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Effect of Organochoride Guest Molecules on the Stability of Homo/Hetero Self-Assembled #,#-Cyclic Peptide Structures: A Computational Study Towards the Control of Nanotube Length Rebeca Garcia-Fandino, and Juan R. Granja Guillan J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/jp400796n • Publication Date (Web): 22 Mar 2013 Downloaded from http://pubs.acs.org on April 13, 2013

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The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Effect of Organochloride Guest Molecules on the Stability of Homo/Hetero Self-assembled α,γ-Cyclic Peptide Structures: A Computational Study Towards the Control of Nanotube Length

Rebeca García-Fandiño* and Juan R. Granja*

Department of Organic Chemistry and Center for Research in Biological Chemistry and Molecular Materials, Campus Vida, Santiago de Compostela University, E-15782 Santiago de Compostela, (Spain) Corresponding author e-mail: [email protected] and [email protected]

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

We present here a molecular dynamics study on peptide nanotubes composed of cis-3aminocyclohexanecarboxylic acid- (γ-Ach) or cis-3-aminocyclopentanecarboxylic acid (γ-Acp)-based α,γ-cyclohexapeptides and also on those formed by heterodimeric pairs from a combination of the two aforementioned peptides (γ-Ach/Acp), being the first time that a self-assembling cyclic peptide nanotube ACS Paragon Plus Environment

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composed of heteromeric units is investigated. The main advantage of these types of nanotube is that they have a partially hydrophobic inner cavity, a property that makes them very exciting alternatives to classical nanotubes. In an effort to analyze the fine details of these ensembles, we investigated the dynamical behaviour of model dimeric structures that mimic the fundamental repeating structural motif of longer nanotubes. In spite of the structural analogy of the β-sheet interaction, our results suggest that extrapolation of the information obtained from those ‘capped’ dimers to nanotube properties has some limitations, since several significant differences have been found between the two systems. This finding is relevant not only for α,γ-cyclic peptide nanotubes, but shows that special care should also be taken when considering other related peptide nanotubes in which N-methylated dimers are used to obtain information about the stability and formation of the nanotubes. The structural and dynamical behaviour of dimers and nanotubes in non-polar (chloroform) and polar protic solvents (water) has been analyzed using state of the art theoretical methods. A marked destabilizing effect on the structure was observed in aqueous solution for all systems studied, suggesting that most of the water molecules that compete for the hydrogen bonds are those that occupy the internal cavity. We show that the introduction of organochloride molecules within the dimers and nanotubes are stabilized in water, and this property opens the door to a large number of possible future applications that are an important challenge in the field of molecular self-assembly, such as in drug delivery processes to control nanotube length by means of appropriate guests. A precise control of the nanotube length can be envisaged from the MD simulations of the encapsulation of 1,2-bis[2-(2,2,2-trichloroethoxy)ethoxy]ethane inside its inner cavity, opening a very interesting possibility in this area.

Keywords Cyclic peptide, Nanotubes, Dimers, Self-assembling, Molecular Dynamics, Encapsulation 1. Introduction Self-assembling cyclic peptide nanotubes (SCPNs) have attracted a great deal of attention from the scientific community in recent years due to their applications in biology, chemistry and material science.1-5 The interest is largely related to their technological possibilities as biosensors, photosensitive ACS Paragon Plus Environment

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materials, antimicrobial agents, selective transporter systems, molecular electronics components, and other potential uses in biology, electronics and optics. 6-15 The first peptide nanotubes derived from cyclic peptides (CPs) were based on alternating D- and L-αamino acids.16 Since then, a variety of CPs based not only on D,L-α-amino acids,17 but also on other structural motifs such as β-amino acids,18-20 γ-amino acids,21-22 δ-amino acids,23 hybrids of α- and εamino acids,24 or α- and γ-amino acids,5 including those consisting of alternating 3aminocycloalkanecarboxylic acid (γ-Aca), such as (1R,3S)-3-aminocyclohexanecarboxylic acid (D-γAch) or (1R,3S)-3-aminocyclopentanecarboxylic acid (D-γ-Acp) and α-amino acid (see Figure 1a),25-37 have been designed and successfully employed to produce tubular structures. In all of these designs the chirality of the amino acids allow the ring to adopt a quasi-planar conformation, in which the amide groups (NH and C=O) of the peptide backbone lie perpendicular to both sides of the ring plane and therefore are able to interact through hydrogen bonds (H-bonds) with those of neighbouring rings, thus leading to the formation of tubular nanotube structures (Figure 1a). One of the limitations of this strategy is the nanotube length control. Some approaches have been developed in the last few years such as the precise deposition of cyclic peptides (one by one) on an AFM tip14 or through the covalently attachment of oligomers.38-40 Another limitation of the SCPNs is the difficulty to modify the nanotube internal cavity properties. This drawback was embarked recently with the use of cyclic γ-amino acids because the β-carbon is projected into the lumen of the ensembly.26-37 The α,γ-SCPNs are a special class of peptide nanotubes derived from cyclic peptides that alternate α- with γ-amino acids. Structurally, they are quite unique because their formation involves two different sets of β-sheet-like H-bonds: one exclusively between the NH and C=O groups of the γ-amino acids (γ-γ interaction, Figure 1a), all of which are projected in the same direction, and the other between the same groups of the α-amino acids (α-α bonding, Figure 1a), which are oriented towards the opposite face. The use of γ-Acas imparts additional properties on the nanotube; for example, while almost all of the cyclic peptide nanotubes that have been developed so far have hydrophilic inner surfaces, thus allowing the permeation of only polar ACS Paragon Plus Environment

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molecules, in the α,γ-SCPN systems the C2 methylene group of each cycloalkane moiety is projected into the lumen of the cylindrical structure to generate a partially hydrophobic cavity. Furthermore, the cavity properties can be modulated by simple chemical modification of the β-carbon of the cyclic γ-Acas and this allows, in principle, finer control of the transport properties of a very wide range of molecules in the nanotube.21,35

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Figure 1: (a) Schematic diagram of SCPNs composed of γ-Ach or γ-Acp and α-amino acid, involving the α-α and γ-γ H-bonding patterns. ‘Capped’ dimers, which are based on N-methylated CPs, used to model longer SCPNs. (b) Schematic representation of the preferential heterodimer formation (over the corresponding homodimers) between CPs made of γ-Ach and γ-Acp. ACS Paragon Plus Environment

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Model nanotube systems based on N-methylated CPs that can form only dimers have been used to study the stability and formation not only of α,γ-SCPNs, but also of other types of nanotubes.41,42 Selective methylation of the backbone amide nitrogen functionalities makes the CP subunit devoid of Hbond donation from one face of the ring, meaning that it is predisposed towards an antiparallel stacked, cylindrical dimer (Figure 1a). The ‘capped’ dimer, which contains the key fundamental repeating structural motif of longer peptide nanotubes, has been studied experimentally.26-35 The resulting dimers have cavities with amphipathic properties and these contain, in the crystal structures, both polar (water) and non-polar (chloroform) molecules inside the cavity. The use of ‘capped’ dimers has allowed the study of their large association constant (kAch-Ach ~ kAcp-Acp : 105–106)31,34 and also the preferential formation of heterodimers (DαAch/Acp) over the corresponding homodimers between CPs made of γamino acids with a five-membered ring (DαAcp) and those whose γ-amino acid is Ach (DαAch) (binding energy of formation is about 2 kcal/mol stronger than the corresponding homodimers, Figure 1b).29 Selfassembly of biomolecules that are capable of selectively forming either homo- and/or heterodimeric entities represents one of the great challenges today because, in general, current procedures do not provide sufficient recognition power for the selective assembly in either a homo- or heteromeric fashion.43-46 These homo- and heterodimerization equilibriums are quite common in nature and constitute an economical means to achieve structural diversity and functional versatility, both of which potentially extend the benefits of this supramolecular process. The preferential formation of DαAch/Acp over homodimers DαAch and DαAcp is caused by the differences in peptide backbone/backbone interactions between CPs and is not due to amino acid side-chain interactions. This constitutes a very opportune property, because it allows side-chain modifications to develop new functions without affecting the self-assembly process, as exemplified by our findings on electron and/or energetic transfer processes.31,32 As mentioned before, this strong heterodimerization preference is independent of the α-


