Special Issue Article Received: 17 March 2014

Revised: 16 April 2014

Accepted: 16 April 2014

Published online in Wiley Online Library: 20 May 2014

(wileyonlinelibrary.com) DOI 10.1002/psc.2648

Aggregation propensity of Aib homo-peptides of different length: an insight from molecular dynamics simulations‡ Gianfranco Bocchinfuso,a* Paolo Conflitti,a Stefano Raniolo,a Mario Caruso,a Claudia Mazzuca,a Emanuela Gatto,a Ernesto Placidi,b,c Fernando Formaggio,d Claudio Toniolo,d Mariano Venanzia and Antonio Palleschia* Interactions between peptides are relevant from a biomedical point of view, in particular for the role played by their aggregates in different important pathologies, and also because peptide aggregates represent promising scaffolds for innovative materials. In the present article, the aggregation properties of the homo-peptides formed by α-aminoisobutyric acid (U) residues are discussed. The peptides investigated have chain lengths between six and 15 residues and comprise benzyl and naphthyl groups at the N- and C-termini, respectively. Spectroscopic experiments and molecular dynamics simulations show that the shortest homopeptide, constituted by six U, does not exhibit any tendency to aggregate under the conditions examined. On the other hand, the homologous peptide with 15 U forms very stable and compact aggregates in 70/30(v/v) methanol/water solution. Atomic force microscopy images indicate that these aggregates promote formation of long fibrils once they are deposited on a mica surface. The aggregation phenomenon is mainly due to hydrophobic interactions occurring between very stable helical structures, and the aromatic groups in the peptides seem to play a minor role. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: α-aminoisobutyric acid; helical fibrils; inversion helical chirality; conformational studies; spectroscopy; atomic force microscopy; role of aromatics; peptide self-aggregation

Introduction

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There is an explosion of interest on design, synthesis, characterization, and applications of peptide-based materials [1]. Peptide nanowires, nanotubes, self-assembled monolayers, and fibers have been obtained by bottom-up self-assembly strategies and tested as nanosensors [2], molecular photodiodes [3,4], and biocompatible platforms for tissue engineering [5]. Amphiphilic peptides are shown to be particularly suitable for the spontaneous construction of ordered nanoscopic and mesoscopic structures via hierarchic self-assembly [6]. Despite the impressive results on the experimental and even applied sides, our understanding at the molecular level of the mechanisms and forces that determine the growth of supramolecular architectures from basic structural peptide motifs is still quite poor. The difficulties originate from the observation that peptide self-assembly is governed by a full spectrum of interactions, from strong electrostatic forces to the multiple contacts of the van der Waals interactions, directional hydrogen bonds, and large-scale hydrophobic effects [7–9]. Among the others, fibrillar aggregates have been deeply investigated in the last decades, because of their role in many important pathologies [10,11]. Nevertheless, the structural features affecting the early stages of the process leading to fibril formation remain unclear. In this context, since the first studies on the role of the FF motif in the aggregation of peptides causing lethal neurodegenerative diseases were published [12], multiple evidence suggest that specific interactions between aromatic

J. Pept. Sci. 2014; 20: 494–507

groups may play a pivotal role [13–17]. Recent works focused on their role in peptide aggregation [18–20]. However, it has been shown that peptide fibrillization can be promoted by building blocks attaining different secondary structures and motifs such as β-sheet arrangements or helical-based coiled-coil conformations [21]. In the plethora of proposed mechanisms for fibril formation, the helical structures seem often to play a key role. Also in the more frequently observed fibrils formed by β-sheet peptides, helical conformations have been evoked as

* Correspondence to: Gianfranco Bocchinfuso and Antonio Palleschi, Department of Chemical Sciences and Technologies, University of Rome ‘Tor Vergata’ via della Ricerca Scientifica 1, 00133 Rome, Italy. E-mail: gianfranco. [email protected]; [email protected] ‡

This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the 1st International Conference on Peptide Materials for Biomedicine and Nanotechnology, Sorrento, October 28–31, 2013, edited by Professor Giancarlo Morelli, Professor Claudio Toniolo and Professor Mariano Venanzi.

a Department of Chemical Sciences and Technologies, University of Rome ‘Tor Vergata’, I-00133 Rome, Italy b Department of Physics, University of Rome ‘Tor Vergata’, I-00133 Rome, Italy c CNR-ISM, Via Fosso del Cavaliere 100, I-00133 Roma, Italy d Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, I-35131 Padua, Italy

Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES in the aggregation properties for peptides with six and 15 U residues, the latter both in the presence and in the absence of aromatic groups (Z-U6-N, Z-U15-N, and U15), are evaluated by MD simulations carried out on systems containing six replica of each peptide.

Materials and Methods Materials Syntheses and characterizations of the peptides investigated in this work were already reported [52]. The peptides are denoted using the acronym Z-Un-N, where Z is benzyloxycarbonyl, U (or Aib) is α-aminoisobutyric acid, N = –O–CH2–CH2–(1)naphthyl, and n is 6, 9, 12, 15. UV Absorption Absorption measurements were carried out on a Cary 100 SCAN (Varian, Palo Alto, CA) spectrophotometer. Molar concentrations were determined in methanol by absorption measurements at λ = 280 nm [naphthyl absorption, ε(280) = 6100 M
1 cm
1]. All experiments were carried out in quartz cells of variable optical length (0.1, 0.5, and 1.0 cm) at micromolar concentrations. Steady-state and Time-resolved Fluorescence Emission spectra at micromolar concentrations were obtained on a Spex-Fluorolog III (Horiba Jobin-Yvon Instruments, Middlesex, UK) spectrofluorimeter, equipped with a 450W Xenon lamp operating in the single-photon counting (SPC) mode. Samples were excited at 280 nm and the fluorescence spectra recorded from 300 to 500 nm using a bandwidth of 2 nm for both excitation and emission slits. Fluorescence time decays were obtained by an EAI Life-Spec-ps (Edinburgh Analytical Instruments, Edinburgh, UK), operating in the SPC mode (λexc = 298 nm; λem = 340 and 400 nm). Excitation at λ = 298 nm was obtained by an IBH NanoLED light emitting diode (Horiba Jobin-Yvon Instrument) with 1-ns pulse duration. Experimental time decays were deconvoluted by the pulsed excitation profile by a standard software provided by EAI. The naphthyl time decay was described in terms of lifetime distributions (100 time components regularly spaced on a logarithmic scale were employed in the best-fitting procedure). Atomic Force Microscopy Atomic force microscopy measurements on peptide films dried on a mica surface were performed in air using a Veeco Multiprobe IIIa (Santa Barbara, CA) instrument. Experiments were carried out at room temperature (20 °C) in the tapping mode by using NanoSensors Si tips with a force constant of about 40 N/m and a typical curvature radius on the tip of 7 nm. The contrast of the AFM images was enhanced for sake of clarity by using standard software supplied by Veeco Instruments. Molecular Dynamics Simulations Molecular dynamics simulations of Z-U6-N, Z-U9-N, Z-U12-N, ZU15-N, and U15 were carried out in 70/30(v/v) MeOH/water solutions, for a total amount of almost 1 μs of simulation time, as specified in Table 1. In the U15 peptide, the N- and C-termini were both set as neutral. In all of the cases, the peptides were initially disposed in the α-helical conformation. In the simulations

