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Solvent Controlled Structural Transition of KI4K Self-Assemblies: from Nanotubes to Nanofibrils Yurong Zhao,† Li Deng,† Jiqian Wang,† Hai Xu,*,† and Jian R. Lu*,‡ †

Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China ‡ Biological Physics Group, School of Physics and Astronomy, The University of Manchester, Manchester M13 9PL, United Kingdom S Supporting Information *

ABSTRACT: The structural modulation of peptide and protein assemblies under well-controlled conditions is of both fundamental and practical significance. In spite of extensive studies, it remains hugely challenging to tune the self-assembled nanostructures in a controllable manner because the self-assembly processes are dictated by various noncovalent interactions and their interplay. We report here how to manipulate the self-assembly of a designed, symmetric amphiphilic peptide (KI4K) via the solvent-controlled structural transition. Structural transition processes were carefully followed by the combination of transmission electronic microscopy (TEM), atomic force microscopy (AFM), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and small angle neutron scattering (SANS). The results show that the introduction of acetonitrile into water significantly affected the hydrophobic interactions among hydrophobic side chains while imposing little impact on the β-sheet hydrogen bonding between peptide backbones. A structural transition occurred from nanotubes to helical/twisted ribbons and then to thin fibrils with the addition of acetonitrile due to the reduced hydrophobic interactions and the consequent weakening of the lateral stacking between KI4K β-sheets. The increased intermolecular electrostatic repulsions among lysine side chain amino groups had little effect on the lateral stacking of KI4K β-sheets due to the molecular symmetry. Complementary molecular dynamic (MD) simulations also indicated the solvation of acetonitrile molecules into the hydrophobic domains weakening the coherence between the neighboring sheets.

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INTRODUCTION Controlled by the combined effect of various noncovalent interactions, peptide self-assembly gives rise to a variety of supramolecular architectures such as vesicles,1,2 nanofibrils,3−5 nanoribbons,6−8 helices,9−11 nanotubes,12−15 and films.16 Due to their biological origin and well-defined nanostructures, nanomaterials based on peptide self-assembly have shown unique biological and physicochemical properties, and therefore hold great promise in many biomedical and biotechnological applications.17−22 To enhance the mechanistic understanding of peptide selfassembly and develop technological applications, more and more studies have been devoted to the structural and morphological transitions of self-assembled architectures. In this aspect, one of the widely used strategies is to change the primary molecular structures of peptides, including terminal modification, sequence variation, and length variation.23−27 For example, the morphological transition from nanotubes to vesicles has been induced by changing aromatic dipeptide FF (F: phenylalanine) to Phg-Phg (Phg: phenylglycine) or introducing a thiol group to its N-terminal.23 Cui et al. have demonstrated that the sequence variations of isomeric tetrapeptide amphiphiles lead to different one-dimensional (1D) nanostructures.25 By increasing the length of the central motif (B) of multidomain ABA peptides, which consists of alternating hydrophobic and hydrophilic residues, Dong et al. © 2015 American Chemical Society

have successfully tuned their self-assembling propensity and self-assembled nanostructures.26 The rationale underlying these investigations is to tune the contributions and interplay of different noncovalent forces involved in peptide self-assembly, such as hydrogen bonding, electrostatic interactions, and hydrophobic/aromatic stacking interactions. Among various self-assembled peptide nanostructures, 1D nanostructures are particularly attractive for several reasons: (1) their entanglement/cross-linking readily leads to the formation of hydrogels (3D scaffolds) applicable to cell culture and cell/ drug encapsulation;20,28,29 (2) they can manipulate or template the fabrication of complex 1D nanostructures of metals and semiconductors under benign conditions, which is of significance for the development of nanoscale electronics, sensors, and plasmonics;30−32 (3) they can serve as a versatile model for the mechanistic investigation of amyloid fibrils due to their similar structures (β-sheet) and morphologies (1D). For most 1D self-assembled peptide nanostructures, peptides adopt β-sheet structures and it is mostly the hydrogen bonding between peptide backbones that drives monomers to pack longitudinally into β-sheets.26,33−35 Along the perpendicular direction of β-sheets, attractions between amino acid side Received: June 23, 2015 Revised: November 3, 2015 Published: November 5, 2015 12975