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amino acid side chains, although the origin of this selectivity is unclear. Based on X-ray data it was suggested that, in addition to the entropy of mixing (∆Smix),29,47,48 the heterodimer is more stable because of an improved alignment of H-bond donors and acceptors.29 Although the ∆Smix is expected to increase, the heterodimer formation should not alter significantly the overall trend in association constants. Theoretical studies into dimer-forming N-methylated CPs have been carried out in recent years.49-52 In a previous publication, we presented density functional theory (DFT) calculations on monomers and dimers of γ-Ach- and γ-Acp-based α,γ-cyclic hexapeptides and α,γ-cyclic octapeptides to investigate the experimentally observed preference for α-α over γ-γ dimerization, which is associated with the two types of stacking patterns present in these peptide nanotubes, as well as the preference for heterodimerization over homodimerization.52 These calculations predicted that the interaction energies in the α-α species are quite similar to those in the γ-γ dimers. However, comparison of dimerization energies (i.e., interaction energies plus deformation energies of monomers) showed that α-α dimerization was energetically favoured over γ-γ dimerization. The calculations strongly suggested that the preference for α-α binding was governed by differences between the deformation energies in the CPα and CPγ monomers, rather than by differences between the relative strengths of the α-α and γ-γ H-bonding patterns. Besides, the results also predicted that the α-α heterodimerization was energetically favoured over α-α homodimerization, although the energy differences calculated for hetero- and homodimerization were very similar to each other. Despite the increasing knowledge on peptide nanotubes, there are many aspects of their microscopic behaviour that remain unclear. Theoretical calculations would largely improve our knowledge concerning these details but, in contrast to the large amount of theoretical work on ‘classical’ D,L-αSCPNs,53-64 very little theoretical work has been performed on α,γ-SCPNs. 65,66

In a previous study66 we carried out theoretical calculations on a SCPN consisting of alternating

L-γ-Ach acid and D-α-amino acid systems formed by six CP units. However, analogous results have not


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been obtained for systems formed by using the L-γ-Acp to build a nanotube consisting of homodimeric units and, in addition, also the possibility of combining both CPs to obtain a heterodimeric nanotube has not been explored. In the work described here we applied the power of Molecular Dynamics (MD) to nanotubes formed by γ-Ach- (SCPNAch) and γ-Acp-based (SCPNAcp) cyclic α,γ-hexapeptides and also studied computationally the dynamical behaviour of a heteromeric α,γ-SCPN composed alternately by γ-Achand γ-Acp-based CPs (SCPNAch/Acp). In order to gain a deeper understanding of these tubular structures, we also investigated the dynamical behaviour of the corresponding ‘capped’ dimers that contain the fundamental repeating structural motif of longer SCPNs. With this aim in mind, we carried out MD simulations on the N-methylated dimers to study the influence of explicit solvent on their stability and to investigate if the results obtained for dimeric systems can be extrapolated to the nanotube structures.

2. Simulation Details Nomenclature: The simulated systems will be denoted as CP, D or SCPN for the cyclic peptide, dimer or nanotube, respectively. A subindex Acp or Ach corresponds to cis-3-aminocyclopentanecarboxylic acid (Acp) or cis-3-aminocyclohexanecarboxylic acid (Ach), respectively, composing the α,γ-cyclohexapeptide. A superindex α or γ refers to the type of interaction between each pair of CPs or, in the case of the Nmethylated monomers, to the type of interaction in which they would participate when forming the corresponding dimer. Preparation of the dimers: The atomic coordinates for the dimers were obtained from a previous X-ray crystal structure with dimer-forming methyl-blocked CP c-[D-Phe-(1R,3S)-γ-MeNAch-]3.26 From this structure, the side chains of the α-amino acid were substituted by methyl groups in such a way that the resulting α-amino acid


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residues were Ala. The Ach unit in DαAch was changed to Acp in both monomers to obtain the homodimer DαAcp, or in only one of the CPs to obtain the heterodimer DαAch/Acp. The γ-γ dimers (denoting DγAch for the Ach-based homodimer, DγAcp for the Acp-based dimer and DγAch/Acp for the heterodimer) were obtained in a similar fashion by changing the position of the methyl group from the N of the γ-amino acid to the N of the α-Ala residue. Preparation of the nanotubes: From the structures of the dimers, the N-methyl groups were removed and changed to NH and the dimers were then replicated four times along the axis perpendicular to the CP planes by a distance equal to that measured between the two original CP units. Therefore, the resulting SCPNs (with SCPNAch denoting the γ-Ach-based nanotube, SCPNAcp the γ-Acp-based and SCPNAch/Acp the heteromeric nanotube) are composed of eight CPs units. Methods: Starting from these structures, MD simulations of a single dimer or SCPN were performed in water and chloroform (at their respective experimental densities) using GROMACS 4.0 software.67 During the solvation step, the solvent molecules present in the dimer and the SCPN inner cavity were removed, so that in the first step of the simulation the channel was completely dry. The initial size of the unit cell was 8 × 8 × 8 nm3 for the dimer and 10 × 10 × 10 nm3 for the nanotube. The water was simulated using the SPC/E model;68 the chloroform model was taken from AMBER parameters.69-70 Concerning the atoms of the SCPN, RESP/6-31G(d) charges were derived as in the original AMBER force-field development, while van der Waals parameters were taken from the GAFF force-field71,72 using standard Lorenz– Bertelot combination rules. Bonded terms were taken as those of standard peptides. All of the systems were partially optimized, thermalized, and equilibrated, followed by unrestrained simulations for at least 40 ns (time step = 2 fs) for each of the systems studied. The constant pressure and temperature canonical ensemble was employed with the pressure of 1 bar controlled using a isotropic Parrinello–Rahman barostat,73 and the temperature of 300 K imposed by a Berendsen74


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thermostat. The LINCS75 algorithm was employed to remove the bond vibrations. The Particle Mesh Ewald method76 coupled to periodic boundary conditions was used to treat the long-range electrostatics using a direct-space cutoff of 1.0 nm and a grid spacing of 0.12 nm. The van der Waals interactions were computed using PBC coupled to a spherical cutoff of 1.0 nm. The potential of mean force (PMF) was used to obtain the free energies of dimerization, using the Umbrella Sampling method, for the homodimers DαAch and DαAch and the heterodimer DαAch/Acp, since experimental data are only available for these systems. The centre of mass of the second ring was pulled away from that of the first ring in the direction parallel to the intersubunit H-bonding (z) direction. For umbrella sampling, the pulled ring was harmonically restrained to subsequent positions on the channel axis (with typical values of the restraining force constant of 1000 kJ mol–1 nm–2) in 50 windows of width ∆z = 0.1 nm. This process restricted its movement to the xy-plane while still allowing diffusion into adjacent windows. Each window was initially minimized. One MD simulation of length 8 ns was carried out for each of the 50 windows. The 50 biased distributions of z positions of the second ring were recombined and unbiased with the Weighted Histogram Analysis Method (WHAM).77-79 The first nanosecond of each window run was discarded as equilibration time, leaving a total of 7 ns per window. Data were analyzed using GROMACS and locally written code. Molecular graphic images were prepared using visual molecular dynamics (VMD).80