J. Pept. Sci. 2014; 20: 494–507 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

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key intermediates in the early stage of fibril formation, in particular for unfolded peptides in solution [22–24]. In these cases, helices would favor the formation of small oligomers seeding the aggregation process. According to this model, these helices should be stable enough to promote formation of the oligomers, but not so stable to impair the evolution toward the large β-ladders nucleating the fibrils. In this connection, it is of interest to evaluate the propensity of short peptide helices to form supramolecular structures. Recently, we characterized the aggregation propensity of oligopeptides formed by the conformationally constrained α-aminoisobutyric acid (denoted as Aib or U in the three- or single-letter code, respectively) in methanol (MeOH) and MeOH/water solutions [25]. The progress of the aggregation process was monitored by spectroscopic (UV absorption, fluorescence, and FT-IR absorption) methods, whereas the morphology of the peptide aggregates on mica was imaged by atomic force microscopy (AFM) experiments. The peptides investigated were functionalized at the C-terminus with a naphthyl (N) chromophore, a well-known spectroscopic probe, to analyze the effect of an extended aromatic group on the aggregation process, and a benzyl group at the N-terminus. Experiments showed that the aggregation propensity of the U homo-oligopeptides, enhanced by hydrophobic effects in water-rich solutions, increases with increasing the length of the peptide chain, i.e. Z-U6-N < Z-U12-N < Z-U15-N (where Z is benzyloxycarbonyl), suggesting that interhelix interactions were ruling the kinetic of the aggregation process and the morphology of the aggregates. In particular, AFM imaging revealed formation of mesoscopic globular and fibrillar structures, the predominance of which is determined by the helix length of the peptide building block. However, the weight and relative contribution of aromatic– aromatic and interhelix interactions in determining the stability and morphology of the peptide aggregates were not fully clarified. Moreover, the structural motifs, which in the early stages of the aggregation process lead to the formation of mesoscopic structures, were only roughly sketched. Because of the difficulties related with the structural characterization of the transient species by experimental methods, the simulation techniques represent an extremely useful tool to face this issue. Since the seminal works of Leach [26] and Scheraga [27], many papers reported theoretical investigations of the conformational features of homo-peptides [28–33]. From these works, and a large body of experimental results [34–46], a clear tendency of U residues to promote helical conformations emerged. Either 310- or α-helices were found to be predominantly populated, depending on peptide length and solvent polarity [33,47–50]. Despite the great number of published works on this topic, only few theoretical studies focused on the aggregation properties of U homo-peptides, and to the best of our knowledge, only dimers were taken into account [47,51]. In this study, we performed extended molecular dynamics (MD) simulations on the conformational and aggregation features of the shortest (Z-U6-N) and longest (Z-U15-N) peptides, previously investigated in MeOH/water solutions. In the first part of this paper, we briefly review the main experimental data of the different propensities of Z-U6-N and Z-U15-N to form aggregates in solution, and how these differences affect the morphology of the peptide aggregates when dried on a mica surface. Next, the MD results obtained from the simulations carried out on isolated peptides (Z-U6-N, Z-U9-N, Z-U12-N, and Z-U15-N) are briefly discussed. Finally, the differences

BOCCHINFUSO ET AL. Table 1. Molecular dynamics simulations discussed in this paper Peptide

No. of peptides in the simulation

No. of independent simulations

Z-U6-N Z-U6-N

1 6

1 2

Z-U9-N

1

2

Z-U12-N

1

2

Z-U15-N Z-U15-N

1 6

1 2

U15

6

2

Simulation code

1ZU6N 6ZU6N-A 6ZU6N-B 1ZU9N-A 1ZU9N-B 1ZU12N-A 1ZU12N-B 1ZU15N 6ZU15N-A 6ZU15N-B 6U15-A 6U15-B

Simulation time (each single run; ns) 40 100 80 80 30 100 100

with six Z-U15-N molecules, the starting helices were introduced three with right- (P-) and three with left- (M-) handed chiralities. The simulations were performed by using GROMACS 4.0.7 [53], with the ffG53a6 force field [54]. The parameters for the torsional angles (ω and χ) of the urethane protecting group present at the N-terminus were evaluated by QM calculations at the HF-6-31G* level on the methyl methylcarbamate, according to a protocol previously used [55], by means of the MacSpartan software package (MacSpartan Pro, Wavefunction Inc., Irvine, CA, 2000) (Figure 1). These parameters were inserted into the force field as Ryckaert–Bellemans torsional angles:

V ðφÞ ¼

5 X i¼0

C i ð cosðφ
180ÞÞi

Table 2. Torsional angle for the urethane: the Ryckaert–Bellemans coefficients (kJ/mol) Angle χ ω

C0

C1

C2

C3

C4

C5

1.6014 42.904

1.6551
9.2149

23.07
4.1551


21.995
21.935


5.509
19.352

1.363 11.368

where the Ci coefficients for the two angles assume the values reported in Table 2. The equilibrium values for the bending angles OE–C–O, O–C–N, and N–C–OE (Figure 1) were set equal to 125°, 124°, and 111°, respectively, obtained as average values from crystal structures of the molecules containing a urethane group in the Cambridge Structural Database [56]. The other parameters were assigned by analogy. The MD settings were adopted according to previously described procedures [57]. Briefly, the peptides were placed (randomly for simulations with six replicas) in cubic boxes with side dimension ranging from 4.9 nm (for the 1ZU6N simulation) to 7.0 nm (for 6ZU15N-A and 6ZU15N-B simulations) and solvated with a solution containing 70/30(v/v) MeOH/water ratio. The SPC model for water [58] and the MeOH model proposed by van Gunsteren and coworkers [59] were used. Following initial energy minimization and a 100 ps MD run during which the peptide atoms were position restrained, the temperature of solute and solvent was raised to 300 K in a stepwise manner, performing four MD runs, 50 ps each, at different temperatures (50, 100, 200, and 250 K). Pressure was kept constant (1 bar) using a Berendsen isotropic barostat (5 ps time constant) [60]. Electrostatic interactions were calculated by using the reaction field method [61], with a cutoff of 1.4 nm. van der Waals interactions were calculated using a cutoff radius (1.4 nm). A time-step of 2 fs was employed. In the simulations with six peptides, the minimum distances between all the possible 15 pairs of peptides were calculated considering all the atoms of each peptide, by

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Figure 1. Energy profile obtained by changing the χ (left) and ω (right) torsion angles of the methyl methylcarbamate. The chemical structure of methyl methylcarbamate is reported above the graphs, where χ and ω are also indicated.

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MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES using the g_mindist tool in the GROMACS suite package. To test the stability of the aggregate formed in the different simulations, for each one of the 15 minimum distance profiles, the root mean square fluctuations (RMSF) were calculated in the last 25 ns of the simulations. The secondary structures were assigned by means of dictionary of protein secondary structures (DSSP) [62]. In the 6ZU6N-A and 6ZU6N-B simulations, peptide molecules were considered in contact when the minimum distance between all the possible couples of atoms belonging to the two molecules falls below 0.4 nm (the contact was considered lost when this distance increases above 1 nm).

