Tuning the Morphology of Nanostructured Peptide Films by Introduction of a Secondary Structure Conformational Constraint: a Case-Study of Hierarchical Self-Assembly

Peptide self-assembly is ubiquitous in nature. It governs the organization of proteins, controlling their folding kinetics and preserving their structure stability and bioactivity. In this connection, model oligopeptides may give important insights on the molecular mechanisms and elementary forces driving the formation of supramolecular structures. In this contribution we show that a single residue substitution, i.e. Aib (α-aminoisobutyric acid) in place of Ala at position 4 of an -(L-Ala) 5 -homo-oligomer, strongly alters the aggregation process. In particular, this process is initiated by the formation of small peptide clusters that promote aggregation at the nanometer scale and, through a hierarchical self-assembly, lead to mesoscopic structures of micrometric dimensions. Furthermore, we show that the use of the well-established Langmuir-Blodgett technique represent an effective strategy for coating extended areas of inorganic substrates by densely-packed peptide layers, thus paving the way for application of peptide films as templates for biomineralization, biocompatible coating of surfaces, and scaffolds for tissue engineering.


INTRODUCTION
Formation of peptide nano-and mesoscopic structures of different morphology (globules, fibrils, nanotubes) is currently under intense investigation for its relevance in biomedicine (development of peptide-based materials for tissue engineering, drug-delivery, antimicrobial coating, etc.), and in bio-inspired nanotechnology (hybrid materials for optoelectronics, nanocatalysis, biosensing, etc.). [1][2][3][4][5][6] Peptide self-assembly depends primarily on the structural and dynamical properties of the peptide building blocks, dictated by their amino acid composition and sequence. Electrostatic, van der Waals, and H-bonding interactions, as well as solvation effects, determine the secondary structure attained by the peptide molecules, and, hence, through hierarchical self-assembly (HSA), their organization into nano-and mesoscopic architectures. 7 Environmental conditions (ionic forces, local dielectric constant, pH) and systemic effects (phase segregation) also play an important role. HSA, i.e. the propagation of the structural properties of the peptide building blocks to the mesoscale, has been highlighted in a series of seminal contributions. [8][9][10][11] A distinctive feature of HSA is represented by the role of aromatic moieties, which, by specific stacking interactions, rule the ordered arrangement of the molecular units giving rise to superstructures of a given morphology. 12 Depending on the amino acid sequence and length of the main chain, a polypeptide can attain several ordered conformations, the most common being the canonical α-helix and β-sheet. Other helical structures (310-, 2.05-helices) are also possible, favored by the presence of particular residues or specific peptide motifs. 13 In this connection, we recently described the unique conformational properties of the αaminoisobutyric acid, i.e. a C α,α -dimethylated -amino acid denoted as Aib or U in the three-or single-letter code, respectively. 14 By use of molecular dynamics simulations it was shown that -(Aib)n-homo-oligopeptides predominantly adopt a 310-helix structure for the shorter components of the series (n≤8), evolving to a more stable α-helix for the longer terms. 15 The Aib β-sheet breaker properties were also recognized, encouraging studies related to the possible utilization of Aibcontaining peptides as pro-drugs for neurodegenerative diseases. [16][17][18] We recently proved that a single Aib substitution at position 4 of an -(L-Ala)n-homo-pentapeptide strongly altered its conformational landscape and the morphology of the aggregates formed in aqueous solutions. 19 A 2-pyrenyl (Py) group, functionalizing both peptides at their N-terminus, was added to the peptide chain to investigate the role of an aromatic moiety in promoting peptide aggregation and to serve as an intrinsic fluorescent probe. We were able to show that aggregation in methanol/water solutions was mainly driven by the hydrophobic effect, but that the morphology of the mesoscopic peptide aggregates strongly depended on the secondary structure attained by the peptide building blocks. In particular, while the -(L-Ala)5-homo-pentapeptide gave rise to amyloidlike fibrillar structures of nanometric thickness and micrometric length, the Aib-substituted peptide formed almost exclusively micrometric globules.
In this contribution, we used the Langmuir-Blodgett (LB) technique on the same pentapeptide and its Aib-analog previously investigated in methanol/water solutions, to analyze how the different conformational features of the two compounds would affect the aggregation process at the air/water (a/w) interface and the final morphology of the mesoscopic aggregates.
Ultrathin films of organic molecules can be easily obtained by well-established LB methodologies, by which a highly diluted solution of amphiphilic molecules in a volatile solvent is spread on the water sub-phase. 20 Once the solvent is evaporated, the surface pressure is increased by closure of the mobile barriers of the trough, forcing the molecules to interact. During the compression of the mobile barriers, the organic layer at the a/w interface undergoes several phase transitions, until a 2D-ordered film is formed. Eventually, at high surface pressures, the system collapses to a so-called solid phase (S), characterized by formation of 3D aggregates. 21 Onset of the film at the a/w surface is controlled by monitoring the surface pressure (π) as a function of the mean area occupied per molecule (Mma). The nature and extent of the intermolecular interactions determine the LB film morphology at intermediate π values and the structure of the supramolecular aggregates at high surface pressures. In addition, the π/Mma isotherms allow one to establish the most suitable conditions for depositing the organic layer on a solid substrate as a dense and homogeneous film, paving the way for applications.
It should be noted that peptide aggregation at the a/w interface has recently been studied for mimicking the toxic interaction between amyloid peptides and lipid membranes. 22 In particular, IR absorption spectroscopy measurements of LB films revealed that amyloid peptides at the a/w interface can adopt a β-sheet conformation, i.e. the precursor of peptide fibrillation. 23 In the same connection, it has been also demonstrated that amphiphilic peptides, i.e. peptides with alternating apolar and polar residues producing amyloid-like fibers in solution, can form β-sheet monolayers at the a/w interface. 24,25 Therefore, investigation of peptide aggregation at the a/w interface may offer significant information on the activity of amyloids at the lipid surface. On the other hand, from the point of view of hybrid materials nanotechnology, LB methods can be applied to control the morphology of peptide films coating extended areas of inorganic substrates. These two aspects are the principal source of inspiration for this work.