3. Results and Discussion The effective radius for the channels generated in the N-methylated dimers and the SCPNs were calculated with HOLE81 after minimization of the corresponding structures (Figure 2). The effective radius is situated in the plane of the CP whereas the maximum radius is located between the two planes of the rings. The largest effective radius, both in dimers and nanotubes, corresponds to the CPs composed of γ-Ach units (0.168–0.175 nm), whereas the heterodimers have an intermediate radius (0.150–0.161 nm) and the structures consisting of only γ-Acp units have the smallest effective radius


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(0.149–0.152 nm). It is also noticeable that the γ-γ dimers have an effective radius slightly longer than that of the α-α dimers. Except for DγAch, in the rest of the dimers there is no significant difference in the maximum radius of the region situated between the two CP planes with respect to the α-α or the γ-γ interaction, following the trend (Figure 2j): r(DαAch) > r(DγAch) > r(DαAch/Acp) ≈ r(DγAch/Acp) > r(DαAcp) ≈ r(DγAcp/Acp). The effective radius in the SCPNs (corresponding to the region of the plane of the CPs, Figure 2k) is larger for the system composed of γ-Ach units (SCPNAch) and smaller when the channel is formed by γAcp (SCPNAcp). In the SCPNAch/Acp two different radii can be appreciated, a smaller one (identical to that observed for the SCPNAcp) and a larger one (identical to that found in SCPNAch). This finding is consistent with the alternation of CPAch and CPAcp units in the heteromeric system. The radius in the region between the two CP planes is larger for the α-α interaction compared to the γ-γ one. This radius is maximum in this region for the SCPNAch and minimum for the SCPNAcp. Once again, the SCPNAch/Acp has an intermediate value between the other two.


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Figure 2: HOLE81 radius for the simulated dimers (a–f, j) and SCPNs (g–i, k). ACS Paragon Plus Environment

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3.1. Dynamical behaviour of N-methylated dimers of cyclopeptides in chloroform and water The N-methylated dimers were simulated in polar protic and non-polar aprotic solvents: i.e. water and chloroform. In chloroform the dimers, in general, are stable and are quite rigid entities, as noted in the values of their root-mean-square deviations (RMSD) and root-mean-square fluctuations by residue (RMSF) (see Suppl. Figure S1a-b). The exception is DγAcp, where four independent replicas were simulated and, in any case, the dimer was not conserved (see Suppl. Figures S1c-d–S2). It is noticeable that both the RMSD and RMSF corresponding to the stable γ-γ dimers are slightly smaller than those corresponding to the α-α dimers, suggesting greater stability for the former ones (see Suppl. Figure S1). Analysis of the radii of gyration (Rg) of the backbone of each CP confirms the lack of any major global or local structural distortion during the trajectories (except for DγAcp), indicating that the rings remain in a flat conformation (see Suppl. Figure S3). However, lower Rgs values are observed for the Acp unit, suggesting a greater tendency for this ring to bend and to lose its flat shape. Actually, experimentally we have observed that while Ach-based cyclic hexapeptide c-[D-MeNAla-(1R,3S)-γ-Ach-]3 dimerizes in chloroform with 230 M-1 the Acp-based analogue c-[D-MeNAla-(1R,3S)-γ-Acp-]3 does not form the corresponding dimer because it folds forming intermolecular H-bonding interactions.82 Molecules of chloroform started to enter or filled the cavities of the stable dimers, while another two molecules blocked the entrance at the edge of the channel (Figure 3). Such findings fit well with the experimental results obtained for this type of dimer: crystallizations carried out in chloroform by vapour-phase equilibration with hexane led to the detection of one chloroform molecule within the internal cavity.26 The velocity at which chloroform enters the cavity differs depending on the dimers, with this process being slow for DαAcp (~90 ns), very fast for DαAch (~300 ps) and DαAch/Acp (~50 ps – when the chloroform molecule enters by the Ach unit side) and relatively fast for DγAch and DγAch/Acp (~2


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ns). This difference in the entrance rate could be related to the greater tendency of the CPAcp to bend, which could in turn make the entrance of molecules into the cavity more difficult.

Figure 3: Snapshots for the last frame of the 40 ns simulation in chloroform for dimers: (a) DαAch, (b) DαAcp, (c) DαAch/Acp, (d) DγAch, (e) DγAcpa and (f) DγAch/Acp. a

Three additional replicas were simulated for this system (see Suppl. Figure S2), but none of them led to a stable dimer.

The distance between the centre of mass (c.o.m.) of the backbone atoms of the two rings in the stable dimers (Table 1 and Suppl. Figure S4) is about 0.463–0.491 nm, depending on the type of interaction and ring used, and these values are quite similar to those found for the {c-[L-Trp-D-MeNLeu-]4}2 in nonane.51


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Distance/nm


DαAch

DαAcp

DαAch/Acp

DγAch

DγAcpa

1.538 (1.105)

between c.o.m.

0.491 (0.010)

0.463 (0.007)

0.473 (0.007)

0.479 (0.006)

0.591 (0.023) 3.166 (1.140)

0.485 (0.007)

1.325 (1.258)

NH…O=C

CHCl3⊂

CHCl3⊂

CHCl3⊂

DαAch

DαAcp

DαAch/Acp

0.746 (0.092)

0.479 (0.020)

0.472 (0.008)

0.565 (0.160)

0.488 (0.032)

0.472 (0.008)

0.495 (0.026)

0.481 (0.028)

0.472 (0.008)

DγAch/Acp

0.219 (0.030)

0.205 (0.020)

0.201 (0.018)

0.199 (0.016)

0.199 (0.021)

0.229 (0.039)

0.231 (0.008)

0.202 (0.002)

0.219 (0.030)

0.205 (0.020)

0.206 (0.019)

0.199 (0.016)

0.198 (0.016)

0.229 (0.040)

0.226 (0.006)

0.218 (0.003)

0.218 (0.029)

0.205 (0.020)

0.202 (0.018)

0.199 (0.016)

0.199 (0.021)

0.245 (0.071)

0.226 (0.006)

0.202 (0.002)

0.219 (0.030)

0.205 (0.020)

0.206 (0.020)

0.199 (0.016)

0.198 (0.016)

0.252 (0.096)

0.226 (0.006)

0.217 (0.003)

0.219 (0.030)

0.205 (0.020)

0.202 (0.018)

0.199 (0.016)

0.199 (0.020)

0.262 (0.108)

0.239 (0.009)

0.202 (0.002)

0.219 (0.030)

0.205 (0.020)

0.207 (0.020)

0.199 (0.017)

0.198 (0.016)

0.246 (0.069)

0.240 (0.009)

0.217 (0.003)

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Table 1: Distance between the c.o.m. of the monomers for all the N-methylated dimers, simulated in chloroform. H-bond distances between units. Values displayed are in nm and correspond to average (from 10–40 ns) and standard deviation. a

Each value corresponds to a different replica.

The number of H-bonds between the two monomers is maximum (6) for most of the structures in the trajectory of the stable dimers, although one (or two – mainly for DαAch) of them is lost along the simulation (Figure 4a). Such a reduction could be attributed, in principle, to the presence of the NACS Paragon Plus Environment

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methyl groups, since it has been reported that the distortion of the N-methylated ring compared to that of the non-alkylated one could be a possible reason for the slight reduction in the number of intersubunit H-bonds for the ‘capped’ versus the ‘uncapped’ dimer.50,51 The H-bond (NH…O=C) distances (Table 1) are slightly smaller in the case of the stable γ-γ dimers compared with their α-α analogues, a finding that is consistent with the lower values found for the aforementioned RMSD and RMSF obtained for these systems. The H-bond distances in all the homodimers are almost identical for the six H-bonds present in the dimer, whereas in the heterodimer there is an alternation between a longer distance (0.206 or 0.199 nm in DαAch/Acp and DγAch/Acp, respectively) and a shorter one (0.202 or 0.198 nm in DαAch/Acp and DγAch/Acp, respectively), as a consequence of the alternation of Ach and Acp rings. In most of the replicas simulated for the unstable DγAcp, there are no H-bonds between the two monomers, although in certain cases some H-bonds are maintained, indicating that despite the loss of the β-sheet structure, an interaction between the two subunits can still remain (Figure 4b).

