AL-CPNT, which has the smallest residue ( ̶ CH3) of Ala (A). The smallest residue of Ser (S) may also reduce the stability of the QS-CPNT. In summary, the RMSD analysis reflects that the structures and the corresponding hydrophilicities/hydrophobicities of side chains both significantly influence the stability of a CPNT at the water/hexane interface.

3.1.3. Deviations from the Interface. Due to the fact that the end-CPs of a CPNT may undergo serious deformations and even fractures, only mid-CPs, 15



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namely P3, P4, P5 and P6, were selected to calculate the COM (center of mass) of a CPNT. The time evolutions of the COMs of seven CPNTs in the z-direction during the 18-ns simulations are shown in Figure 8. It can be clearly found that four hydrophobic CPNTs (AL, VL, IL and WL) are at or near the water/hexane interface, whereas three hydrophilic CPNTs (QT, QS and QC) are completely out of the interface region and enter the water phase. MD simulation trajectories show that water molecules can enter the inner cavities of all seven CPNTs. Nevertheless, hexane molecules are too large to get into any CPNTs. Water molecules entering the inner cavity of a CPNT may form H-bonded interactions with the backbone of the CPNT. Such interactions may significantly weaken the original hydrophobicity of a hydrophobic CPNT. As a result, four hydrophobic CPNTs wander near the water/hexane interface without going deep into the hydrophobic hexane phase. Nevertheless, the H-bonded interactions between water molecules and the inner cavity of a hydrophilic CPNT may further strengthen the original hydrophilicity of the CPNT. Such synergistic effect leads three hydrophilic CPNTs to move away from the water/hexane interface and enter the water phase.

3.1.4. Surface Tensions. The pressure tensors Pxx , Pyy and Pzz obtained from the 18-ns simulation can be used to calculate the surface tension (σ) of the water/hexane interface in a water/CPNT/hexane system according to the following equation,51

σ = Lz  Pzz − ( Pxx + Pyy ) 2 2 1

(2) 16


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where Lz is the z-direction dimension of a steady water/hexane system, i.e., 106.64 Å in this work. Pzz is the local pressure normal to the interface, and Pxx and Pyy are the local pressures tangential to the interface. The surface tensions in seven systems separately implanted with the AL, VL, IL, WL, QT, QS and QC-CPNTs calculated using the above equation are 43.04, 43.27, 43.45, 43.32, 42.84, 43.27 and 43.19 mN/m, respectively. Namely, the surface tensions of the water/hexane interface in seven systems at 310 K are about 42∼43 mN/m. In general, surface tension may decrease with the increase of the temperature. Thus, the above results are reasonable compared with the experimental result (about 50 mN/m) of the water/n-Hexane system at 298 K.52 In addition, the values of the surface tensions in individual systems are roughly the same, which results from the fact that the force balance of an interface may lead to the same tension at each position of a system, regardless of the presence or absence of a CPNT.

3.2. H-bonded interactions. 3.2.1. Intersubunit Backbone H-bonds and Side Chain H-bonds. The probability distributions of the H-bond numbers between neighbouring backbones and between neighbouring side chains of seven CPNTs during the last 10-ns trajectories are collectively displayed in Figure 9. As can be seen from Figure 9(a), all seven CPNTs consisting of eight CP subunits have less than 56 (a theoretically value for an intact CPNT) intersubunit backbone H-bonds, indicating that the water/hexane interface significantly affects the 17



