Behavior of a Peptide During a LangmuirâBlodgett Compression

Article Cite This: J. Phys. Chem. C XXXX, XXX, XXX−XXX

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Behavior of a Peptide During a Langmuir−Blodgett Compression Isotherm: A Molecular Dynamics Simulation Study Benedetta Di Napoli, Claudia Mazzuca, Paolo Conflitti, Mariano Venanzi, and Antonio Palleschi* Department of Chemical Sciences and Technologies and CSGI, University of Rome “Tor Vergata”, Via della Ricerca Scientifica snc, 00133 Rome, Italy S Supporting Information *

ABSTRACT: A detailed characterization of the behavior of the amphiphilic antimicrobial peptide Trichogin GA IV (TRIC) at the air/water interface during a Langmuir−Blodgett (LB) isotherm is reported. By means of molecular dynamics simulations, experimental data are explained in terms of the conformational changes and aggregate features adopted by TRIC. We show that, due to compression, different structural changes occur: initially formed drop-like aggregates coalesce, forming nanofibers; on increasing the surface tension further, these nanofibers constitute a web-like structure in which meshes are filled by water pools. During these transitions, the peptide chains lie almost parallel to the surface mostly adopting a helical conformation. At high peptide concentration, reaching the maximum of the allowed surface pressure, a monolayer of TRICs in nonhelical conformation and vertically aligned with respect to the air/water interface is formed. “butterfly shapes”, irregular rectangles, dots,17 nanofibers, or nanoribbons.18−20 It should be noted, however, that the molecular characterization of peptide during the LB isotherm is always limited to surface analyses, as up to now, to the best of our knowledge, no molecular dynamics (MD) simulations have been performed in this context. In this paper, we have studied the behavior at the air/water interface of the antimicrobial peptide Trichogin GA IV (TRIC), a peptide that has been deeply characterized by our group, in solution, using physicochemical techniques and MD simulation.21−27 TRIC belongs to the lipopeptaibol family and was isolated from Trichoderma longibrachiatum in 1992;28 its primary structure is n-Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-AibGly-Ile-Lol, where Aib is α-aminoisobutyric acid, n-Oct is noctanoyl, and Lol is leucinol. Interestingly, this peptide is amphiphilic and, even if relatively short (11 amino acids long), adopts a helical structure in both the solution and membrane phase, with a flexible hinge formed by two consecutive glycine residues21,22 in positions 5 and 6. Moreover, this peptide is able to form aggregates, both in membrane and in water, characterized by small conformational rearrangements.25 Aggregation studies of this peculiar peptide at the air/water interface have not yet been reported in the literature. To study the molecular behavior of TRIC at the air/water interface, during an LB isotherm, we applied both experimental

1. INTRODUCTION The Langmuir−Blodgett (LB) technique is a simple and lowcost method for the production of layered structures at the air/ water interface, the ordering of which can be controlled by mechanical forces.1,2 The LB method is typically applied to amphiphilic compounds, detergents, and lipids,3−5 whose conformational and aggregation behavior at the air/water interface has been deeply characterized by experimental methods and molecular dynamics (MD) simulations.6−8 Detailed analyses concerning the conformation and the different phases that molecules experience at the air/water interface during an LB isotherm are usually reported by considering amphiphilic molecules like lipids and fatty acid. However, extensive experimental research has also been carried out on Langmuir films formed by “unusual” molecules, like polymers, DNA, liquid crystals, proteins, carbon nanotubes, and inorganic nanostructures.1,2,8 In this connection, for example, the formation of peptide monolayers by the LB technique is a common experimental procedure to study the conformation and the self-assembly monolayer (SAM) of peptides and other macromolecules upon surface pressure changes.1,9,10 In addition, peptide LB monolayers could be particularly informative as models for studying the organization of antimicrobial peptides at the air/ water interface. However, despite the great interest in potential applications of LB films of peptides,1,11−13 the comprehension at a molecular level of the peptide aggregation mechanism at the air/water interface14−16 is still poor. SAMs of peptides obtained by the LB technique show a rich morphology like © XXXX American Chemical Society