Figure 4: H-bonds between the monomers in the stable dimers (a) and in the unstable DγAcp, all simulated in chloroform, during 40 ns of simulation. ACS Paragon Plus Environment

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The behaviour of the dimers was computationally studied not only in chloroform, but also in aqueous solution. Dimer formation has been observed experimentally in aqueous media for γ-Acp-based peptides bearing a pyrene group and the inclusion of this unit resulted in a significant decrease in the association constant.31,34 However, all attempts to obtain a dimer in water experimentally without using aminoacidic side chains, which aid the dimerization process, have been unsuccessful to date. With the aim of understanding the effect that water has on the system, four replicas of each one of the six dimers previously studied in chloroform were simulated but immersed into a box of water. The DαAch system loses its β-sheet structure (Figure 5a and Suppl. Figure S5a) at the same time, as most of the H-bonds between the two monomers are lost. Curiously, in some of the simulations, the cyclohexyl moiety or the methyl group of the α-Ala of one of the CPs is projected into the cavity of the other ring and this reflects the partially hydrophobic character of the cavity, which in most cases prefers to be filled by a non-polar molecule rather than by water molecules. The monomers of DαAcp separated completely in some of the replicas, whereas in others a new type of dimer was formed in which the N-methyl groups were packed together and hidden from the water, thus showing a preference for a more hydrophobic environment (Figure 5b and Suppl. Figure S5b). Interestingly, the starting β-sheet structure of the heterodimer DαAch/Acp was conserved in half of the replicas simulated (Suppl. Figure S5c), whereas in other cases the monomers were completely separated after 40 ns or one of the methyl groups of the α-Ala was projected towards the interior of the other CP (Figure 5c and Suppl. Figure S5c). On the other hand, none of the dimers in which a γ-γ interaction was established led to a stable system in water, reflecting the lower tendency of the α-N-methylated monomers to form dimers (Figure 5d-f and Suppl. Figure S6). The RMSDs for the simulations of all of these systems in water (Suppl. Figure S7) confirm higher fluctuations for the γ-γ dimers compared to the α-α ones. However, even in the latter case the RMSDs are very large, suggesting that in aqueous solution there is a great deal of competition between the Hbonds formed between CPs and those between monomers and the solvent. The exposure of the


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hydrophobic methyl groups of the ‘capped’ dimers to the hydrophilic water molecules could be another reason for the greater instability of these systems.50,83 The competition between intersubunit H-bonding and H-bonding with water molecules has also been reported previously.83 In that study, it was observed that, under rigorously anhydrous conditions, the association constant for c-[(L-Phe-D-MeNAla-)4]2 in deuterochloroform approximately doubled, suggesting competition by water for intermolecular Hbonding sites. Besides, a previous computational study carried out on the aforementioned dimers in nonane and water also showed that they were unstable in a hydrophilic solvent whereas they showed stability in nonane.51

Figure 5: Snapshots for the last frame of the 40 ns simulation in water for dimers: (a) DαAch, (b) DαAcp, (c) DαAch/Acp, (d) DγAch, (e) DγAcp and (f) DγAch/Acp.a a

Three additional replicas were simulated for each one of these systems (see Suppl. Figures S5 and S6).


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Comparison of the interaction energies obtained from the simulations of the α-α dimers in chloroform (Table 2), calculated along the last 30 ns of the trajectory, suggests that although the standard deviations are quite significant, the trend is that the heterodimer DαAch/Acp is the most stable ensemble (interaction energy of –203.9 kcal/mol versus –191.0 and –196.5 kcal/mol for DαAch and DαAcp, respectively). The difference between the homodimeric DαAch and DαAcp is not very high, although there is a small preference for the DαAcp – a finding that is consistent with the experimental results obtained with analogous CPs.29 Theoretical calculations previously carried out using DFT on these systems also predicted that the α-α heterodimerization was energetically favoured over α-α homodimerization, although the energy differences found for hetero- and homodimerization were very similar.52 MD calculations, including the presence of explicit solvent, also show a similar trend that reproduces the experimental results quite well. Interestingly, higher interaction energies were obtained for DγAch and DγAcp compared to those involved in the α-α interaction. The homodimer DγAcp presents much lower interaction energies, since the monomers separate along the trajectory (as mentioned above). However, experimental work on hexameric and octameric γ-Ach- and γ-Acp-based α,γ-CPs led to the suggestion that the α-α interaction is stronger than the γ-γ one.26,27,29 For example, at 298 K, c-[(1R,3S)-γ-Ach-DMe

NAla-]3 forms dimers through the γ face, with an association constant of 230 M–1 in CDCl3 and 2.5 ×

104 M–1 in 2:3 CDCl3/CCl4. The association constant for dimerization of c-[D-Phe-(1R,3S)-γ-MeNAch]3, i.e. for the formation of α-α dimers, was estimated to be 106 M–1 in chloroform.31 Our previous computational work, using DFT methods on these systems, revealed similar interaction energies for both dimeric species, suggesting that the strength of the α-α stacking (due to H-bonding and van der Waals interactions) was rather similar to that for γ-γ stacking. The lower association constant observed for the γ-γ dimers was attributed to stabilization (folding) of the γ-monomers due to intramolecular H-bonding, which resulted in a significant increase in their deformation energies and consequently to a decrease in the absolute values of the dimerization energies.


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DαAch

DαAcp

DαAch/Acp

DγAch

Water

–84.0 (13.3) –98.5 (39.9) –59.9 (50.0) -107.3 (54.2)

–52.3 (28.6) –155.4 (16.1) –0.002 (0.02) –26.2 (45.4)

–74.3 (79.3) –48.3 (42.5) –190.5 (17.7) –192.1 (17.4)

–0.2 (2.3) –79.5 (30.9) –50.4 (48.5) –7.0 (13.6)

Chloroform

–191.0 (12.4)

–196.5 (9.9)

–203.9 (9.9)

–260.5 (11.5)

DγAcpa

–120.7 (36.2) –4.5 (16.3) –10.0 (22.8) –0.03 (0.06) –44.5 (61.2) –145.1 (10.5) –0.1 (0.6) –85.7 (69.6)

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CHCl3⊂

CHCl3⊂

CHCl3⊂

DαAch

DαAcp

DαAch/Acp

–124.6 (34.5) –31.3 (33.6) –65.3 (36.8) –45.3 (55.1)

–86.9 (12.5) –125.7 (33.4) –169.9 (32.6)

–181.6 (21.1) –174.4 (28.3) –181.5 (23.4)

–254.8 (12.3)

-

-

DγAch/Acp

–204.4 (11.3) –204.2 (11.5) –204.0 (11.6)

-

Table 2: Interaction energies between the monomers of the ‘capped’ dimers simulated in water and chloroform. Values displayed are in kcal/mol and correspond to average (from 10–40 ns) and standard deviation. Each value corresponds to a different replica.