MATERIALS
The synthesis and characterization of the two pentapeptide analogs investigated, denoted in the following as PyA5 and PyA3UA, have already been published elsewhere. 19 Molecular formulas and acronyms are reported in Schematic 1 for clarity.

MOLECULAR DYNAMICS
Molecular dynamics (MD) simulations for PyA5 and PyA3UA monolayers at the a/w interface were performed using the software GROMACS 4.6.7 27 and the GROMOS53a6 force field. 28 To this aim, two symmetrical monolayers, each formed by 16 peptide molecules, were created in a 9x9x27 nm 3 simulation box. The two peptide monolayers were separated by a 3-nm thick slab comprising 8715 water molecules (SPC model). 29 A 27 nm long (z) dimension was taken into account to eliminate interactions between the periodic images of the system. The peptides, initially attaining an extended conformation, were positioned in the simulation box in a perpendicular arrangement with respect to the water surface. Energy minimization was therefore carried out by a two-step procedure, minimizing the energy of the solvent molecules at first and then the solute energy, before starting with the MD simulations. The solvent was equilibrated using a 150 ps MD at 50 K, while the 16 peptide molecules were restrained. The system was gradually heated to 300 K through a 1 ns MD simulation before the 50 ns production run. The equilibration step, the heating step, and the production simulation were carried out using a 2 fs time step, the PME algorithm 30 for electrostatics with a double cut-off at 1.4 nm for van der Waals interactions, and the Berendsen algorithm 31 for both temperature (coupling constant: τT = 0.2 ps) and pressure (τP = 1 ps, semiisotropic conditions) couplings.
The topology needed for the 2-methylpyrenyl group was obtained from the Automated Topology Builder and Repository, 32 while the topology for Aib was developed using the Ala parameters as reference. 15 Both peptides were capped at the C-and N-termini with a tert-butoxyl (OtBu) and a 2carboxymethylpyrenyl group, respectively.