Received: October 5, 2017 Revised: December 7, 2017 Published: December 7, 2017 A

DOI: 10.1021/acs.jpcc.7b09850 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

proteins) GROMACS tool.41 The visual molecular dynamics (VMD) program42 was used for the structure visualization. Model. Two vacuum/water interfaces separated by a 3 nm thick water slab to minimize the interference between the two interfaces were created in the simulation box of 9 nm × 9 nm × 27 nm (x, y, and z axes, respectively). The high value of the z axis, perpendicular to the vacuum/water interface, is required to eliminate interactions between the system periodic images. For each simulation, the peptides were placed at the vacuum/water interface, lying perpendicularly to the water surface and inhibiting interchain peptide interactions. To reproduce the experimental LB isotherm, 13 systems at different Mma values, ranging from 3600 to 76 Å2/molecule, were simulated. To mimic compression, MD simulations have been performed using a water box with fixed dimensions but changing the number of peptides (n varying from 1 to 100) in the box, thus reproducing the LB isotherm (indicated by lines in Figure 1; n = 1 and 2 are not reported for clarity). In Table 1

and theoretical approaches. To reproduce LB data, we ran several MD simulations, fixing the system dimension and varying the number of peptides inserted in the simulation box. By using this approach, we were able to characterize in detail the conformational changes of the single peptide chain, the peptide rearrangements, and the molecular interactions that occur during an LB experiment on increasing the surface pressure, at the air/water interface.

2. EXPERIMENTAL SECTION Materials. The synthesis of TRIC has been performed according to a procedure reported elsewhere.29,30 Spectrograde solvents (Acros Organics, Thermo Fisher Scientific Geel − Belgium) were exclusively used. Water was distilled and passed through a Milli-Q (Millipore, Billerica, MA, USA) purification system before use. Langmuir−Blodgett Compression Isotherms. Measurements of surface pressure (π) vs mean molecular area (Mma) isotherms were carried out using a computer controlled KSV LB apparatus (KSV MiniMicro, Helsinki, Finland). The trough and the barriers were made of Teflon, and the pressure was measured using a Wilhelmy plate. Monolayers were carried out on a deionized water subphase (resistivity of 18.2 MΩ·cm). TRIC chloroform solution (0.2 mg/mL) were carefully deposited on the water surface, using a Hamilton microsyringe. After solvent evaporation (15−20 min), the surface pressure vs the mean molecular area (π-Mma) isotherm was recorded at a constant compression rate of 5 mm min−1. The water subphase temperature was kept constant at 25.0 ± 0.5 °C. Further experimental details concerning the LB isotherm are reported in the SI. Molecular Dynamics Simulations. Force Field and MD Parameters. Molecular dynamics simulations were performed for peptides at the vacuum/water interface using the GROMACS 4.6.731 software and the GROMOS53a6 force field,32 with a slab of 8715 water molecules (SPC model).33,34 All simulations were carried out in a constant pressure and temperature ensemble (NPT), using periodic boundaries conditions. The topology and the charge distributions needed for noctanoyl and leucinol termini were derived selecting the more suitable GROMOS standard parameter in accordance with results obtained using the restrained electrostatic potential (RESP) method and the software package RESP ESP charge Derive (RED),35 while the topology of Aib amino acid was already reported.36 Lennard-Jones parameters, bond, angle, and dihedral parameters for n-octanoyl and the leucinol group are listed in Table S1. Following our protocol, for each system there was a two-step energy minimization: first, only the solvent was energy minimized and after that the solute. Then, the peptide was restrained, and the solvent was equilibrated for 150 ps at 50 K. All the systems were gradually heated from 50 to 300 K in a 1 ns MD, and then 50 ns production runs were performed. A 2 fs time step was used in all steps and simulations, using the particle mesh ewald (PME) algorithm37,38 for electrostatic interactions with a double cutoff39 at 1.4 nm for van der Waals and Berendsen algorithm40 for both temperature (coupling constant τT = 0.2 ps) and pressure (τP = 1 ps, semi-isotropic condition) coupling. All the parameters reported have been obtained during the last 10 ns of simulation time, when the systems are equilibrated. Secondary structure analyses were performed using the DSSP (define secondary structures of