Results Spectroscopy The aggregation propensities of Z-U6-N and Z-U15-N in MeOH/ water solutions were characterized by UV absorption and fluorescence spectroscopies. The absorption spectra are dominated by the absorption bands of the N chromophore, i.e. the n → π* [λmax = 280 nm, ε(280) = 6100 M
1 cm
1] and the π → π* [λ = 220 nm, ε(220) = 70 000 M
1 cm
1] transitions. Figure 2A shows that in MeOH solution, the spectra of Z-U6-N and Z-U15-N almost overlap and reproduce the typical profile of the naphthyl monomer absorption. On the contrary, although the absorption spectrum of Z-U6-N in 70/30(v/v) MeOH/water solution (Figure 2B) maintains the monomer spectral features observed in pure MeOH, that of Z-U15-N appears to be strongly affected by water addition. In particular, a wide broadening and a significant reduced intensity of the π → π* transition can be observed. Furthermore, both the π → π* and n → π* absorption maxima are only slightly redshifted with respect to the wavelength maxima measured in MeOH, i.e. from 223.8 to 224.3 nm and from 281.3 to 281.7 nm, respectively. These results suggest the formation of N···N aggregates, driven by the combination of aromatic stacking interactions and hydrophobic effects, and triggered by the water addition to the MeOH solution. Interestingly, no significant variations of the measured absorption spectra were observed in 1 month aged solutions, indicating long-term stability of the Z-U6-N monomers and Z-U15-N aggregates in the aqueous solution investigated.

Steady-state fluorescence experiments provided further insights on the different propensity of the Z-U6-N and Z-U15-N peptides to aggregate in aqueous solutions. In agreement with the UV absorption results, also the fluorescence spectra of Z-U6-N and Z-U15-N in MeOH almost overlap and are typical of the naphthyl monomer emission (Figure 3A). In contrast, whereas Z-U6-N in 70/30(v/v) MeOH/water maintains the spectral features typical of the monomer emission, the fluorescence spectrum of Z-U15-N appears to be strongly redshifted and almost devoid of vibronic structure (Figure 3B). The redshifted component of the Z-U15-N emission band can be assigned to the formation of intermolecular excimer species, i.e. fluorescent excited-state complexes involving naphthyl groups at relatively close distances. Interestingly, the excitation spectra of Z-U15-N taken at λem = 340 and 400 nm were found to overlap the absorption spectrum, which suggests that the naphthyl groups involved in such interaction are separated by no less than 3–4 Å, the typical distance for the onset of short-range aromatic– aromatic interactions. Also in this case, the fluorescence emission spectra of aged (1 month) solutions of Z-U15-N did not show significant changes, confirming the stability of the peptide aggregates in MeOH/water solutions. Time-resolved fluorescence experiments also provided important insights on the aggregation process. In Figure 4, we report the lifetime distributions obtained from the experimental time decays of Z-U6-N and Z-U15-N in MeOH and 70/30(v/v) MeOH/ water, respectively. It can be easily seen that in MeOH solution, the lifetime distributions of the two peptides, both peaked at around 15 ns, almost perfectly overlap, which indicates that in this environment, the two compounds attain a similar distribution of conformers, typical of monomeric species. A very small long-time component peaked at 74 ns was also detected for Z-U15-N (Figure 4A), signaling the presence of a small fraction of excimer species. On the contrary, in 70/30(v/v) MeOH/water, whereas the Z-U6-N lifetime distribution shows essentially the same shape measured in pure MeOH, the time decays of Z-U15-N reveal a complex structure, indicative of the formation of heterogeneous aggregates, characterized by a relative large distribution of N···N distances and orientations. Multiple time decays have already been observed for excimers in organic polymeric glasses, where the short-time decay components have been associated to the blueshifted region of the emission band

J. Pept. Sci. 2014; 20: 494–507 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

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Figure 2. UV absorption spectra of Z-U6-N (solid line) and Z-U15-N (dotted line) in MeOH (A) and 70/30(v/v) MeOH/water (B) solutions. All absorption bands are normalized to unit areas.

BOCCHINFUSO ET AL.

Figure 3. Fluorescence spectra of Z-U6-N (solid line) and Z-U15-N (dotted line) in MeOH (A) and 70/30(v/v) MeOH/water (B) solution. The fluorescence emission bands are all normalized to unit areas.

Figure 4. Fluorescence lifetime distribution (λem = 340 nm) of Z-U6-N (solid line) and Z-U15-N (dotted line) in MeOH (A) and 70/30(v/v) MeOH/water (B) solution. Fluorescence lifetime distributions are all normalized to unit areas. In the inset (part B), the fluorescence lifetime distribution of Z-U15-N in 70/ 30(v/v) MeOH/water (λem = 400 nm).

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and the long-time tail to the redshifted region of the spectrum [63]. The former is characteristic of a sandwich-like arrangement of the aromatic moieties (H aggregates), whereas the relaxed (redshifted) component of the emission spectrum is usually assigned to parallel slipped-out stacked chromophores (J-aggregates) [20]. The number and width of the lifetime distributions testify on the number of different conformers, the heterogeneity of the local environment embedding the fluorophore, and the number of fluorescent species. This finding reflects a continuum of aromatic–aromatic interactions that gives rise to the formation of both ground-state and excited-state complexes (excimers). Interestingly, time-resolved experiments carried out on Z-U15-N in 70/30(v/v) MeOH/water at λem = 400 nm, i.e. selecting the redshifted component of the N emission spectrum, mainly ascribable to excimer-like species, exhibited a rather narrow-time distribution peaked at 73 ns (inset in Figure 4B). The position of this band matches the small long-time distribution found for Z-U15-N in MeOH (Figure 4A) and in 70/30(v/v) MeOH/water (Figure 4B) at λem = 340 nm. The evolution from an excimer-like (pure excited state complex) to a J-type emission (weakly bound fluorescent ground-state complex) is governed by the onset of short-range (3–4 Å) π–π interactions and the organization of the solvent molecules in proximity of the aromatic moieties (hydrophobic effect).

Clearly, the peptide secondary structure affects both these factors, by determining the relative positioning of the aromatic moieties and the surface exposed to solvent interactions. AFM Imaging Striking differences between Z-U6-N and Z-U15-N were revealed by AFM measurements carried out on dried peptide films obtained by overnight incubation of 70/30(v/v) MeOH/water micromolar peptide solutions on mica. In the case of the shorter peptide, micrometric globular structures were predominantly observed (Figure 5A). This morphology is characteristic of nonspecific aggregation of apolar molecules driven by hydrophobic effects. On the other hand, for Z-U15-N, micrometric filaments with a width of 100 ± 10 nm were almost exclusively found (Figure 5B). The different aggregation propensity of these peptides can be ascribed to the different structural and dynamic properties of the peptide backbone in the two cases and, in particular, to the stabilization of the helical conformations as the peptide chain is elongated and the onset of helix–helix interactions. To shed more light on these points and have a deeper understanding of the aggregation mechanism at the molecular level, we performed MD simulations.

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MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES

Figure 5. AFM imaging of Z-U6-N (A) and Z-U15-N (B) on a mica surface after 18 hs incubation of a 70/30(v/v) MeOH/water solution.