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compact of individual CP subunits in a CPNT. Among four hydrophobic CPNTs, the number of the backbone H-bonds in the WL-CPNT is the highest, reaching a maximum of 33, with a peak at 17, which is in line with the phenomenon shown in Figure 4 (note that the shedding of the P1 subunit was merely caused by the application of periodic boundary conditions in the MD simulation). The π-π stacking interactions and weak H-bonded interactions between indole rings of Trp (W) side chains synergistically make the WL-CPNT relatively stable. The number of the backbone H-bonds of the IL-CPNT is the least with a maximum value no more than 22, which is in conformity with the result of three CP subunits (P1, P7 and P8) falling off presented in Figure 4. Relatively, three hydrophilic CPNTs, especially the QT- and QC-CPNTs, exhibit more backbone H-bonds compared to the hydrophobic CPNTs, reaching maxima of 32 and 36, respectively. This fact accords with the phenomenon that the two CPNTs substantially remain tubular structures shown in Figure 4. Among three hydrophilic CPNTs, the QS-CPNT displays the most dramatic fluctuation of the backbone H-bond number. The amino acid Ser (S) has a small but very active residue of ̶ CH2OH. The presence of Ser may weaken the stability of a CPNT, causing the backbone H-bonds to become breakable. In fact, it can be seen that the number of the CP subunits shedding from the QS-CPNT in Figure 4 is the largest, reaching four. Figure 9(b) shows that there are no H-bonded interactions at all between neighbouring side chains of the AL-, VL- and IL-CPNTs, and the WL-CPNT has occasional H-bonded interactions between the side chains. Nevertheless, distinct 18


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H-bonded interactions between the side chains exist in three hydrophilic CPNTs. The total numbers of H-bonds between the side chains in the QT- and QS-CPNTs are much higher than that in the QC-CPNT, reaching maxima of 16 and 14, respectively. Except the jointly owned residue Gln (Q) with an acylamino moiety capable of forming H-bonds, the residues Thr (T) and Ser (S) in the QT- and QS-CPNTs, respectively, both have a hydroxyl group on their side chains, while the residue Cys (C) in the QC-CPNT does not. Figure S6 illustrates the forms of the H-bonded interactions between the side chains of three hydrophilic CPNTs (QT, QS and QC), respectively. Obviously, there are three types of H-bonds between the side chains of the QT- and QS-CPNTs, namely Q-Q, T-T, Q-T and Q-Q, S-S, Q-S, respectively, while the QC-CPNT only has one form of H-bond, that is Q-Q. The sum number of the backbone H-bonds and side chain H-bonds of the QT-CPNT reaches the most among seven CPNTs, which is in consistent with the phenomenon that this CPNT maintains the most intact tubular structure at the water/hexane interface shown in Figure 4.

3.2.2. CPNT-water H-bonds. The probability distributions of the H-bonded interactions of seven CPNTs' backbones and side chains with water molecules are given in Figure 9. It is apparent that the backbone of the AL-CPNT forms the highest number of H-bonds with water molecules compared to other three hydrophobic CPNTs (VL, IL and WL). MD simulation study on a similar system47 has proved that the existence of a small side chain, such as Ala (A), may make water molecules more accessible to the backbone carbonyl groups by 19



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penetrating from the crevices between the side chains, resulting in more H-bonds formed between the backbone carbonyls and water. For the QS-CPNT, the side chain of the residue Ser (S) is also relatively small compared to the other residues. So similar to the AL-CPNT, more H-bonds between the backbone and water molecules were formed, reaching a maximum of 41. The backbones of the VL-, IL-, WL-, QT- and QC-CPNTs form small amounts of H-bonds with water molecules due to the presences of large side chains, which hinder water molecules from closing to the surfaces of the tubes, resulting in the weakening of the H-bonded interactions between the backbone carbonyl groups and water molecules. The QT-CPNT forms the smallest amount of H-bonds with water, no more than 19, being the weakest competition to the backbone H-bonded interactions. Thus, the number of the backbone H-bonds of the QT-CPNT is relatively large, resulting in the firmness of this CPNT. Figure 9(d) shows that there are no H-bonded interactions between the side chains of the AL-, VL- and IL-CPNTs and water molecules, and few H-bonded interactions occur in the WL-CPNT system. Nevertheless, the side chains of three hydrophilic CPNTs (QT, QS and QC) form a larger number of H-bonds with water molecules, reaching maxima of 54, 57 and 30, respectively. The QT- and QS-CPNTS both have two groups capable of forming H-bonds on their side chains, leading to relatively large numbers of H-bonds with water, respectively.

20


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In a word, the structures of the amino acid residues in a CPNT significantly influence the H-bonded interactions among backbones, side chains and solvent water.

3.3. Electrostatic and VdW Interactions. The electrostatic and vdW interactions between a CPNT, water and hexane phases in individual systems have been investigated based on the last 3.0-ns trajectories and the results are presented in Figures S7 and 10.