Figure 1. Experimental LB isotherm of TRIC. The numbers on top of the vertical lines indicate the number of peptides in the simulation box, corresponding to the mean molecular area reported on the abscissa.

the number of peptides present in the simulation box and the corresponding mean molecular area in the LB isotherm are reported. Surface Pressure. The surface pressure of the system at each Mma value was obtained from Π(A) = γaw − γm(A)

(1)

where A is the surface area per peptide, i.e., the mean molecular area; γaw is the surface tension of water at 300 K; and γm(A) is the corresponding surface tension of the system at the surface area A and 300 K. The surface tension γm(A) was calculated from the average surface tension given by the difference of the normal (PN) and lateral (PL) pressure in the box, according to the equation L γm(A) = (PN − PL) · z (2) 2 where Lz is the box normal size and

PL = (Pxx + Pyy)/2

(3)

γaw is calculated as γm(A) but after a simulation performed using only water (simulation time: 50 ns).43 Interface Thickness and Position of Peptides at the Interface. The interface thickness was estimated from the density profile along the normal to the interface (z axis) by B


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The Journal of Physical Chemistry C Table 1. Number of Peptides (n) Placed on Each Water Surface and the Corresponding Mean Molecular Area Mma [Å2/peptide]

76.1

90.6

126.9

158.6

217.5

271.9

380.7

475.6

951.8

1269

1903

2441

3622

n

100

84

60

48

35

28

20

16

8

6

4

2

1

means of the g_density GROMACS tool. The depth was defined as the distance between the position where the density is 10% and 90% of the bulk water density.43−45 Symmetry Factor. To characterize the shape of peptide aggregates we have determined a symmetry factor (Is) defined as

Is =

2I1 − I2 − I3 I1 + I2 + I3

To get insight at the molecular level of the peptide features during an LB experiment, first of all, we have considered the effect of the surface peptide concentration on the thickness of the air/water interface.44,45 As reported in Figure 3, the interface thickness calculated up to the first phase transition (n < 16) increases on increasing the

(4)

where I1, I2, and I3 are the major, intermediate, and minor tensor components of the second moment of the mass distribution. According to this definition, for example, Is = 0 if the aggregate has a spherical form, while Is = 2 in the case of a linear aggregate.

3. RESULTS AND DISCUSSION 3.1. LB Isotherm. The π/Mma LB isotherm of TRIC, reported in Figure 1, shows that the first transition starts at Mma = 520 Å2/peptide, while the second one occurs at about 160 Å2/peptide. These findings cannot be explained only taking into account a uniform monolayer in which peptides pass, due to compression, from a parallel (in which the hydrophilic side is in water and the hydrophobic one faces air) to a perpendicular arrangement with respect to the surface.17 The molecular areas, calculated considering a helical peptide in a parallel and perpendicular arrangement, are indeed about 184 Å2 and 59 Å2, respectively. We have therefore carried out MD simulations to explain the experimental result reported in Figure 1 in terms of molecular features like peptide position and orientation at the air/water interface, peptide conformations, and aggregation at several steps during compression. 3.2. MD Simulations. As reported in Figure 2, comparison between the isotherm obtained experimentally with the theoretically predicted surface pressure values shows a very good agreement; this result attests the validity of our simulation studies.46

Figure 3. Interface thickness (blue), z-distance of the peptide center of mass (light green), of the deepest (red), and outermost (deep green) peptide atoms at the air/water interface as a function of the number of peptides in the simulation box. All the distances are referred to the innermost z-value of the interface facing the bulk side (z = 0 in the figure). Lines are only a guide for the eyes.