Molecular Dynamics Simulations Simulations of the peptide monomers To investigate the influence of the chain length on peptide secondary structure, in the conditions adopted in the experiments, we carried out MD simulations of monomers in 70/30(v/v) MeOH/water solutions for Z-U6-N (40 ns of simulation time), Z-U9-N and Z-U12-N (two simulations for each peptide, each 80 ns long), and Z-U15-N (30 ns). In all cases, the simulations started from an α-helical conformation. For Z-U9-N and Z-U12-N, two (and longer) simulations were performed because no simulations with peptide replicas were carried out. Figure 6 reports the DSSP analyses of these trajectories. Because the results of the Z-U9-N and Z-U12-N simulations are very similar, only one of them is shown. The DSSP analysis indicates that the α-helix is definitely the most populated conformation for Z-U9-N, Z-U12-N, and Z-U15-N (for these peptides, unfolding events during the simulation time were not registered). As a consequence, despite

the absence of chiral centers in the molecules, only P-helices (the starting conformation) were populated. In the cases of Z-U9-N and Z-U12-N, Figure 6 suggests the presence of sporadic molecules in partial (especially at the C-terminus) 310-helical conformations, but they did not exceed a few percentage of the total simulation time. On the other hand, the starting α-helical structure of Z-U6-N was lost after roughly 6 ns. The peptide populated mainly 310- and α-helices and, to a minor extent, β-turn conformations as well. Frequent switches between these conformations took place during the simulation. Overall, this peptide, even if it exhibited a marked preference for the helical structures, is much more flexible than its longer homologous. In Figure 7, the Ramachandran plots for the U residues 2, 3, 4, and 5, as obtained from the 1ZU6N simulation, are reported. Conformers populating the region corresponding to left-handed helices are present, particularly for residues 2 and 5. Although a full M-helical conformation was never populated during this (short) simulation, these data suggest that transitions between helices of different chirality are operative for Z-U6-N in the nanosecond timescale. This aspect will be further discussed in the next section.

Simulations of the peptide aggregates The spectroscopic data clearly indicated that the Z-U6-N and Z-U15-N peptides feature different aggregation properties. Furthermore, AFM measurements of peptides dried on the mica surface revealed the formation of fibrillar aggregates only for Z-U15-N (Figure 5). At the same time, MD simulations of monomers showed a different helix stability for the two peptides. To investigate how the different helix stability plays a role on the formation of mesoscopic structures, we studied the early stages of the aggregation process by simulating systems formed by six replicas of the Z-U6-N and Z-U15-N peptides. In parallel, to study the influence of the aromatic moieties present in our peptides, we also simulated the aggregation process for peptide U15, in which the terminal Z and N groups are removed. In the starting structures, six helical peptides were randomly placed in the simulation boxes (the so-called minimum bias approach). We have already shown that this approach is particularly useful to explore the conformational space in systems

J. Pept. Sci. 2014; 20: 494–507 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

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Figure 6. Peptide secondary structures as obtained by DSSP analyses of the simulations of Z-(U)n-N monomers. (A) 1ZU6N; (B) 1ZU9N-A; (C) 1ZU12N-A; (D) ZU15N.

BOCCHINFUSO ET AL.

Figure 7. Ramachandran plots as obtained from the simulations of Z-(U)6-N monomer for residues U(2), U(3), U(4), and U(5).

500

formed by different molecules for which no detailed structural information is available [64–67]. The inspection of the trajectories, besides confirming the different helix stabilities, also showed very different aggregation properties between the hexa- and pentadeca-U homooligopeptides (Figure 8). In fine agreement with the experimental results, the former compound did not exhibit any tendency to form stable aggregates. At the high concentration simulated, more than 350 contacts between couples of Z-U6-N molecules have been detected during the two simulations, but these contacts resulted very fleeting, i.e. only 28 of them lived for more than 10 ns (16 in the first simulation and 12 in the second one) and none more than 25 ns. Notably, these contacts did not

contribute to stabilize the helical structures, as suggested in the literature [32]. On the contrary, the contacts between molecules of the longer peptides, once occurred, became immediately stable and persisted for the remaining time of the simulations. This behavior resulted in the formation of stable aggregates in the simulation time, irrespectively from the presence or not of the aromatic groups. As an example, the steps leading to the aggregate in the 6ZU15N-A simulation are reported in Figure 9. In the first 5 ns of simulation, a disordered aggregate formed, which involved five of the six peptides. This aggregate became more and more ordered, and it incorporated also the sixth molecule. Roughly at 40 ns, it reached the final structure. More in general, all the obtained aggregates are characterized by a

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MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES

Figure 8. Stability of the aggregates formed. The graph reports the RMSF values calculated on the minimum distance profiles of all the 15 possible pairs of peptides, during the last 25 ns of the simulations: 6ZU6N-A (filled red squares), 6ZU6N-B (empty red squares), and 6ZU15N-A (filled blue circles), 6ZU15N-B (empty blue circles), 6U15-A (filled green triangles), and 6U15-B (empty green triangles). The numbers labeling the x-axis indicate the specific pair of molecules that the plotted RMSF values refer to.

highly regular packing of the peptides. In all four MD simulations, two planes were obtained, each one formed by three almost parallel peptides. The angle between these planes is not the same in the four simulations: In three cases, the planes are almost parallel (simulations 6ZU15N-B, 6U15-A, and 6U15-B), whereas in the fourth simulation (6ZU15N-A), they are perpendicular to each other. Figure 8 reports the RMSF values of the minimum distances between all of the possible couples of peptides calculated in the last 25 ns of the MD simulations. In the simulations with Z-U15-N and U15, the minimum distances between the different peptides were found to be essentially frozen in the timescale considered, and almost in all cases, an RMSF lower than 0.1 nm was obtained. Interestingly, no detectable differences

were observed between peptides containing or not containing the aromatic groups at the N- and C-termini. On the contrary, in the two simulations with Z-U6-N, stable aggregates did not form, and RMSF values for the minimum distances were measured in the range of 1 nm, one order of magnitude higher with respect to those obtained for Z-U15-N and U15. The latter compounds populated mainly α-helical secondary structures. Unfolding of peptides, and consequently, reversal of the helical chirality, was never observed in the four 100-ns-long simulations. Therefore, we considered that, under this point of view, it is more interesting to analyze the simulations with six molecules of Z-U6-N peptides, because of its greater flexibility (Figure 6) and also because no stable aggregates, which could influence the peptide conformations, formed during the simulations (Figure 8). As for the 1ZU6N simulation, we investigated the propensity to reverse helix chirality, favored in homo-U peptides by the absence of chiral centers. In the 40-ns-long monomer simulation, only reversals of chirality of single residues were observed (Figure 7). In this case, the presence of six molecules in the 6ZU6N-A and 6ZU6N-B simulations and the longer time of the MD simulations generated a better sampling of the conformational space. This procedure is feasible because (i) the many contacts between peptides registered during the simulations were quickly lost and (ii) each peptide seems to behave independently from the others for a large part of the simulations. To test the handedness of the helices formed, we measured the ϕ torsion angles of residues 3 and 4 (in the central region of the peptide). These angles assumed positive values (indicative of left-handed helices) in the 43% and 36% of the total time, for the 6ZU6N-A and 6ZU6N-B simulations, respectively, which suggest only a little bias of the starting (right-handed) conformation. Figure 10 illustrates the values of these angles in the 6ZU6N-A simulation for each peptide [a similar behavior was obtained from the 6ZU6N-B simulation (not shown)]. The ϕ angle was chosen because, being less flexible than the ψ angle, it is more diagnostic of stable changes of the helix chirality. For the same reason, only the values of the residues in the middle of the peptides were taken into account. Figure 10 shows that during the simulation, both ϕ angles assume many times positive values (regions highlighted in gray in the figure). In these tracts of the simulation, M-helices

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Figure 9. Aggregate formation during the 6ZU15N-A simulation. The snapshots are taken at different times during the simulation. The backbones of the peptides are represented as cyan ribbons, and the atoms of the aromatic groups at the N- and C-termini are reported as sticks (H white, C cyan, N blue, and O red).