3.3.1. Water-hexane Electrostatic and VdW Interactions. Two black squared lines in Figure S7 indicate that the main form of the non-bonded interactions between water and hexane phases is the vdW interaction term. Evidently, the electrostatic and vdW interaction energies between two phases in each system are both reduced after the addition of a CPNT. Moreover, the energy reduction caused from the addition of a hydrophobic CPNT is larger than that from a hydrophilic CPNT. It is mainly because a hydrophobic CPNT tends to stay at the water/hexane interface, closely contact with two solvent phases from two sides, thus resulting in weakening of the interactions between two phases. Nevertheless, the effect of a hydrophilic CPNT completely entering water phase on the interphase interactions is relatively weak.

3.3.2. CP-hexane Electrostatic and VdW Interactions. Figure 10(a) visually exhibits the electrostatic and vdW interactions between each CP subunit of individual CPNTs and the solvent hexane. First, it is clear that there is scarcely electrostatic interaction between any CP subunit and hexane, while at the same 21



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time, the vdW interaction exists between them within a narrow distribution range from -40 to 0 kcal/mol. Also, it seems that there is no variation regularity for the fluctuation of the vdW interactions versus the positions of the CP subunits. Second, the one-to-one correspondences of the vdW interaction energies versus different CP subunits indicate that the vdW interactions of three hydrophilic CPNTs are generally weaker than those of four hydrophobic CPNTs, which is determined by the inherent properties of the hydrophilic CPNTs easily penetrating the aqueous phase, away from the hexane phase and the water/hexane interface.

3.3.3. CP-water Electrostatic and VdW Interactions. Careful observation of Figure 10(b) discloses that the vdW interaction energy between each CP subunit and the solvent water is relatively weak and fluctuates around -20 kcal/mol in a small range. Nevertheless, the electrostatic term varies in a wide range from 0 to -260 kcal/mol, namely strong and volatile. By comparing with the interaction energies of individual CPNTs with the hexane phase [shown in Figure 10(a)], it can be deduced that the electrostatic interactions between CP subunits and the water phase play a major role in influencing the behavior of a hydrophilic/hydrophobic CPNT at the water/hexane interface. The following two points can be summarized through further in-depth analysis of the electrostatic interactions. (1) The electrostatic interaction of a hydrophilic CPNT with the water phase is stronger than that of a hydrophobic CPNT, due to the fact that more water molecules around a hydrophilic CPNT and more H-bonds of the CPNT 22


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backbone and side chains with the surrounding water molecules. (2) The electrostatic interactions of the end-CP subunits are significantly stronger than those of the mid-CPs in all systems, as the electrostatic interaction energies of the P1 and P8 subunits in individual CPNTs can reach -90 ∼ -250 kcal/mol, while those of the mid-CPs hover between -5 ∼ -110 kcal/mol. This phenomenon may be resulted from the joint contributions of relatively more water molecules locating in the entrance regions at both ends of a CPNT and the susceptibilities of the bare carbonyl groups at the entrances. These findings are precisely consistent with the phenomenon that the end-CPs are more likely to break and distort.

3.3.4. CP-CP Electrostatic and VdW Interactions. By comparing the upper and lower layers of Figure 10(c), it is easy to derive that the electrostatic and vdW interactions both exist between individual CP subunits in all simulation systems and the former fluctuates more tensely. What is more, the electrostatic interaction energies (close to zero) in gaps 1 and 7 of four hydrophobic CPNTs are much lower than those in five mid-gaps, which is exactly the opposite of the information given by the CP-water electrostatic interactions shown in Figure 10(b). That is, there are competitions between the CP-water and CP-CP electrostatic interactions in four hydrophobic CPNTs. The fluctuation trend of the CP-CP electrostatic interactions versus the gap positions in a hydrophilic CPNT is different from that in a hydrophobic CPNT. For the QS-CPNT, the electrostatic interaction energies in gaps 1, 2, 6 and 7 are much lower than those in mid-gaps. In fact, this CPNT suffers a serious fracture at the interface, where the four CPs at 23