PEPTIDE FILM FORMATION AT THE AIR/WATER INTERFACE
The aggregation process at the a/w interface of the two pentapeptides was monitored by recording the  vs. Mma LB isotherms ( Figure 1). It can be seen that, while those obtained by adding  It has been suggested that horizontally oriented helices partially changed their orientation to vertically oriented upon compression in the plateau region of the isotherm. 33 Alternatively, it can be hypothesized that the floating film at first forms a multilayer structure or small peptide clusters, evolving to mesoscopic aggregates upon further compression. 34 It should be noted that for all the added volumes, the onset of the LE→LC phase transition occurs at much lower Mma values for PyA5 than for PyA3UA. These results strongly suggest that PyA5 can be easily packed as a dense film at the a/w interface, giving rise to the same compact structure independently of the peptide concentration on the water surface.
In the case of PyA5, a fast rising of the surface pressure with lowering of Mma is observed, until at π higher than 35 mN/m a steep rise of the LB isotherm takes place, signaling the 3D collapse of the film (solid phase, S) at π =65 mN/m. At this surface pressure, the Mma value is less than 10 Å 2 /molecule, indicating the formation of 3D peptide aggregates. On the basis of these results, we decided to deposit the PyA5 film on the solid substrate at π = 30 mN/m, i.e. at the end of the LE→LC phase transition.
In the case of PyA3UA, in the region of the LB isotherm comprised between 80 and 40 Å 2 /molecule, and depending on peptide concentration, a definitely slower increase of the surface pressure is seen, with a characteristic inflection of the curve, suggesting that, in a certain surface  These results indicate that PyA5 aggregation at the a/w interface can be considered a quasireversible process, following the same aggregation pathway at each compression/expansion cycle, and, most likely, attaining the same compact structure. The remarkable hysteresis observed in the case of PyA3UA is suggestive of irreversible changes caused by compression of the peptide film, probably due to formation of heterogeneous 3D aggregates. 35 Further differences are made evident by considering the variation of the surface compressibility modulus of the two compounds during formation of the peptide films. The surface compressibility factor (K) is defined as (1) Basically, the higher the value of K, the tighter is the lateral packing of the molecules. The K vs.
Mma isotherms of PyA5 and PyA3UA are reported as Supporting Information ( Figure S1).

SPECTROSCOPIC STUDIES OF PEPTIDE FILMS ON QUARTZ
Mono-and multilayer films of PyA5 and PyA3UA were deposited by the LB technique on a hydrophilic quartz substrate for spectroscopic studies at π = 30 and 15 mN/m, respectively.

FLUORESCENCE EXPERIMENTS
Steady-state fluorescence studies were carried out on PyA5 and PyA3UA multilayers immobilized by LB deposition on a quartz substrate (Figure 3). Interestingly, the spectrum of PyA5 is dominated by the excimer emission, characteristic of an ordered array of stacked pyrene chromophores. 36 Notably, the excimer emission intensity steadily increases as the number of peptide layers increases, suggesting an ordered stratification of the PyA5 layers on the solid substrate for successive LB emersion/immersion steps.
It should be noted that such excimer emission was found to be definitely less intense in the fluorescence spectrum of PyA5 in methanol/water solutions under aggregating conditions (see for  On the contrary, the pyrene emission in PyA3UA is characterized by a monomer-like spectrum, with only a minor contribution from the excimer species. In addition, the emission intensity changes randomly for the PyA3UA films formed through a different number of LB emersion/immersion steps, pointing to a not homogenous coating of the substrate.