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Figure 10. The ϕ angles (C(n
1)-Nn-C αn-Cn) of the third (red points) and fourth (blue points) residues for each one of the six peptides in the simulations 6ZU15N-A. In the regions where both angles assume positive values, the left-handed helices dominate (regions highlighted with a gray background). Three structures, extracted from the conformations sampled by the first peptide, are also shown on top. From the right: a right-handed α-helix (frame at 88 ns), a left-handed 310-helix (frame at 50 ns), and a mixed helix conformation (frame at 15 ns). The backbone is represented as a cyan ribbon, and the atoms of the aromatic groups at the N- and C-termini are reported as sticks (H white, C cyan, N blue, and O red).

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are populated, like that extracted at 50 ns and shown on top of the figure. During the simulation, both ϕ angles pass almost 40 times between the positive and negative regions or vice versa. Considering a total simulation time of 600 ns (a 100-ns-long simulation for each of the six peptides), the average transition time between P- and M-helices can be estimated in the range of a few tens of nanoseconds. By inspecting the trajectory during

these transitions, it is evident that most transitions proceed through a population (often for short time) of unfolded conformations. Furthermore, many times during the simulation, the two ϕ angles assume values of opposite sign. Here, conformations like that extracted at 15 ns and reported on top of Figure 10, in which single helical residues with different chirality occur, are populated.

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MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES

Figure 11. Distances between couples of N groups during the last 25 ns of the two simulations 6ZU15N-A and 6ZU15N-B (on the left). The relative distribution is also reported (in the center). On the right, the final structures obtained from the two simulations are also reported. The N groups are highlighted as red sticks. The arrows represent the couples taken into account (the color is the same used in the distance and distribution graphs for that couple).

Figure 8 already showed that the presence of aromatic groups is not necessary for the onset of aggregates. On the other hand, it is interesting to analyze the mobility of the aromatic groups, once the aggregates form. In particular, we focused on the behavior of the N group at the C-terminus, the features of which were tested by fluorescence experiments. For the 6ZU15N-A and 6ZU15N-B simulations, Figure 11 describes the distances between couples of N groups, which are on the same side in the aggregates. The distances are plotted in the last 25 ns of simulations, when the aggregates were already formed. The distributions of these distances are also reported. The trends of the distances reflect a significant mobility of the N groups in the timescale of tens of nanoseconds. Also when two groups became very close to each other, the contact were not stable for times longer than a few nanoseconds. In the figure, the two final structures obtained by the simulations are also reported. The main features of these structures, well representative of the two aggregates, are evident, i.e. the almost parallel arrangement assumed by nearest neighbor peptides, the two planes formed by three peptides each, and the different relative orientation between these planes in the two simulations.

Discussion

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In the last few years, fibril aggregates formed by peptide molecules received great attention by the chemical and biochemical communities. Initially, this observation originated from the role of β-sheet fibrils on the development of important pathologies (i.e. Alzheimer’s disease), but currently, it spans different fields

including potential applications as scaffolds in 3D cell culture and tissue engineering and as templates for the assembly of polymers and inorganic materials [21,68,69]. This wide spectrum is essentially due to the great flexibility of the peptide building blocks (amino acids), the most impressive lego bricks available in the nature. A deep understanding of the preliminary aggregation stages needed to form mesoscopic structures not only would give the possibility to define the rules of the game in fibril formation but also could shed further light on the first principles that govern protein folding. In this connection, here we investigated the aggregation properties of a family of homo-peptides constituted by the nonproteinogenic U amino acid, which is known to induce stabilization of helical structures. The peptides examined contain two aromatic groups, one benzyl and one N group at the N- and C-termini, respectively. It is well established that the process leading to fibrillization of the β-structured Aβs peptides is strongly dependent on the intermolecular interactions between the benzyl groups of the FF motif. Indeed, a relatively minor change in these two residues (i.e. an F to W mutation) has been reported to modify its aggregation propensity [70]. One of the goals of this work was to define the role of the aromatic–aromatic interactions (i.e. π–π) in these peptide systems. On the other hand, the presence of aromatic residues seems not to be crucial in the formation of fibrils formed by helically structured peptides. In this case, the key characteristic in the stabilization of the protofibrillar aggregates is the interaction between different side chains, which define specific structural patterns grafted on the helical scaffold. They are usually obtained by starting from the standard abcdefg motif, which consists of

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sequences of HPPHPPP residues (where H and P indicate hydrophobic and polar residues, respectively) [71,72]. The homo-U peptides investigated here form uniform helices and do not contain side-chain groups able to give rise to specific and complementary interactions. We will evaluate the driving force of almost-naked helices in the formation of aggregates in the mesoscopic scale. At first, we investigated these systems by means of spectroscopic techniques. In this connection, the presence of the N group at the C-terminus provided us the opportunity to also use fluorescence methods. The data summarized in Figures 2 and 3 show that for Z-U6-N, no interactions between the N chromophores take place, suggesting that these peptides do not aggregate under the conditions examined. On the other hand, for Z-U15-N, the changes in the UV and fluorescence spectra in 70/30(v/v) MeOH/water solution clearly indicate that the increased polarity and hydrophobic effects promote aggregation of these peptides. The different behavior of Z-U6-N and Z-U15-N also suggests a minor role for the aromatic–aromatic interactions in the aggregation process. The aromatic groups not only are present in both peptides but also are expected to play a more relevant role in the shorter compound. As a consequence, other effects seem to be more prominent in driving the aggregation process. The close chemical similarity between the two systems suggests that the different properties could come from 3D structural and/or dynamical differences between the two peptides induced by the different chain length. Previous studies on U homo-peptides underscore their strong tendency to adopt helical structures. In the crystalline state, they adopt a 310-helix structure, regardless of the chain length [34,37,38,40,42]. In solution, numerous data confirm their strong propensity to fold into helical structures. Furthermore, a certain degree of structural flexibility is evident, as reversals between P- and M-helices were observed by NMR [73–76]. Interestingly, the time of conversion between helices of different screw sense was found to depend on peptide length and solvent polarity. Unfortunately, these investigations were carried out under different conditions with respect to those employed in the present study. Therefore, it is important to investigate whether these chain-dependent differences are also retained in 70/30(v/v) MeOH/water solution. From the experimental point of view, the technique of choice used to study the secondary structure content of peptides in solution, i.e. CD spectroscopy, cannot be used because of the achiral nature of the U homo-peptides. Therefore, the conformational and dynamic features of these peptides in 70/30(v/v) MeOH/water solutions were examined by means of MD simulations. We started by simulating peptides with different chain length at infinite dilution (Figure 6). Our data show a marked discontinuity in the secondary structure stability between Z-U6-N and the longest three peptides. Z-U6-N is the only peptide for which transitions between different secondary structures were observed. Despite a clear preference for 310and α-helical conformations, it is evident that other structures are also frequently sampled. Furthermore, combining the results obtained from simulations with only one (Figures 6 and 7) and with six replicas of the peptide (Figure 10), we can reasonably predict that under these conditions transitions between P- and M-helices take place in the timescale of tens of nanosecond. Notably, helix screw-sense reversal is usually considered a twostate process [74]. However, in our simulations, intermediates with different conformations are significantly populated, often promoted by the initial change of the ψ torsion angle.