number of peptides in the simulation box (corresponding experimentally to a decrease of the mean molecular area). In particular, this increase is greater at low peptide concentration (n = 2−8), passing from 3.6 Å (very close to the air/water interface thickness in the absence of peptides) to 6 Å.47 A close inspection of the z distance of the center of mass (COM) of peptides indicates that they tend to move to the surface on increasing peptide concentration. At low peptide concentration, indeed, TRIC molecules are closer to the bulk, while they reach the surface in correspondence of the beginning of the second transition (n = 48), where the area accessible to peptides sensibly decreases. Moreover, during this transition, peptides tend to stay out of the interface, pointing to the air side; this can be caused by several effects, i.e., an orientation and/or conformational change or aggregation and/or formation of multilayers. This finding is confirmed by an inspection of the position of the deepest and outermost atoms of TRICs; interestingly, the deepest atom lie always at the same distance with respect to the interface independently on the peptide concentration, while the z-distance of the outermost atom tends to increase on increasing compression. The thickness increase could be ascribable to the change of the angle that the peptides assume with respect to the surface. As reported in Figure 4, indeed, during the first transition (at about n = 16), TRICs lie parallel to the air/water interface independently on the peptide concentration, as the mean tilt angle (that is the average of the angles of the peptide axes with respect to the z axis of the simulation box) is close to 90°. It should be noted that although the peptides at the beginning of the simulation are put oriented perpendicular to the surface (see Materials and Methods section) in a few picoseconds they

Figure 2. Comparison between the isotherm obtained experimentally (continuous line) and the surface pressure values obtained from molecular dynamics data (circles). C


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Figure 4. Mean tilt angle that TRIC assumes with respect to the normal to the interface, during the compression.

Figure 6. Last MD frames of TRIC peptides at mean molecular area of 951.7 (A), 475.6 (B), 271.9 (C), and 126.9 (D) Å2/peptide (boxes contain 8, 16, 28, and 60 peptides, respectively). Yellow ribbons indicate helical structures, while thicker tubes represent random coil (red) and all other conformations (blue) in new cartoon representation (VMD). For the sake of clarity, only the peptide backbone is shown.

reorient themselves parallel to the water surface, leading to the hydrophilic moieties of this peptide in contact to water (see Figures S1 and S2). Starting from the second transition, instead (n > 48), peptides vary their orientation, assuming angles greater than 90° (up to 150°). This is because the peptides are too bulky to lie parallel to the water surface, when the surface occupied by peptides is higher than 80%, as shown later. All these results indicate that a transition from parallel to vertical orientation of the peptide chains could be responsible for the increase of the air/water interface thickness at high TRIC concentration (for n > 48). With regard to the analysis of conformations adopted by TRIC during compression, three groups of secondary structures have been taken into account: helices (including α-, 310-, and 5helix), turn/β (including turn, β-bend, and β-bridge), and random coil. The relative population of these groups as a function of the number of peptides in the simulation box is reported in Figure 5.

each), while aggregation leads TRIC to prefer helical conformations.26,27 Conversely, at high peptide concentration (n > 48), during the second transition, the variation in the percentage of the helical structures adopted by TRIC tends to decrease abruptly in a cooperative way, reaching values less than 10%. Concomitantly there is a sudden increase in the percentage of random coil and turn/β structures that reach values close to 80%. All the results can be visualized by the side-view snapshots showing the peptides at the air/water interface (see Figure 7).

Figure 7. Side-view snapshots of systems containing 8 (A), 16 (B), 48 (C), and 100 (D) peptides in the simulation box, respectively. Glycine moieties are colored in green, the octanoyl groups in red, and all other groups in blue.

Figure 5. Percentage of helices (black), random coil (blue), and turn/ β (red) secondary structures adopted by TRIC during compression. Lines are only a guide for the eyes.