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both ends are significantly off. As for the QC-CPNT, there is an end-CP shedding at the interface, which is in agreement with a significantly lower electrostatic interaction in gap 1. The phenomenon that the QT-CPNT always keeps a tubular structure at the interface just coincides with the high electrostatic interaction energies in individual gaps. Summarily, the behavior of a CPNT at a biphase interface is the comprehensive manifestation of all the interactions of individual CP subunits with the neighbourings. The CP-water and CP-hexane interactions are dominated by the electrostatic and vdW interactions, respectively, and the former is overwhelming for the end-CPs, which facilitates the end-CPs being easily pulled by the surrounding water molecules and simultaneously separating from the adjacent CPs. The electrostatic and vdW interactions both exist between CP subunits, and the sum of them is visibly stronger for a mid-CP of a hydrophobic CPNT than the interactions with two solvent phases, thus guaranteeing a hydrophobic CPNT to maintain its original tubular structure at the water/hexane interface. The CP-CP electrostatic interaction basically reflects the structural characteristics of a hydrophilic CPNT at the interface, i.e., maintaining a good tubular structure of the QT-CPNT, serious fracture of the QS-CPNT and fracture at one end of the QC-CPNT. The strong CP-water electrostatic interaction is in agreement with the formation of many H-bonds between a hydrophilic CPNT and the water phase.

4. CONCLUSIONS

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The present study has substantially investigated the structural characteristics, dynamical

properties

and

deformation

mechanisms

of

seven

hydrophilic/hydrophobic CPNTs at the water/hexane interface. The surface tensions of the water/hexane interface in seven systems are almost the same, about 42∼43 mN/m, resulted from the force balance of the interface. MD simulations disclose that the hydrophilic QT-CPNT and hydrophobic VL-CPNT almost maintain perfect tubular structures, but the former thoroughly enters the water phase and seriously tilts. The rest five CPNTs exhibit certain degrees of fractures, in which the QS-CPNT most severely raptures. A hydrophobic CPNT tends to stay at the water/hexane interface while a hydrophilic CPNT completely enters the water phase, resulted from the formation of many H-bonds with neighbouring water molecules. The presence of small groups, i.e., –CH3 in Ala and –CH2OH in Ser may increase the flexibilities of the side chains, thus reducing the stability of a CPNT. Furthermore, the end-CPs with overwhelming electrostatic interactions with water molecules are more susceptible to the surroundings than the mid-CPs and are therefore more prone to deform or even to shed. The CP-CP electrostatic interaction energies are in good agreement with the deformation and fracture of a CPNT at the water/hexane interface. Summarily, through the comprehensive analysis of the CP ellipticities, RMSD, position deviation from the interface, H-bond formation, electrostatic and vdW interactions, the behavior of a hydrophilic/hydrophobic CPNT at the water/hexane interface was recognized and understood primarily, which may 25



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help to further explore and expand the CPNT’s property and potential application at a water/oil interface.

ASSOCIATED CONTENT Supporting Information Figures S1, S3 and S5: Time evolutions of the intersubunit distances of the IL- and QT-CPNTs, the total energies of individual "water/CPNT/hexane" systems, Rcen and θ between two neighbouring benzene rings of the WL-CPNT. Figure S2: Average pressures and the corresponding standard deviations in individual “water/CPNT/hexane” systems. Figure S4: Schematic diagram of the π-π stacking interaction. Figure S6: Snapshots of the side chain H-bond forms in three hydrophilic CPNTs. Figure S7: Water-hexane electrostatic and vdW interaction energies.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] and [email protected]. Tel: 0086-0512-65883271.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work has been supported by the National Natural Science Foundation of China (Grant 21173154) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. It was further supported by the National 26


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Basic research Program of China (973 program, Grant 2012CBB25803). The authors are grateful to Mr. Jian Liu and Miss Yi Yu for their insightful suggestions.