Concerning the time required between two consecutive transitions, it appears to be extraordinarily shorter (thus reflecting the high flexibility of Z-U6-N under these conditions) than the value measured for the unprotected U6 peptide in dichloromethane (tens of microseconds) [74]. For comparison, in the other systems investigated (Z-U9-N, Z-U12-N, and Z-U15-N), we did not observe any transition between the two different helical screw sense. In summary, the other systems were simulated for few microseconds (per single peptide, also considering the simulations with six replicas of the peptides containing 15 U residues), which indicates that reversal for these systems does require longer times. Apart from these marked differences in terms of flexibility, other variations between Z-U6-N and Z-U15-N are evident if one looks at the nature of the helices populated (Figure 6). For the hexapeptide, no preference between 310- and α-helices could be spotted. On the contrary, for the 15-residue-long peptide, the α-helical conformation largely predominates. This issue has been widely debated in the literature, in particular the role of α-helical structures for U homo-peptides [28,77,78]. The results reported here agree well with the general view that α-helices are favored by increasing solvent polarity and for peptides with longer chains. Notably, chemical groups present at the peptide termini can influence this equilibrium [46]. Although we have not conclusive data in this respect, we observed that in the 3% of the simulation time of Z-U15-N, the amide hydrogen of residue 3 or 4 is located at distances lower than 0.4 nm from a phenyl ring, suggesting that a sort of modest capping effect could take place. This effect should stabilize the N–H not involved in intramolecular H-bonds near the N-terminus, and in turn, it is expected to favor the α-helical conformation. Despite a bias from the conditions we used in the simulations cannot be completely ruled out, in particular the choice of the force field [79–83] and, in a few simulations, the simultaneous presence of six peptides in the box, we can reasonably conclude that Z-U6-N differs from its longer homologous peptides for its conformational and dynamic features even in very diluted solutions. To investigate the relation between these differences and the diverging propensity to aggregate, we performed MD simulations with six replicas of the peptide (Figures 8–11). During the MD simulations, Z-U6-N retains the flexibility shown with only one molecule. Moreover, the occurrence of sporadic interactions with other peptide molecules does not induce any tendency to stabilize the helical structures. The final result of this flexibility is that Z-U6-N does not form stable aggregates. On the contrary, both Z-U15-N and U15, which populate very stable α-helical structures, form stable aggregates after a few tens of nanoseconds, in good agreement with the experimental results. Therefore, these data suggest a close relationship between the intrinsic helical stability and the ability to form aggregates. Interestingly, no differences in the aggregation propensity are evident between peptides containing or not containing aromatic groups. These findings confirm that the presence of the aromatic group is not a precondition for aggregation of the peptides. Furthermore, when these groups are present and stable aggregates form (like for Z-U15-N), it seems that the aromatic moieties are unable to establish persistent interactions between them (Figure 11). This result is coherent with the broad time decay distribution of N in 70/30(v/v) MeOH/water solution (Figure 4B), which indicates that numerous conformers are accessible. Our MD results suggest that the main driving force in fibril formation observed for Z-U15-N is not due to specific interactions,

wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 494–507

MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES such as those between aromatic groups for the Aβs peptides or between complementary side chains in coiled-coil aggregates. On the contrary, in our pentadecapeptide, aggregation is the consequence of hydrophobic interactions between very stable helices. Obviously, these interactions are amplified by increasing solvent polarity (Figures 2 and 3). The absence of specific interactions generates more compact and ordered aggregates (Figure 11) than those proposed for other types of fibrils formed by helix coiled-coil helix or β-sheet motifs. In our aggregates, the helical peptides are almost parallel, making the formation of fibrils formed by overlapped planes possible, in which the peptides are perpendicular to the fibrils axis. This unusual superstructure, already proposed for other fibrils on the basis of helical peptides [68,84], could explain the bigger diameters measured for our fibrils (100 ± 10 nm) (Figure 5), as compared with the few tens of nanometers reported in the literature.

Conclusions In this paper, we report the results of our computational and spectroscopic studies on the conformational and aggregation properties of U homo-peptides with different chain length and aromatic protecting groups at the N- and C-termini. Our findings indicate that the peptide with only six U residues, because of its higher intrinsic flexibility, is not able to form stable aggregates in solution. On the other hand, the peptide with 15 U residues forms highly regular and compact aggregates in 70/30(v/v) MeOH/water solution and, if deposited on a mica surface, gives rise to micrometer fibrils with a large diameter (100 nm). The mechanism leading to fibrils is peculiar of this peptide. We have shown that it is guided by hydrophobic interactions between peptide molecules in helical conformation. The known fibrillization mechanisms are not applicable in this case, as specific interactions between amino acid side chains are not operative with U homo-peptides and a major contribution from the aromatic groups is very unlikely. Acknowledgements The authors gratefully acknowledge the Italian Ministry of Education University and Research (MIUR) for the financial support and CINECA for the computational resources. Part of this work was supported by MIUR with the grant PRIN 2010-2011 No. 2010FM738P.

References

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1 Aleman C, Bianco A, Venanzi M. (Eds). Peptide Materials: From Nanostructures to Applications, Wiley: Chichester, UK,2013. 2 Looger LL, Dwyer MA, Smith JJ, Hellinga HW. Computational design of receptor and sensor proteins with novel functions. Nature 2003; 423: 185–190. 3 Yasutomi S, Morita T, Imanishi Y, Kimura S. A molecular photodiode system that can switch photocurrent direction. Science 2004; 304: 1944–1947. 4 Gatto E, Caruso M, Porchetta A, Toniolo C, Formaggio F, Crisma M, Venanzi M. Photocurrent generation through peptide-based selfassembled monolayers on a gold surface: antenna and junction effects. J. Pept. Sci. 2011; 17: 124–131. 5 Shah RN, Shah NA, Lim MMDR, Hsieh C, Nuber G, Stupp SI. Supramolecular design of self-assembling nanofibers for cartilage regeneration. Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 3293–3298. 6 Hamley IW. Self-assembly of amphiphilic peptides. Soft Matter. 2011; 7: 4122–4138. 7 Zhang S. Fabrication of novel biomaterials through molecular selfassembly. Nat. Biotechnol. 2003; 212: 1171–1178.