When the number of peptides in the system is less than n = 48, peptides retain their helical structure and lie parallel to the surface. They tend to expose to the solvent the more hydrophilic amino acids (glycine, colored in green in Figure 7), while the octanoyl moieties (colored in red in Figure 7) are always pointing to the air subphase. However, when the number of peptides in the box exceeds n ∼ 50, the system becomes too bulky (surface area of the simulation box is ∼8000 Å2, while the surface occupied by a peptide in a helical conformation is at least 160 Å2). Consequently, a fraction of the helical peptides must vary their secondary structures in order to realize a good packing while maintaining the hydrophilic moieties in contact with water.

As can be seen, the percentage of helical structure increases on increasing peptide from n = 1 to n = 8, reaching percentages close to 80%. This increase parallels the strong decrease in the percentage of both the peptide random coil and turn/β structures, thus indicating that a (cooperative) transition from random coil and/or turn/β to helices occurs during the first steps of the LB isotherm. This is because, as shown in Figure 6, TRIC tends to form aggregates also at very low concentrations (n = 2), and the packing is favored by helical conformations; thus, if TRIC is in a monomeric form, it could unfold easily (for n = 1, the helices turn/β, and random coil percentages are 33% D


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assume a prolate or rod-like shape. On the basis of data reported in Figures 6, 8, and 9, TRICs at 8 < n < 20 tend to form asymmetric multiaggregate structures like nanofibers, due to the coalescence instead to the symmetric growth of the droplike little aggregates. On increasing compression (n > 28), another change in shape occurs, and Is assumes values around 1, suggesting the formation of more symmetrical aggregates. Indeed, the previously formed elongated nanofibers interconnect to form a more symmetrical web-like structure. More in detail, as shown in Figures 3, 4, and 6, this web-like structure lies on the interface with water pools. TRICs are oriented parallel to the surface, and the COMs of the aggregates are well inserted in the interface. Interestingly, in such aggregates, TRICs form both head−tail and head−head aggregates (data not shown) as found in solution and in membrane.26 Finally, when the number of peptides in the system is higher (n > 48), Is remains stable, while the projected area of each peptide decreases abruptly on increasing concentration. This evidence indicates that TRICs form an extended aggregate like a monolayer, and due to the bulkiness, the peptides lose helical conformation, forming predominantly elongated chains, and do not lie parallel to the interface. It should be noted, however, that in this condition, even if the relative population of the helix group (see Figure 4) structures is less than 10% (see Figure 4), the percentage of random coil structures is about 40%. This indicates that the presence of the other structures, belonging to the turn-beta group, is responsible for an almost “ordered” tightly packed aggregate. Noteworthy, also in the case of minimum area per molecule (n = 100), TRIC multilayer aggregates were not observed. To obtain a fully detailed description of the processes occurring during the LB isotherm, we have also investigated the role of water, in terms of the amount of water presents on the surface in the several steps of the compression. As shown in Figure 10, at the LB isotherm starting point, that is, the beginning of the first transition (n = 16 corresponding to

At higher concentrations, peptides are preferentially nonhelical, very tightly packed together, and are forced due to the increasing bulkiness to be no longer oriented parallel to the surface. In this case the octanoyl group residues are exposed to the air and form a hydrophobic sheet (see Figure 7D). To get a deeper insight into the aggregate features we have also analyzed the area projected by TRICs on the water surface as a function of the peptide concentration. The aim is to determine the extent of peptide close packing during aggregation. As shown in Figure 8, when the number of

Figure 8. Projected area per molecule of the TRIC aggregates on the water surface as a function of the number of peptides.

peptides is low (2 < n < 8), the area per peptide decreases quickly, indicating that peptides form compact aggregates characterized by a drop-like shape (as shown in Figure 6). As reported earlier, close packing is favored by the fact that TRIC adopts helical structures. On increasing the peptide concentration (from n = 16 to n = 48), the area projected by each TRIC in the aggregates decreases much more slowly. This finding suggests that aggregation could be caused by the coalescence of previously formed smaller aggregates that in this process retain their compactness. This hypothesis has been confirmed analyzing the symmetry factor (Is) of the TRIC aggregates (Figure 9). From these data, it should be noted that TRIC peptides at low concentration (n = 6−8) form oblate or drop-like aggregates as Is ≤ 1. On increasing the concentration, a dramatic change in aggregate shapes takes place. Indeed, Is becomes about 1.5, thus indicating that TRIC aggregates

Figure 10. Percentage of surface occupied by water molecules during compression. The 3-D features of the aggregates that TRIC forms at the several stages of the isotherm are reported.