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Table 1. Basic Information of Seven CPNTs, Including the Symbols, Short Forms, Outer-wall Features and Tube Lengths No.

symbol of CPNTa

1

8 × cyclo-(AL)4

2

short form

outer-wall feature

tube length (Å)

AL

hydrophobicity

33.56

8 × cyclo-(VL)4

VL

hydrophobicity

34.20

3

8 × cyclo-(IL)4

IL

hydrophobicity

34.66

4

8 × cyclo-(WL)4

WL

hydrophobicity

33.68

5

8 × cyclo-(QT)4

QT

hydrophilicity

33.52

6

8 × cyclo-(QS)4

QS

hydrophilicity

33.30

7

8 × cyclo-(QC)4

QC

hydrophilicity

33.40

a

The underlined letters in the symbols of CPNTs refer to D-amino acids. The symbols A, L, V, I, W, Q, T, S and C refer to alanine, leucine, valine, isoleucine, tryptophan, glutamine, threonine, serine and cysteine, respectively. See Figure 1 for details.

Figure 1. Structural formulas of individual amino acid residues and the corresponding seven kinds CP subunits, i.e., (AL)4, (VL)4, (IL)4, (WL)4, (QT)4, (QS)4 and (QC)4 which were used to further construct seven CPNTs with diverse hydrophilicities/hydrophobicities.

31



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Figure 2. Profiles of the density distributions of water and hexane components in a water/hexane interfacial system along the z-direction (normal to the interface) at 310 K. The green arrow representing the intersection of two lines refers to the position of the water/hexane interface.

Figure 3. Snapshots of the front and the top views of the WL-CPNT simulation system in which the CPNT was inserted at the center of the water/hexane interface with the tube axis parallel to the interface on the xy plane. For clarity, the side chains of the CPNT are omitted.

32


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Figure 4. Snapshots of the backbone structures of seven CPNTs obtained from the last frames of 18-ns MD simulations (thick lines), compared with the initial configurations (thin lines) inserted at the center of the water/hexane interface with the tube axes parallel to the interface. For clarity, the water/hexane interface is omitted. CPs with obvious deformations or sheddings are specially marked. The numberings of individual CP subunits and gaps between neighbouring CP subunits are illustrated in the sub-graph at the right bottom. Each CP subunit was assigned a specific color, i.e., green, yellow, violet, magenta, orange, blue, red and silver are assigned to P1, P2, P3, P4, P5, P6, P7 and P8, respectively.

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Figure 5. Average ellipticities of individual CP subunits of seven CPNTs at the water/hexane interface during the last 10-ns trajectories, respectively. The numberings of individual CP subunits are shown at the right bottom of Figure 4. The embedded images exhibit the snapshots of the deformed CP subunits with the ellipticities of 1.1, 1.2, 1.4, 1.5, 1.7 and 2.5, respectively.

Figure 6. Probability distributions of the total numbers of the backbone and side chain H-bonds in individual gaps of seven CPNTs during the last 10-ns trajectories, respectively.

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Figure 7. Average RMSDs and the corresponding standard deviations of the backbones and heavy atoms of the side chains of four hydrophobic CPNTs (AL, VL, IL and WL symbolized as upper triangles) and three hydrophilic CPNTs (QT, QS and QC symbolized as lower triangles).

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Figure 8. Time evolutions of the COMs of individual CPNTs in the z-direction. The red lines indicate the z-coordinates of the water/hexane interface in individual simulation systems.

Figure 9. Probability distributions of the H-bond numbers: (a) between neighbouring backbones; (b) between neighbouring side chains; (c) between backbones and water; (d) between side chains and water. 36


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Figure 10. Electrostatic and vdW interaction energies of each CP subunit of individual CPNTs with the hexane phase (a) and water phase (b), and between adjacent CP subunits of individual CPNTs (c). The numbering of individual CP subunits and gaps between neighbouring CP subunits can be found at the right bottom of Figure 4.

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Molecular

Dynamics

Simulations

Page 38 of 38

on

the

Behaviors of Hydrophilic/Hydrophobic Cyclic Peptide

Nanotubes

at

the

Water/Hexane

Interface Huifang Lin, Jianfen Fan,∗ Peipei Weng, Xialan Si, and Xin Zhao College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China

∗ E-mail: [email protected] and [email protected]. Tel: 0086-0512-65883271. 38


Molecular Dynamics Simulations on the Behaviors of Hydrophilic

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