8 Loo Y, Zhang S, Hauser CA. From short peptides to nanofibers to macromolecular assemblies in biomedicine. Biotechnol. Adv. 2012; 30: 593–603. 9 Hamley IW. Peptide fibrillization. Angew. Chem. Int. Ed. 2007; 46: 8128–8147. 10 Harper JD, Lansbury PT. Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem. 1997; 66: 385–407. 11 Caughey B, Lansbury PT. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 2003; 26: 267–298. 12 Wood SJ, Wetzel R, Martin JD, Hurle MR. Prolines and amylodoigenicity in fragments of the Alzheimer’s peptideβ/A4. Biochemistry 1995; 34: 724–730. 13 Findeis MA, Musso GM, Arico-Muendel CC, Benjamin HW, Hundal AM, Lee JJ, Chin J, Kelley M, Wakefield J, Hayward NJ, Molineaux SM. Modified-peptide inhibitors of amyloid β-peptide polymerization. Biochemistry 1999; 38: 6791–6800. 14 Gordon DJ, Sciarretta KL, Meredith SC. Inhibition of β-amyloid(40) fibrillogenesis and disassembly of β-amyloid(40) fibrils by short β-amyloid congeners containing N-methyl amino acids at alternate residues. Biochemistry 2001; 40: 8237–8245. 15 Fernandez-Escamilla AM, Rousseau F, Schymkowitz J, Serrano L. Prediction and sequence-dependent effects on the aggregation of peptides and proteins. Nat. Biotechnol. 2004; 22: 1302–1306. 16 Rousseau F, Schymkowitz J, Serrano L. Protein aggregation and amyloidosis: confusion of the kinds? Curr. Opin. Struct. Biol. 2006; 16: 118–126. 17 Lansbury PT, Lashuel HAA. Century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 2006; 443: 774–779. 18 Tu L-H, Raleigh DP. Role of aromatic interactions in amyloid formation by islet amyloid polypeptide. Biochemistry 2013; 52: 333–342. 19 Lakshmanan A, Cheong DW, Accardo A, Di Fabrizio E, Riekel C, Hauser CAE. Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis. Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 519–524. 20 Caruso M, Gatto E, Placidi E, Ballano G, Formaggio F, Toniolo C, Zanuy D, Alemán C, Venanzi M. A single-residue substitution inhibits fibrillization of Ala-based pentapeptides. A spectroscopic and molecular dynamics investigation. Soft Matter 2014; 10: 2508–2519. 21 Zhang S. Emerging biological materials through molecular selfassembly. Biotechnol. Adv. 2002; 20: 321–339. 22 Apostolidou M, Jayasinghe SA, Langen R. Structure of helical membrane-bound human islet amyloid polypeptide and its implications for membrane-mediated misfolding. J. Biol. Chem. 2008; 283: 17205–17210. 23 Abedini A, Raleigh DP. A role for helical intermediates in amyloid formation by natively unfolded polypeptides? Phys. Biol. 2009; 6: 015005. 24 Hauser CAE, Deng R, Mishra A, Loo Y, Khoe U, Zhuang F, Cheong DW, Accardo A, Sullivan MB, Riekel C, Ying JY, Hauser UA. Natural tri- to hexapeptides self-assemble in water to amyloid β-type fiber aggregates by unexpected α-helical intermediate structures. Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 1361–1366. 25 Caruso M, Placidi E, Gatto E, Mazzuca C, Stella L, Bocchinfuso G, Palleschi A, Formaggio F, Toniolo C, Venanzi M. Fibrils or globules? Tuning the morphology of peptide aggregates from helical building blocks. J. Phys. Chem. B 2013; 117: 5448–5459. 26 Burgess AW, Leach SJ. An obligatory α-helical amino acid residue. Biopolymers 1973; 12: 2599–2605. 27 Paterson Y, Rumsey SM, Benedetti E, Némethy G, Scheraga HA, Sensitivity of polypeptide conformation to geometry. Theoretical conformational analysis of oligomers of α-aminoisobutyric acid. J. Am. Chem. Soc. 1981; 103: 2947–2955. 28 Zhang L, Hermans J. 310-Helix versus α-helix: a molecular dynamics study of conformational preferences of Aib and alanine. J. Am. Chem. Soc. 1994; 116: 11915–11921. 29 Wang Y, Kuczera K. Multidimensional conformational free energy surface exploration: helical states of Alan and Aibn peptides. J. Phys. Chem. B 1997; 101: 5205–5213. 30 Improta R, Barone V, Kudin KN, Scuseria GE, Structure and conformational behavior of biopolymers by density functional calculations employing periodic boundary conditions. I. The case of polyglycine, polyalanine, and poly-α-aminoisobutyric acid in vacuo. J. Am. Chem. Soc. 2001; 123: 3311–3322.

BOCCHINFUSO ET AL.

506

31 Improta R, Antonello S, Formaggio F, Maran F, Rega N, Barone V. Understanding electron transfer across negatively-charged Aib oligopeptides. J. Phys. Chem. B 2005; 109: 1023–1033. 32 Kameda T, Takada S. Secondary structure provides a template for the folding of nearby polypeptides. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17765–17770. 33 Grubišić S, Brancato G, Barone V. An improved AMBER force field for α,α-dialkylated peptides: intrinsic and solvent-induced conformational preferences of model systems. Phys. Chem. Chem. Phys. 2013; 15: 17395–17407. 34 Shamala N, Nagaraj R, Balaram P. The 310-helical conformation of a pentapeptide containing α-aminoisobutyric acid (Aib): X-ray crystal structure of Tos–(Aib)5–OMe. J. Chem. Soc., Chem. Commun. 1978; 996–997. DOI is 10.1039/C39780000996 35 Toniolo C, Bonora GM, Barone V, Bavoso A, Benedetti E, Di Blasio B, Grimaldi P, Lelj F, Pavone V, Pedone C. Conformation of pleionomers of α-aminoisobutyric acid. Macromolecules 1985; 18: 895–902. 36 Karle IL, Balaram P. Structural characteristics of α-helical peptide molecules containing Aib residues. Biochemistry 1990; 29: 6747–6756. 37 Pavone V, di Blasio B, Santini A, Benedetti E, Pedone C, Toniolo C Crisma M The longest, regular polypeptide 310-helix at atomic resolution. J. Mol. Biol. 1990; 214: 633–635. 38 Toniolo C, Crisma M, Bonora GM, Benedetti E, Dl Blasio B, Pavone V, Pedone C, Santini A. Preferred conformation of the terminally blocked (Aib)10 homo-oligopeptide: a long, regular 310-helix. Biopolymers 1991; 31: 129–138. 39 Toniolo C, Benedetti E. Structures of polypeptides from α-amino acids disubstituted at the α-carbon. Macromolecules 1991; 24: 4004–4009. 40 Toniolo C, Crisma M, Formaggio F Peggion C Control of peptide conformation by the Thorpe-Ingold effect (Cα-tetrasubstitution). Biopolymers 2001; 60: 396–419. 41 Bellanda M, Peggion E, Burgi R, van Gunsteren WF, Mammi S. Conformational study of an Aib-rich peptide in DMSO by NMR. J. Peptide Res. 2001; 57: 97–106. 42 Gessmann R, Brückner H, Petratos K. Three complete turns of a 310-helix at atomic resolution: the crystal structure of Z-(Aib)11OtBu. J. Pept. Sci. 2003; 9: 753–762. 43 Crisma M, Formaggio F, Moretto A, Toniolo C. Peptide helices based on α-amino acids. Biopolymers (Pept. Sci.) 2006; 84: 3–12. 44 Maekawa H, Formaggio F, Toniolo C Ge NH Onset of 310-helical secondary structure in Aib oligopeptides probed by coherent 2D IR spectroscopy. J. Am. Chem. Soc. 2008; 130: 6556–6566. 45 Brown RA, Marcelli T, De Poli M, Solà J, Clayden J. Induction of unexpected left-handed helicity by an N-terminal L-amino acid in an otherwise achiral peptide chain. Angew. Chem. Int. Ed. 2012; 51: 1395–1399. 46 Ceccacci F, Mancini G, Rossi P, Scrimin P, Sorrenti A, Tecilla P. Deracemization and the first CD spectrum of a 310-helical peptide made of achiral α-aminoisobutyric acid residues in a chiral membrane mimetic environment. Chem. Commun. 2013; 49: 10133–10135. 47 Aleman C, Subirana JA, Perez JJ. A molecular mechanical study of the structure of poly(α-aminoisobutyric acid). Biopolymers 1992; 32: 621–631. 48 Smythe ML, Huston SE Marshall GR The molten helix: effects of solvation on the α- to 310-helical transition. J. Am. Chem. Soc. 1995; 117: 5445–5452. 49 Bellanda M, Mammi S, Geremia S, Demitri N, Randaccio L, Broxterman QB, Kaptein B, Pengo P, Pasquato L, Scrimin P. Solvent polarity α controls the helical conformation of short peptides rich in C tetrasubstituted amino acids. Chem. Eur. J. 2007; 13: 407–416. 50 Crisma M, Saviano M, Moretto A, Broxterman QB, Kaptein B, Toniolo C. Peptide α/310-helix dimorphism in the crystal state. J. Am. Chem. Soc. 2007; 129: 15471–15473. 51 Breed J, Kerr ID, Sankararamakrishnan R, Sansom MSP. Packing interactions of Aib-containing helices: molecular modeling of parallel dimers of simple hydrophobic helices and of alamethicin. Biopolymers 1995; 35: 639–655. 52 Pispisa B, Stella L, Venanzi M, Palleschi A, Viappiani C, Polese A, Formaggio F, Toniolo C. Quenching mechanisms in bichromophoric, 310-helical Aib-based peptides, modulated by chain length dependent topologies. Macromolecules 2000; 33: 906–915. 53 van der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC. GROMACS: fast, flexible and free. J. Comp. Chem. 2005; 26: 1701–1718. 54 Oostenbrinck C, Soares TA, van der Veght NF, van Gusteren WF. Validation of the 53A6 GROMOS force field. Eur. Biophys. J. 2005; 34: 273–284.