FTIR-ATR ABSORPTION SPECTROSCOPY
FTIR-ATR absorption measurements were carried out on LB films of PyA5 and PyA3UA to obtain information on the secondary structure adopted by the peptide building blocks immobilized in the multilayer coating the quartz/ZnSe substrate. In the case of the -(L-Ala)5-homo-pentapeptide, the FTIR-ATR spectrum ( Figure 4A) shows very sharp absorption bands centered at 3300 cm -1 (N-H stretching), 1620 cm -1 (amide I), and 1530 cm -1 (amide II). These spectral regions are close to those already reported for β-sheet forming (L-Ala)n n = 5-7 sequences with different terminals in the solid state. 37,38 The FTIR-ATR absorption bands of the PyA3UA LB film ( Figure 4B) appear definitely broader than those obtained for the PyA5 film, indicative of a higher conformational heterogeneity in the former.
The Amide I band is widely used to analyze the peptide conformational properties, as different secondary structures give rise to characteristic absorption bands in this region. Typically, the αhelix absorption band is observed in the 1650-1660 cm -1 range, while absorption bands in the 1615-1627 cm -1 range are ascribed to intermolecular β-sheets. 310-helices and turns are generally observed at higher energies, i.e. at 1660-1670 and 1680-1690 cm -1 , respectively. 39 Deconvolution of the Amide I absorption band shows that for PyA5, the component centered at 1626 cm -1 largely predominates, the other components observed at 1650 and 1685 cm -1 accounting for less than 10% of the Amide I total area (Supporting Information, Figure S2). On the contrary, for PyA3UA, several components of comparable intensities, centered at 1627 (β sheet), 1650 (α-helix), 1668 (310helix) and 1687 cm -1 (β-turn), were detected by deconvolution of the FTIR absorption spectrum in the same region. Besides that, in the amide A region, an absorption band (≈3440 cm -1 ) typical of free NH groups, i.e. NH groups not involved in H-bonding interactions, was also measured, confirming the heterogeneous conformational landscape of the Aib-substituted pentapeptide. 40 Spectroscopic data, therefore, suggest that, while PyA3UA adopts multiple conformations, causing some heterogeneity of the LB film morphology, PyA5 forms well-ordered β-sheet domains under the surface pressure conditions of LB deposition. These findings are of some interest because βsheet ladders are well-known to nucleate the growth of fibrils and micrometric filaments, precursor of the formation of the large amyloid plaques responsible for several neurodegenerative diseases.

AFM IMAGING OF PEPTIDE FILMS
PyA5 and PyA3UA LB monolayers were deposited on mica for AFM characterization of the peptide film morphology with nanometric resolution. A gallery of AFM images obtained for a PyA5 monolayer deposited at π=30 mN/m on mica are reported in Figures 5 and 6. Interestingly, Figure 5A shows the presence of both micrometric globular structures and long peptide fibers of nanometric thickness. Figure 5B, taken in a different region of the same specimen, exhibits a densely-packed array of peptide fibers, the details of which is shown with higher resolution in Figure 5C. AFM height profiles, taken along the long white line section in Figure 5C, and reported in Figure 5D, reveal that the imaged peptide fibers have thicknesses ranging from 10 to 30 nm and micrometric lengths.
The AFM images reported in Figure 5 also show that the packing of the peptide fibers occurs with a remarkable orientation order, as a result of both attractive interactions between the peptide fibers and the compression exerted by the mobile barriers of the LB equipment. Recently, we have shown that PyA5 in water/methanol solutions forms amyloid-like aggregates because of the combined effect of hydrophobic interactions, the ordered secondary structure of the peptide chains, and the J-type stacking interactions between the Py groups. 19 In that case, AFM imaging of PyA5 dried on mica after overnight deposition from a methanol/water solution under aggregative conditions only showed spaghetti-like structures, i.e. intertwined micrometric filaments without apparent ordering (see Figure 8 in Ref. 19).
A very interesting feature, giving some insight into the process of fiber growth and formation, is represented by the AFM images reported in Figure 6, where, like pearls in a necklace, nanometric peptide globules are chained by micrometric filaments, indicative that peptide fibers grow directly from globular structures. It should be considered that formation of fibers requires the asymmetric growth of peptide aggregates and that the conformational properties of the peptide strongly affect the formation of the small peptide clusters seeding the aggregation process. Interestingly, in a kinetic study of protein aggregation carried out by super-resolution fluorescence techniques, it was found that globular species, first observed after two hours, continued to be seen after longer times in apparent coexistence with fibers and fiber assemblies, suggesting the continuous growth of fibers upon addition of protein monomers. 41 In the AFM experiments carried out on PyA3UA LB films immobilized on mica, only micrometric globular structures could be imaged, as evident from Figure 7. In particular, neither mature

MOLECULAR DYNAMICS SIMULATIONS
MD calculations were carried out to investigate formation of the small peptide clusters nucleating the aggregation process of the two peptide analogs under study. In Figure 8   PyA5 (blue) and PyA3UA (red) molecules at the a/w interface.