It is worth mentioning that the energy differences in the interaction energy for the α-α interaction arise due to the increase in the Coulombic and Lennard–Jones (LJ) energy for DαAch/Acp (Table 3). The LJ contribution for DαAch is similar to that of the heterodimer but its Coulombic contribution is lower. The opposite behaviour is observed for DαAcp, where the Coulombic contribution is very similar to that found in the heterodimeric system but the LJ contribution is slightly lower. The LJ contribution for the γ-γ interaction is not too different to that found in the α-α interaction (even for the unstable DγAcp), but the main difference undoubtedly concerns the Coulombic contribution, which is much bigger than that corresponding to the α-α interaction for the dimers that remain stable throughout the simulation. The interaction energies were always lower when the dimers were simulated in water (Table 2), even for the replicas where the dimers were maintained along the simulation, and this is due to the competition between intersubunit H-bonding and H-bonding with water molecules. ACS Paragon Plus Environment

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α-α interaction DαAch

DαAcp

DαAch/Acp

γ-γ interaction DγAch

DγAcp

DγAch/Acp

Coulombic

–101.61 –111.70

–112.97

–168.86 –40.50 a

–166.32

Lennard– Jones

–89.41

–91.04

–91.83

–89.14a

–88.22

–84.87

Table 3: Contribution of the different terms to the interaction energy between the monomers of the ‘capped’ dimers in chloroform. Values displayed are in kcal/mol and correspond to average (from 10–40 ns). a

This value was calculated for replica 1.

The free energy of dimerization was studied for the N-methylated dimers that interact through their αamino acids in chloroform (DαAch, DαAcp and DαAch/Acp). This parameter was evaluated according to the protocol indicated in the Methods section. The free energy profile of the distance between the two CPs as a function of the z coordinate of the pulled ring is shown in Figure 6. The initial position of the pulled ring corresponds to a distance of ~0.4 nm between the z coordinates of the c.o.m. of the two rings. Since no restraint in xy is imposed during the Umbrella Sampling simulations, the rings do not remain parallel after 8 ns of each window and, as a result, an extension of the separation distance between the rings up to 5 nm was applied. The free energy difference between the initial state of the two rings when they are H-bonded to each other and when they are separated is larger in the case of the heterodimer DαAch/Acp and also the homodimer DαAcp (~9.4 and ~9.3 kcal/mol, respectively) compared to the corresponding homodimer DαAch (~7.5 kcal/mol). However, the differences between the three cases are small. This finding is not unexpected since the energetic differences obtained experimentally are also very small, i.e. around 2 kcal/mol, which is close to the limit of the error of the technique. The important fact is that all of these findings fit well with the experimental results obtained for analogous systems in chloroform (binding energies of 6.9–8.2 kcal/mol for the homodimers and energies 2 kcal/mol higher in the case of ACS Paragon Plus Environment

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the heterodimer)31,34 and are in the same range as those obtained by Khurana et al. on using a similar technique with Ghadiri ‘capped’ dimers.51

Figure 6: Potentials of Mean Force (PMF) for the corresponding ‘capped’ dimers as a function of the separation of one of the monomers with respect to the others.

3.2. From dimers to nanotubes: Dynamical behaviour of SCPNs in chloroform and water The study of the dynamical behaviour of the ‘capped’ dimers leads one to question the relationship between the stability of the dimeric systems and the nanotubes. An understanding of this aspect is also important not only for our systems, but also in all studies that concern the N-methylated CPs containing the key fundamental repeating structural motif of longer peptide nanotubes.41,42 One of the aims of the present computational study was to address several aspects, such as the influence of the N-methyl groups on the interaction between monomeric CPs (unprotected in the SCPNs), or the relative stability, α-α versus γ-γ, of each interaction in the SCPN. In that sense, according to the results obtained for dimers, one would expect that the most stable nanotube would consist of heterodimeric pairs of CPs,


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SCPNAch/Acp, but one clear question arises; can the results obtained for ‘capped’ dimers be extrapolated to SCPNs? In an effort to answer these questions MD simulations in water and chloroform were carried out on three different SCPNs: one composed only by CPs containing Ach units (SCNPAch),84 another one formed exclusively by CPs with Acp units (SCNPAcp) and the last one composed of CPs containing alternated Ach and Acp units (SCNPAch/Acp). All of these systems start with a γ-γ interaction to prevent the formation of intramolecular H-bonds between the free γ-amino acids, as observed in previous works.66 Analysis of the intramolecular H-bond pattern (Figure 7c) reveals that the three nanotubes are very well maintained in chloroform, since most of the structures of the trajectory conserve the maximum number of H-bonds (42). The homomeric SCNPAch and SCNPAcp present similar RMSD values (Figure 7a) for the whole simulation (0.189±0.016 and 0.184±0.026 nm, respectively), whereas the values for the heteromeric SCNPAch/Acp are slightly lower (0.131±0.014 nm). The RMSF plots (Figure 7b) also show the difference between the central (more rigid) and terminal parts (more flexible) in all three types of SCPNs studied, indicating that the fluctuation observed for the Ach units in SCNPAch is smaller than for the other two systems. Analysis of the Rg of the backbone of each CP confirms the absence of any major global or local structural distortions during the trajectories, indicating that the rings stay in a flat conformation (Suppl. Figure S8). However, it was observed that SCPNAcp presents slightly lower values for its terminal monomers compared to the internal ones. Besides, in SCPNAch/Acp the Rgs of the Acp monomers are lower than those corresponding to the Ach units, thus confirming that the Acp unit has a greater tendency to bend and lose its flat shape compared to the Ach one, as had been observed with the N-methylated monomers (see above).


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Figure 7: (a) RMSD, (b) RMSF and (c) normalized distribution of the number of H-bonds between the monomers in nanotubes SCPNAch, SCPNAcp and SCPNAch/Acp along 40 ns simulated in chloroform.

The distance between the c.o.m. of the backbone atoms of each pair of rings in SCPNAch is very similar for the α-α and γ-γ interaction (Table 4), a characteristic that is not extrapolated from the Nmethylated dimers, where the distance between the c.o.m. of DαAch was slightly longer than in DγAch (by about 0.01 nm, Table 1). The difference between the two types of interactions is apparent, however, in SCPNAcp and SCPNAch/Acp – where the distances between the c.o.m. of the monomers in the nanotube are practically identical to that found in the corresponding ‘capped’ dimers. The decrease in both the average and standard deviations of the NH…O=C distances in SCPNAch (Suppl. Table S1) compared to those found in DαAch and DγAch (Table 1), suggests a closer approach of the monomers in the nanotube and also indicates that the main difference between nanotubes and ‘capped’ dimers corresponds to the system formed by Ach units (DαAch versus the α-α interaction in SCPNAch).

Monomers pair SCPNAch and type of

SCPNAcp

7CHCl3⊂

7CHCl3⊂

7CHCl3⊂

SCPNAch

SCPNAcp

SCPNAch/Acp

SCPNAch/Acp

interaction 1–2 (γγ)

0.475 (0.005) 0.498 (0.007) 0.485 (0.007) 0.478 (0.007) 0.500 (0.011) 0.482 (0.008)

2–3 (αα)

0.472 (0.007) 0.465 (0.007) 0.471 (0.007) 0.477 (0.008) 0.470 (0.007) 0.473 (0.007)


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3–4 (γγ)

0.477 (0.005) 0.494 (0.007) 0.482 (0.006) 0.480 (0.006) 0.496 (0.008) 0.483 (0.007)

4–5 (αα)

0.475 (0.007) 0.466 (0.007) 0.472 (0.007) 0.477 (0.008) 0.470 (0.007) 0.473 (0.007)

5–6 (γγ)

0.477 (0.006) 0.494 (0.007) 0.482 (0.006) 0.480 (0.006) 0.496 (0.008) 0.483 (0.007)

6–7 (αα)

0.472 (0.007) 0.465 (0.007) 0.471 (0.007) 0.477 (0.008) 0.470 (0.007) 0.472 (0.007)

7–8 (γγ)

0.475 (0.005) 0.498 (0.007) 0.480 (0.006) 0.478 (0.007) 0.500 (0.011) 0.483 (0.007)

Table 4: Distance between the c.o.m. of the monomers of the SCPNs composed of CPs containing Ach, Acp and Ach/Acp units, simulated in chloroform (SCPNAch, SCPNAcp and SCPNAch/Acp) or simulated in water with seven molecules of chloroform pre-encapsulated into their inner cavity (7CHCl3⊂ SCPNAch, 7CHCl3⊂SCPNAcp and 7CHCl3⊂SCPNAch/Acp). Values displayed are in nm and correspond to average (from 10–40 ns) and standard deviation.