55 Chiessi E, Branca M, Palleschi A, Pispisa B. Copper(II) complexes immobilized on a polymeric matrix. thermodynamics, spectroscopy, and molecular modeling. Inorg. Chem. 1995; 34: 2600–2609. 56 Allen FH. The Cambridge Structural Database: a quarter of a million crystal structures and rising. Acta Crystallogr. B 2002; 58: 380–388. 57 Martinelli S, Torreri P, Tinti M, Stella L, Bocchinfuso G, Flex E, Grottesi A, Ceccarini M, Palleschi A, Cesareni G, Castagnoli L, Petrucci TC, Gelb BD, Tartaglia M. Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes. Hum. Mol. Genet. 2008; 17: 2018–2029. 58 Berendsen HJC, Postma JM, van Gunsteren WF Hermans J. Interaction models for water in relation to protein hydration. In Intermolecular Forces, (ed). Reidel Publishing Company: Dordrecht, The Netherlands,1981; 331–342. 59 Walser R, Mark AE, van Gunsteren WF, Lauterbach M, Wipff G. The effect of force-field parameters on properties of liquids: parametrization of a simple three-site model for methanol. J. Chem. Phys. 2000; 112: 10450. 60 Berendsen HJC, Postma JPM, van Gunsteren WF, Di Nola A, Haak JR. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984; 81: 3684–3690. 61 Tironi IG, Sperb R, Smith PE, van Gunsteren WF. A generalized reaction field method for molecular dynamics simulations. J. Chem. Phys. 1995; 102: 5451–5459. 62 Kabsch W, Sander C. Dictionary of protein secondary structure. Pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 1983; 22: 2577–2637. 63 Winnik FM. Photophysics of preassociated pyrenes in aqueous polymer solutions and in other organized media. Chem. Rev. 1993; 93: 587–614. 64 Orioni B, Bocchinfuso G, Kim JY, Palleschi A, Grande G, Bobone S, Venanzi M, Park Y, Kim JI, Hahm KS, Stella L. Membrane perturbation by the antimicrobial peptide PMAP-23: a fluorescence and molecular dynamics study. Biochim. Biophys. Acta, Biomembranes 2009; 1788: 1523–1533. 65 Bocchinfuso G, Palleschi A, Orioni B, Grande G, Formaggio F, Toniolo C, Park Y, Hahm KS, Stella L. Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations. J. Pept. Sci. 2009; 15: 550–558. 66 Bocchinfuso G, Bobone S, Mazzuca C, Palleschi A, Stella L. Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides. Cell. Mol. Life Sci. 2011; 68: 2281–2301. 67 Bobone S, Bocchinfuso G, Park Y, Palleschi A, Hahm KS, Stella L. The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5. J. Pept. Sci. 2013; 19: 758–769. 68 Woolfson DN, Ryadnov MG. Peptide-based fibrous biomaterials: some things old, new and borrowed. Curr. Opin. Chem. Biol. 2006; 10: 559–567. 69 Banwell EF, Abelardo ES, Adams DJ, Birchall MA, Corrigan A, Donald AM, Kirkland M, Serpell LC, Butler MF, Woolfson DN. Rational design and application of responsive α-helical peptide hydrogels. Nat. Mater. 2009; 8: 596–600. 70 Xie L, Luo Y, Wei G. Aβ(16–22) Peptides can assemble into ordered β-barrels and bilayer β-sheets, while substitution of phenylalanine 19 by tryptophan increases the population of disordered aggregates. J. Phys. Chem. B 2013; 117: 10149–10160. 71 Woolfson DN. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 2005; 70: 79–112. 72 Dong H, Paramonov SE, Hartgerink JD. Self-assembly of αhelical coiled coil nanofibers. J. Am. Chem. Soc. 2008; 30: 13691–13695. 73 Hummel RP, Toniolo C, Jung G. Conformational transitions between enantiomeric 310-helices. Angew. Chem. Int. Ed. Engl. 1987; 26: 1150–1152. 74 Kubasik M, Kotz J, Szabo C, Furlong T, Stace J. Helix–helix interconversion rates of short 13C-labeled helical peptides as measured by dynamic NMR spectroscopy. Biopolymers 2005; 78: 87–95. 75 Kubasik M, Blom A. Acceleration of short helical peptide conformational dynamics by trifluoroethanol in an organic solvent. Chem. Bio. Chem. 2005; 6: 1187–1190.

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MOLECULAR DYNAMICS SIMULATION ON THE AGGREGATION OF AIB HOMO-PEPTIDES 76 Solà J, Morris GA, Clayden J. Measuring screw-sense preference in a helical oligomer by comparison of 13C NMR signal separation at slow and fast exchange. J. Am. Chem. Soc. 2011; 133: 3712–3715. 77 Basu G, Kitao A, Hirata F, Go N. A collective motion description of the 310-/α-helix transition: implications for a natural reaction coordinate. J. Am. Chem. Soc. 1994; 116: 6307–6315. 78 Carlotto S, Cimino P, Zerbetto M, Franco L, Corvaja C, Crisma M, Formaggio F, Toniolo C, Polimeno A, Barone V. Unraveling solventdriven equilibria between α- and 310-helices through an integrated spin labeling and computational approach. J. Am. Chem. Soc. 2007; 129: 11248–11258. 79 Todorova N, Legge FS, Treutlein H, Yarovsky I. Systematic comparison of empirical force fields for molecular dynamics simulation of insulin. J. Phys. Chem. B 2008; 112: 11137–11146.

80 Matthes D, de Groot BL. Secondary structure propensities in peptide folding simulations: a systematic comparison of molecular mechanics interaction schemes. Biophys. J. 2009; 97: 599–608. 81 Patapati KK, Glykos NM. Three force fields’ views of the 310-helix. Biophys. J. 2011; 101: 1766–1771. 82 Li J, Lakshminarayanan R, Bai Y, Liu S, Zhou L, Pervushin K, Verma C, Beuerman RW. Molecular dynamics simulations of a new branched antimicrobial peptide: a comparison of force fields. J. Chem. Phys. 2012; 137: 215101. 83 Cao Z, Liu L, Zhao L, Li H, Wang J. Comparison of the structural charac2+ teristics of Cu -bound and unbound α-syn12 peptide obtained in simulations using different force fields. J. Mol. Model. 2013; 19: 1237–1250. 84 Lazar KL, Miller-Auer H, Getz GS, Orgel JP, Meredith SC. Helix-turnhelix peptides that form α-helical fibrils: turn sequences drive fibril structure. Biochemistry 2005; 44: 12681–12689.

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