Mma = 475.6 Å2/peptide), the surface occupied by water is remarkable, about 70%. On the contrary, at n ≥ 48 (corresponding to Mma ≤158.6 Å2/peptide), the surface occupied by water is less than 20%. These data confirm that in the first steps of the isotherm drops of TRIC aggregates lie surrounded by water, while at the end of the transition a peptide monolayer is formed in which water drops are still present. Moreover, it is possible to understand why the

Figure 9. Symmetry factor of TRIC aggregates obtained according to eq 4 (see the Experimental Section). Line is only a guide for the eyes. E



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interface thickness does not increase at n ≥ 48 (see Figure 3); in these conditions, indeed, the water surface is almost entirely covered by a peptide monolayer and is not affected significantly by the little changes of water loss. At intermediate concentration the increasing peptide concentration leads to a progressive loss of water on surface leading to the coalescence of drop-like TRIC aggregates into nanofibers and, in turn, into web structures. Summarizing, it is possible to explain every single step during the LB experiment and assign, on the basis of molecular details obtained, the transitions that occur during the LB isotherm. Results show that during compression, in the Mma ranging from 2500 to 950 Å2/peptide (n = 2−8), TRIC aggregates increase the amount of helical conformation and form, on the water surface, bidimensional drops that tend to increase in dimension. On increasing concentration, in the molecular area range of about 500−400 Å2/peptide (n = 16−20), when the percentage of surface occupied by water is around 70%, a first transition occurs in which peptide drops coalesce into nanofibers. In the second transition, that takes place in the range 150−250 Å2/peptide (28 ≤ n ≤ 48), a web due to the interconnection of the peptide nanofibers appears. The meshes of the web are occupied by water pools that tend to decrease in dimension on increasing compression. It should be noted that this transition occurs when the percentage of the surface occupied by water becomes less than 50% and the surface coverage due to peptides becomes predominant. Moreover, in this concentration range, the peptide remains always almost parallel to the surface and predominantly in helical conformation. Finally, at higher concentration (n > 48, corresponding to Mma < 150 Å2/peptide) a transition occurs in which the web becomes a monolayer; that is, the meshes of the web are so little that the surface of the box looks like an inhomogeneous layer of peptides in which little holes are filled by water. In this range the water present on the surface is less than 15%. When n > 100, due to the bulkiness, TRICs lie almost perpendicular to the surface and are predominantly in nonhelical conformation.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +39 0672594466. ORCID

Antonio Palleschi: 0000-0002-3626-1873 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank the University of Rome “Tor Vergata”, “Consolidate the Foundations 2015” funding.

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ABBREVIATIONS TRIC, Trichogin GA IV; LB, Langmuir−Blodgett; MD, molecular dynamics; SAM, self-assembly monolayer; COM, center of mass; Mma, mean molecular area

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REFERENCES

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CONCLUSION Using molecular dynamics simulations, we have explained, at a molecular level, the experimental Langmuir−Blodgett isotherm of the amphiphilic peptide Trichogin GA IV, in terms of peptide orientation, aggregation, conformational features at the air−water interface, and role of water. To mimic compression, MD simulations have been performed using a fixed water box dimension and varying the number of peptides in the simulation box, thus reproducing the LB isotherm. Based on our results, we are able to propose for peptides the first definition of the transitions that explain the LB isotherm, in a more appropriate way than that traditionally used to describe the lipid LB isotherm.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b09850. Details about the LB isotherm experiments and MD parameters used for the terminal groups and the values of tilt angles for n = 48 and 100 during MD simulations have been reported (PDF) F


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