A very interesting aspect of SCPNs is their ability to act as selective transporters (channels) or containers for small molecules. In all of the nanotubes studied in this work, molecules of chloroform start to enter the cavities (Figure 8). However, the diffusion inside is slow (multinanosecond scale), probably because of the small size of the pore and the polar nature of its external borders – even extending the simulations up to 120 ns (Suppl. Figure S9) did not lead to an appreciable displacement of the inner chloroform. This behaviour involves the creation of temporally hollow pores and suggests that the hydrophobic environment of the α,γ-SCPN interior can act as a good container for non-polar molecules such as chloroform, but in the absence of external forces it does not seem to be a good transporter.85 The entrance of chloroform molecules into the pore is not arbitrary as they penetrate very rapidly and almost simultaneously after ~120 ps in SCPNAch (Figure 8 and Suppl. Figure S10), and periods of 2.5 and 6.5 ns, respectively, for each molecule of chloroform are required for their insertion in SCPNAcp. This different rate of entry can be seen very clearly into SCPNAch/Acp, where a faster entrance can be observed through the Ach unit side (~200 ps) compared to the corresponding entry through the Acp end (~30 ns). This observation suggests the preferential entry of chloroform at the side


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of the CPs bearing the Ach units, presumably due to its larger effective radius and its lower tendency to bend and lose its flat shape.

Figure 8: Snapshots for the last frame of the 40 ns trajectory obtained for SCPNAch, SCPNAcp and SCPNAch/Acp simulated in chloroform, and z coordinate of the carbon of both molecules of chloroform entering the channel. Nanotube is comprended between ~3.1 and ~6.9 nm. ACS Paragon Plus Environment

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Monomers pair SCPNAch

SCPNAcp

7CHCl3⊂

7CHCl3⊂

7CHCl3⊂

SCPNAch

SCPNAcp

SCPNAch/Acp

SCPNAch/Acp

and type of interaction 1–2 (γγ)

–264.8 (8.9)

–243.9 (9.7)

–255.9 (9.1) –248.1 (13.7) –231.3 (18.1) –245.7 (13.0)

2–3 (αα)

–198.5 (8.2)

–188.9 (8.9)

–196.5 (8.5)

3–4 (γγ)

–266.7 (9.0)

–248.0 (9.0)

–259.2 (8.5) –248.7 (13.0) –241.3 (11.2) –248.7 (10.3)

4–5 (αα)

–197.2 (8.6) –187.78 (9.2)

–196.3 (8.6)

5–6 (γγ)

–266.7 (9.1)

–247.9 (9.0)

–258.8 (8.5) –249.2 (13.1) –241.5 (11.0) –248.5 (10.5)

6–7 (αα)

–198.5 (8.1)

–188.8 (8.9)

–196.4 (8.4)

7–8 (γγ)

–264.7 (8.9)

–243.9 (9.6)

–258.8 (8.5) –248.4 (13.6) –233.1 (16.2) –246.6 (12.1)

–198.0 (9.4)

–199.1 (9.3)

–198.1 (9.5)

–186.3 (10.3)

–186.5 (10.3)

–186.5 (10.2)

–198.4 (9.2)

–198.1 (9.2)

–196.2 (9.3)

Table 5: Interaction energies between each two units of the peptide nanotubes SCPNAch, SCPNAcp and SCPNAch/Acp simulated in chloroform, and of 7CHCl3⊂ SCPNAch, 7CHCl3⊂ SCPNAcp and 7CHCl3⊂ SCPNAch/Acp simulated in water, starting with nanotubes with seven molecules of chloroform pre-encapsulated in their inner cavities. Values displayed are in kcal/mol and correspond to average (from 10–40 ns) and standard deviation.

The γ-γ interaction energy between the CPs that form the three SCPNs is stronger than the α-α interaction (Table 5). Once again, as seen for the dimers, it can be appreciated that although experimentally the γ-γ dimers present lower association constants, presumably due to the deformation energy of the monomers, this interaction is stronger than the α-α one in the nanotube. Furthermore, SCPNAch, and not the heteromeric system, shows the highest interaction energies. While the α-α interaction energy is quite similar to that found in SCPNAch/Acp, the γ-γ interaction is stronger, which translates into an overall stabilization for this nanotube. Both α-α and γ-γ interactions are also stronger 27 ACS Paragon Plus Environment


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in this SCPN than in the ‘capped’ dimers, suggesting a large destabilization effect due to the N-methyl groups present in DαAch and DγAch On the other hand, the least stable nanotube is SCPNAcp, where both the α-α and the γ-γ interactions present the lowest values of all the studied SCPNs. The relationship dimer/nanotube is opposite to that found for the SCPNAch, i.e. the interaction energies between the monomers containing γ-Acps in dimer-forming CPs are lower than those observed in the nanotube. The heteromeric SCPNAch/Acp combines both effects; the α-α interaction is weaker in the nanotube whereas the γ-γ interaction is stronger. Simulations of the three α,γ-SCPNs in water (three replicas in each case) showed that any system that contains Acp units (SCPNAcp and SCPNAch/Acp) was not stable after 40 ns (Figure 9 and Suppl. Figure S11).86 Large RMSD and RMSF values support this instability (Suppl. Figures S12-13) and, although the structure of the nanotube is not conserved, the CPs establish H-bonds between them in a disordered manner in an effort to accommodate the hydrophobic groups of the rings far away from water. SCPNAch remains more conserved, although the CPs at the edges were also disassembled. The RMSD increases with time, with the most markedly fluctuating units being those located at the edge of the channel (Suppl. Figure S14). There is an average loss of several H-bonds per CP with respect to the theoretical maximum (42) and this is due to competition with water molecules. Indeed, structures are found along the simulation that conserve about 30 H-bonds, which corresponds to six internal CPs from the original nanotube. Recent studies on classical SCPNs with different surface polarity, {c-[D-Trp-L-Leu-]4}8 and {c-[L-Gln-D-Ala-L-Glu-D-Ala-]2}8, also suggest that intermolecular H-bonding interactions are more consistent in the nonpolar solvent medium than in the polar solvent.64


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Figure 9: Last snapshots at 40 ns obtained for SCPNAch, SCPNAcp and SCPNAch/Acp simulated in water.a a

Two other replicas were simulated for each system and the results are shown in the Supporting Information, Figure S11.