DISCUSSION
The results reported above demonstrate that, like hydrostatic pressure in solution, 43 the surface pressure can be used for modulating the aggregation process at the a/w interface of self-assembling peptides.
The hysteresis observed in the PyA3UA compression/expansion isotherm cycles strongly suggests formation of 3D structures at relatively low surface pressures. 22check Such hysteresis was not observed in the case of PyA5, indicating that the peptide secondary structure strongly affects the aggregation process, and specifically the LE-LC transition and the collapse to the solid-like phase. This view is reinforced by the AFM imaging results, that revealed definitely different morphologies of the mesoscopic structures formed by the two pentapeptide analogs. The link between the subnanometer scale of the secondary structure attained by the single peptide chains and the morphology of the micrometric structures formed by peptide aggregates indicates the hierarchical nature of peptide self-assembly.
In the case of PyA5, the hierarchy develops at the sub-nanometer scale through stacking of the Py aromatic groups and β-sheet-like alignment of the peptide chains, at the nanometer scale by ordering within small peptide fibrils, and at the micrometer scale by the intertwining of the fibrils into peptide fibers.
The globular structure of PyA3UA is determined at the mesoscopic scale by the predominant role of hydrophobic interaction, and, at the sub-nanometer scale by the conformational constraint of the Aib residue that inhibited the regular arrangement of Aib-substituted peptide chains in a β-sheet array, precursor of peptide fibrillation. These differences are clearly illustrated by the MD simulations, which for PyA5 show that aromatic interactions promote the ordered stacking of the Py groups, and hence formation of structured peptide clusters that seed the directional growth of the peptide fibers.
A significant difference in the aggregation process of the two peptide analogs at the macroscale of the LB experiment is the collapse of the PyA5 films to a solid (3D) phase, that occurs at surface pressures definitely higher (>60 mN/m) than those observed for PyA3UA (<30 mN/m). This finding strongly suggests that PyA5 is tightly packed in layered β-sheet structures, as confirmed by FTIR-ATR measurements. AFM images clearly showed that PyA5 fibrils can tightly pack to form amyloid plaque-like clumps.
The morphological differences between the micrometric structures formed by PyA5 and PyA3UA, highlighted by the AFM measurements, are undoubtedly striking and reminiscent of the analogous structures obtained driving the peptide aggregation in solution by the hydrophobic effect. More specifically, fibrillation was observed only for PyA5, while the Aib-substituted pentapeptide formed globular structures only. 19 However, it should be stressed that the densely-packed layers obtained for PyA5 are a specific product of the LB methodology. Interestingly, Maltseva and Brezesinski 44 reported that the surface fibrillation rate is much faster than the rates of fibril formation observed in solution, with nucleation occurring at a much lower concentration. Our conclusion is that surface pressure can be used to modulate the morphology of self-assembling peptides and to obtain ordered arrays of densely-packed peptide fibers over extended areas.

CONCLUSION
LB experiments, fluorescence and IR absorption spectroscopy results, AFM imaging data, and MD simulations, all substantiate the same conclusion, namely that peptide aggregation is a hierarchical process, so that the morphology of the micrometric structures is dictated by the secondary structure properties of the peptide building blocks.
The results described here open two interesting perspectives. On the biomedical side, as studies on neurodegenerative diseases point to a toxic interaction between amyloid peptides and lipid membranes, 45 the evidence that a single Aib substitution can inhibit formation of peptide fibrils at the a/w interface could represent a promising approach toward the development of therapeutic peptides against the insurgence of those diseases.
On the bionanotechnology side, we showed that the LB methodology represents an excellent strategy for the preparation of peptide nano-and mesostructures coating extended areas of hydrophilic substrates. Therefore, these results may find application for the production of biocompatible platforms for biomineralization, antimicrobial coating of devices, and tissue engineering.