3.3. Effect of guest molecules on the stability of dimers and nanotubes. Towards control of the length of the SCPN The results presented previously indicate that both dimers and SCPNs are more stable in chloroform than in aqueous solution. However, the formation of dimers and nanotubes in water is very important, since it opens the door to interesting applications, mainly in the biological area. Simulations in water suggested that the systems are not stable because of competition with the solvent for amide H-bond donors and acceptors, a phenomenon that destroys supramolecular entities. However, our investigation suggests that both in dimers and nanotubes, the entrance of a molecule of chloroform between two CPs is favourable and presumably could stabilize the system. To further investigate the effect of guest molecules on the stability of the stacked CPs, dimers and SCPNs with chloroform molecules preencapsulated in their inner cavities were built, immersed into a box of water and simulated using MD simulations under the same conditions used for the analogous empty systems in pure aqueous solvent. ACS Paragon Plus Environment

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As it has been published recently,87 perpendicular halogen bonding can become competitive when the πelectron system of carbonyl groups is enriched by electron-donating groups, which is the case for peptides. In this case, macromolecular confinement and steric hindrance arising from orthogonal hydrogen bonds may readily favour this nonconventional supramolecular interaction. None of the three replicas simulated for CHCl3⊂ DαAch (with a pre-encapsulated chloroform molecule) led to stable systems (Figure 10a and Suppl. Figure S15). However, in the case of the Acpbased homodimer (CHCl3⊂ DαAcp) and the heterodimer (CHCl3⊂ DαAch/Acp) the presence of a chloroform molecule pre-encapsulated inside their cavities was sufficient to stabilize the systems and prevent their destruction in water (Figure 10b-c and Supp. Figure S15). Much lower RMSD (Figure 10) and RMSF (Suppl. Figure S16) values were obtained after chloroform pre-encapsulation compared to those obtained for the analogous empty dimers in pure water (Suppl. Figure S7). The stabilization can also be deduced from the number of H-bonds between the monomers of the three dimers investigated (Figure 10). The heterodimeric system is the one in which more H-bonds are conserved between the monomers, followed by DαAcp where water is competing for amide interactions, as can be observed from the increase in the number of structures H-bonded by four or H-bonds or fewer. In the case of DαAch there is a replica in which the dimer is quite stable, since it conserves the six H-bonds in a proportion of the structures of the trajectory. It must be noted, however, that a large number of H-bonds are lost as a consequence of the destruction of the dimer.


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Figure 10: Top: Last snapshot after 40 ns of the simulation of the dimers simulated in water with a molecule of chloroform pre-encapsulated into their inner cavities (CHCl3⊂ DαAch, CHCl3⊂ DαAcp and CHCl3⊂ DαAch/Acp). The results for the other two replicas are represented in the Supporting Information, Figure S15. Middle and bottom: RMSD and normalized distribution of the number of inter-monomer Hbonds, respectively, along the simulation of the aforementioned dimers. Three replicas were simulated in each case and each replica is represented by a colour.


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The pre-encapsulation of chloroform by CHCl3⊂ DαAcp in water led to a slight increase in the distance of the c.o.m. between the monomers (and its corresponding standard deviation) in comparison to the corresponding dimer simulated in pure chloroform (Table 1). The H-bonds in the former case are also longer, although they show less marked fluctuations in the standard deviation. The stabilization of CHCl3⊂ DαAcp in water due to the pre-encapsulation of chloroform is also reflected by the interaction energy between its two CPs, which is slightly lower but of the same order as that obtained from analogous hollow systems in chloroform (Table 2). The standard deviation is higher – probably due to the higher exchange of H-bonds with water. However, the most stable system upon guest encapsulation (chloroform) is the heterodimeric one (CHCl3⊂ DαAch/Acp), where both the distances between the c.o.m. of the monomers and the H-bonding distances are almost identical when the simulation is carried out in pure non-polar solvent or in water with a chloroform molecule in the inner cavity of the dimer. The interaction energy between the monomers is almost identical to that found for the simulation in chloroform, with a similar standard deviation. It can be concluded that the encapsulation of a single chloroform molecule inside the dimers stabilizes them in a similar way to the case where they were surrounded by a chloroform medium, except for DαAch – where the encapsulation of chloroform is not sufficient to compete with the H-bonds of the water, presumably due to an easier exit of the guest molecule from the cavity. These results suggest that the disruption of the nanotube takes places mainly through the internal cavity in which water molecules must have more accessible the carbonyl and NH groups that are involved in the stabilization of the ensemble. Analogous MD simulations were carried out on SCPNs, in which the cavities were filled with chloroform molecules (seven CHCl3, one of them between each two rings) and they were then immersed into a box of water. These simulations successfully led to stable nanotubes in aqueous solution (Figure 11). The RMSD values (Figure 11a) corresponding to the simulation of the SCPNs with the ACS Paragon Plus Environment

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encapsulated chloroform in water are even lower than those corresponding to analogous hollow nanotubes in pure chloroform (0.145 ± 0.015, 0.135 ± 0.018 and 0.110 ± 0.013 nm for 7CHCl3⊂ SCPNAch, 7CHCl3⊂ SCPNAcp and 7CHCl3⊂ SCPNAch/Acp, respectively). Once more the lowest value for the RMSD corresponds to the heteromeric nanotube. The RMSF plots (Figure 11b) also show the difference between central (more rigid) and terminal parts (more flexible) in all three types of SCPNs studied. The fluctuation observed for the Ach units in SCPNAch is larger than for the other two systems, in contrast to the situation when the nanotubes were empty and simulated in a chloroform box. Analysis of the intramolecular H-bond scheme (Figure 11c) also reveals that the SCPN is very well maintained in all three cases, since there are a large number of structures in the trajectory that conserve the maximum number of H-bonds (42). However, comparison of the number of H-bonds calculated for analogous empty nanotubes in chloroform (Figure 7c) shows that there are a larger number of structures that form between 35–41 H-bonds in the water simulation of the 7CHCl3⊂ SCPNAch. This phenomenon is due to the β-sheet H-bond exchange with water, a process that did not take place when the simulations were carried in pure chloroform.


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Figure 11: Top: Last snapshot of the 40 ns simulation of α,γ-SCPN simulated in water with seven molecules of chloroform pre-encapsulated within their inner cavities (7CHCl3⊂ SCPNAch, 7CHCl3⊂ SCPNAcp and 7CHCl3⊂ SCPNAch/Acp). Bottom: (a) RMSD, (b) RMSF and (c) normalized distribution of the number of H-bonds between the CPs of the three mentioned nanotubes along 40 ns simulated in water.


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The Rg of the backbone of the CPs stays close to the initial values for all the monomers in the three SCPNs studied (Suppl. Figure S17), although 7CHCl3⊂ SCPNAcp presents slightly lower values for its terminal monomers compared to the internal ones, a consecuence of their higher bending tend in contact with water. Besides, in 7CHCl3⊂ SCPNAch/Acp, the Rg values of the Acp monomers are slightly lower than those corresponding to the Ach units – as occurred with the analogous empty systems simulated in chloroform. The distances between the c.o.m. of the backbone atoms of each pair of rings are practically identical to those obtained for the hollow nanotubes in chloroform (Table 4), suggesting that the stabilization caused by the chloroform is very similar to the effect of replacing water by chloroform in the box of solvent. The interaction energies obtained for the α-α interaction in the SCPNs pre-filled with chloroform and simulated in water are also practically identical to those obtained when the simulation was carried out in pure chloroform (Table 5). However, a decrease in the γ-γ energy interaction occurred, suggesting that it is the most properness to the water exchange. The introduction of chloroform into the inner cavity of the SCPNs has an effect on the interaction energy between the monomers similar to that observed when the SCPNs are simulated in pure chloroform, indicating that a clear stabilization is achieved with the guest (organochlorated) molecules. In order to ascertain whether two molecules of chloroform are sufficient to stabilize the SCPN in water, with each CHCl3 placed at one edge of the tube, an analogous nanotube made with CPsAcp and with only two chloroform guest molecules (2CHCl3⊂ SCPNAcp) was studied. The destruction of the SCPN can be nicely visualized when the two molecules of chloroform start to penetrate into the hollow cavity created in the channel. In this case the CPs at the edges disassemble and after 40 ns the structure of the nanotube is completely lost (Figure 12). This experiment showed that the stabilization of the ACS Paragon Plus Environment

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SCPN introduced by the pre-encapsulated guest at the edges is not transmitted to the rest of the nanotube. Water is able to destroy the parts of the SCPN with no stabilizing effect introduced by the guest organochloride molecules. The pressure of the solvent molecules to fill the empty space leaved at the inner part of the nanotube must induce the diffusion of the water molecules into the ensemble that gives rise to compete with β-sheet structures triggering nanotube deassembly.

Figure 12: Different snapshots of the simulation in water of SCPNAcp with only two molecules of chloroform situated at the edges of the channel (2CHCl3⊂ SCPNAcp).

The encapsulation of chlorinated molecules inside the SCPNs could be extrapolated to applications such as, for example, to control the length of the channel by means of a chlorinated oligomer. Precise morphological control is an important challenge in the field of molecular self-assembly. For nanotubular objects, the ultimate challenge is to control their length, diameter and wall thickness together. In reality, there are very few examples of rational control of these size parameters from a bottom-up approach.88 The control of the growth of SCPN using chlorinated guests would offer a very interesting approach in this area. MD simulations of 1,2-bis[2-(2,2,2-trichloroethoxy)ethoxy]ethane (bteee) encapsulated in a SCPN composed of CPsAcp (bteee⊂ SCPNAcp) units were carried out in chloroform and in aqueous


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solution, leading to stable systems in both cases (Figure 13). The polymer does not leave the nanotube and contributes to its stabilization in aqueous solution. Our results predict great potential for the use of this type of guest to control the length of SCPNs.

Figure 13: First and last snapshots of the simulation of SCPNAcp with a chlorinated polymer (bteee) encapsulated within the inner cavity (bteee⊂ SCPNAcp), in water and chloroform (40 ns). ACS Paragon Plus Environment

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4. Conclusions The microscopic details and dynamical behaviour of SCPNs formed by α,γ-cyclohexapeptides based on γ-Acp and γ-Ach have been studied using MD simulations. Heteromeric nanotubes, composed alternately by γ-Ach- and γ-Acp-based CPs, have also been investigated and this is the first time that an SCPN nanotube composed of heteromeric units has been investigated. In addition, in order to gain a deeper knowledge of these tubular structures, we also studied the dynamical behaviour of the corresponding N-methylated dimers that contain the fundamental repeating structural motif of longer SCPNs. With this aim in mind, MD simulations on the N-methylated dimers were carried out to study the effect of explicit solvents on their stability and to investigate if the results obtained on dimeric systems can be extrapolated to nanotubes. As far as the single dimers are concerned, the most stable organization is that composed of heterodimeric units, one of which contains γ-Ach and the other one γAcp-based α,γ-CPs. However, the most stable SCPN, according to the interaction energy between its monomeric CPs, is not the one formed by heterodimeric pairs of ‘uncapped’ CPs, but the one formed exclusively by CPs containing Ach units. Our results suggest that the N-methyl groups introduced into the dimer destabilize the system compared to their analogous unmethylated monomers in the SCPN. Analogous differences could be present in other systems in which discrete dimers are used to study the formation and stabilization of longer nanotubes, so special care should be taken before extrapolating the conclusions obtained from the dimers when studying SCPNs. It was also found that although the α-α interaction gives a higher association constant experimentally, the γ-γ interaction is the stronger one, once formed, in both the dimer and the nanotube. Both dimers and SCPNs were simulated in polar and nonpolar solvents (water and chloroform, respectively) and it was concluded that the aqueous solution has a destabilizing effect on the structure of all the systems studied and that the main water molecules that compete for the H-bonds are those occupying their inner cavity. Our calculations suggest that the introduction of guest molecules that fit


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within the ensembles, such as chloroform and other chlorinated molecules, leads to their stabilization in water. This possibility opens the door to a large number of possible future applications, such as control of the length of the nanotube using, for example, chlorinated oligomers. A precise control of the nanotube length using 1,2-bis[2-(2,2,2-trichloroethoxy)ethoxy]ethane as guest has been envisaged from out MD simulations, suggesting very good advantages for this methodology to be employed in the area of molecular self-assembly. These potential applications are under investigation. The results obtained allow the full characterization of the structural, energetic, dynamic, and transport properties of this interesting new class of SCPNs and suggest interesting possibilities for the design of new nanotube derivatives and guest molecules to introduce into their inner cavities.

Acknowledgment We are grateful for funding from the Spanish Ministry of Science and Innovation MICINN and the ERF (CTQ2010-15725, Consolider Ingenio 2010 CSD2007-00006) and the Xunta de Galicia (GRC2006/132 and EM 2012/117). R.G.F. thanks the Spanish Ministry of Education and Science for financial support from the program Juan de la Cierva. All calculations were carried out on the MareNostrum supercomputer at the Barcelona Supercomputer Center and on the Centro de Supercomputación de Galicia (CESGA).

Supporting Information Available

Figures S1-S17 and Table S1 including simulated structures and the partial charges of the cyclic α,γpeptide rings including N-methylated and unmethlyated ones. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES: ACS Paragon Plus Environment

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69. Fox, T.; Kollman, P. Application of the RESP Methodology in the Parametrization of Organic Solvents J. Phys. Chem. B 1998, 102, 8070-8079. 70. Cieplak, P.; Caldwell, J.; Kollman, P. Molecular mechanical models for organic and biological systems going beyond the atom centered two body additive approximation: aqueous solution free energies of methanol and N-methyl acetamide, nucleic acid base, and amide hydrogen bonding and chloroform/water partition coefficients of the nucleic acid bases. J. Comput. Chem. 2001, 22, 10481057. 71. Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graphics Modell. 2006, 25, 247-260. 72. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 2004, 25, 1157-1174. 73. Parrinello, M.; Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method . J. Appl. Phys. 1981, 52, 7182-7190. 74. Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684-3690. 75. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 1997, 18, 1463-1472. 76. Essman, U.; Perera, L.; Berkowitz, M.; Darden, T.; Lee, H.; Pedersen, L. A smooth particle mesh Ewald method. J. Chem. Phys. 1995, 103, 8577-8593. 77. Kumar, S.; Rosenberg, J. M.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A. Multidimensional free-energy calculations using the weighted histogram analysis method. J. Comput. Chem. 1995, 16, 1339-1350. 78. Grossfield, A, WHAM: an implementation of the weighted histogram analysis method, http://membrane.urmc.rochester.edu/content/wham/. 79. Kumar, Sm.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A.; Rosenberg, J. M. THE weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comp. Chem. 2002, 13, 1011-1021. 80. Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular dynamics. J. Mol. Graphics 1996, 14, 33-38. 81. Smart, O. S.; Neduvelil, J. G.; Wang, X.; Wallace, B. A.; Sansom, M. S. P. HOLE: A program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 1996, 14, 354360. 82. Unpublished results. 83. Clark, T. D.; Buriak, J. M.; Kobayashi, K.; Isler, M. P.; McRee, D. E.; Ghadiri, M. R. Cylindrical beta-sheet peptide assemblies. J. Am. Chem. Soc. 1998, 120, 8949-8962. 84. The nanotube composed of Ach is analogous to that used in a previous study (see reference 66) with the difference that now another two cyclic units have been added and eight CPs instead only six monomers were used in the simulation. Besides, constant pressure instead of constant volume was employed, to reproduce better the conditions employed in the experiments. 85. In a previous communication (see reference 66) where we simulated an analogous SCPN composed of γ-Ach units (six CPs) we did observe diffusion of chloroform along the channel after extending the simulation. It should be noted that those simulations had been carried out at constant ACS Paragon Plus Environment

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volume, whereas the present calculations have been carried out at constant pressure, to simulate conditions more similar to the experimental ones. 86. In the case of SCPNAch/Acp, five of its inner rings were still assembled in one of the replicas. 87. El-Sheshtawy, H. S.; Bassil, B. S.; Assaf, K. I.; Kortz, U.; Nau, W. M. Halogen Bonding Inside a Molecular Container. J. Am. Chem. Soc. 2012, 134, 19935-19941. 88. Valery, C.; Artzner, F.; Paternostre, M. Peptide nanotubes :Molecular organisations, self-assembly mechanisms and applications Soft Matter 2011, 7, 9583-9594.


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Effect of Organochloride Guest Molecules on the ... - ACS Publications

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