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Sample Preparation. KI4K showed high solubility in water and the mixture of acetonitrile/water. The peptide solutions were obtained by directly dissolving the KI4K fluffy powder in the solvents at a concentration of 16 mM and then incubating for 7 days at room temperature for instrumental characterizations. CD. CD measurements were performed on a MOS-450 spectrometer (Biologic, France) under ambient conditions. During the measurements, the relative higher peptide concentration of 16 mM produced CD signals too strong for detection even using a 0.1 mm path-length quartz cuvette. Instead, a small volume of peptide solution was sandwiched between two flat quartz slides and a very thin layer was thus formed for the measurements. Note that all the tested peptide solutions were viscous and the thin layers formed remained unchanged during our measurements for a constant path-length. The spectra were recorded at room temperature with wavelengths ranging from 190 to 250 nm and a scan speed of 50 nm min−1. The Xe lamp was used as the light resource and the bandwidth was set to 0.5 nm. By using the Biokine software package, the solvent background was subtracted and the spectra were smoothed. The CD signals obtained in these measurements were expressed as millidegree rather than [θ] (deg cm2 dmol−1) versus wavelength and each spectral profile was the average of at least six scans. FTIR. FTIR experiments were performed on a Nicolet 6700 FT-IR spectrometer equipped with a DGTS detector in absorbance mode. In order to eliminate the interference of H2O to the amide I band, samples for this experiment were prepared by dissolving the peptide in D2O or the mixture of D2O and acetonitrile. The peptide solutions were also incubated for 7 days at room temperature before further characterizations. The CaF2 cell with 0.1 mm spacer was used for the measurements. For each measurement, the spectra were collected from 4000 to 400 cm−1 with resolution of 4 cm−1 and 128 scans were collected to increase the signal-to-noise ratio. By using the OMINC software (v 3.0, Nicolet), the background was subtracted and the spectra could be smoothed. Cryo-TEM. Samples for cryo-TEM characterization were prepared in a controlled environment vitrification system (CEVS). The aged peptide solution with a small volume of about 5 μL was dropped onto the TEM copper grid coated with a laced support film and then wiped away with the filter paper on both sides for about 3 s. The resulting thin film suspended on the mesh holes was quickly plunged into a reservoir of liquid ethane (cooled by the nitrogen) at −165 °C. Then, the vitrified samples were stored in the liquid nitrogen before transferring to the sample holder (Gatan 626) and examined at about −174 °C on a JEOL JEM-1400 TEM with the accelerating voltage of 120 kV. Negative-Staining TEM. TEM micrographs were recorded on a JEOL JEM-2100 UHR electron microscope with an accelerating voltage of 200 kV. For TEM sample preparation, the aged peptide solution with a small volume of about 5 μL was dropped onto the TEM copper grid coated with a carbon support film and allowed to adsorb for about 1 min before excess peptide solution was wiped away by the filter paper. Then the grid was negatively stained with uranyl acetate (2%, w/v) aqueous solution for about 1 min and excess solution was removed with a filter paper. AFM. AFM measurements were performed on a commercial Nanoscope IVa MultiMode AFM (Digital Instruments, Santa Barbara, CA) in tapping mode. Samples for AFM characterization were prepared by dropping a small volume (∼10 μL) of aged peptide solution onto freshly cleaved mica surface and allowed to adsorb for about 30 s before rinsing with water. The resulting surface was gently dried with nitrogen gas. The scan rate was set to 1.50 Hz with the scan angle at 0°. All images were flattened by using a first-order line fit (the flatten function in AFM software) to correct for piezo-derived differences between scan lines. SANS. SANS measurements were performed on LOQ, ISIS Neutron Facility, Rutherford Appleton Laboratory (Oxford, UK). The samples for the experiment were prepared by directly dissolving the peptide in D2O or its mixtures with CD3CN and incubated for 7 days at room temperature. Disc-shaped silica cells with a 2.0 mm path length were used for the measurements. Neutron incident wavelengths

chains promote their lateral stacking, which is however constrained by the repulsive forces between them and the natural twisting of the β-sheets themselves.6 These interactions, geometrical constraints, and their interplay dictate the final selfassembled morphologies, and our aim is to explore how to harness these interactions through peptide design and environmental conditions to achieve better structural and morphological controls. Accordingly, we have recently designed a series of short amphiphilic peptides such as I3K, I4K, I4K2, and KI4K (I-isoleucine, K-Lysine).34−36 Due to the high hydrophobicity and β-sheet forming propensity of isoleucine, these designed peptides undergo self-assembly in aqueous solution to form various 1D nanostructures. Among them, the bola-like peptide KI4K with the two charged lysine residues at the two termini displays unique self-assembled characteristics in aqueous solution by forming long and wide nanotubes with diameters over the range of 80−160 nm, suggesting significant lateral stacking of β-sheets.35 Relative to varying the molecular structures of peptides, physical treatments, and changing solution conditions such as ultrasound, variations in pH, ionic strength, and solvent polarity, as well as the introduction of organic dye molecules, are simpler and less expensive means in tuning peptide secondary structures and self-assembled nanostructures, particularly useful for practical applications.6,26,37−41 Because the lateral stacking of β-sheets of the KI4K self-assembly in water is primarily caused by the hydrophobic contact of isoleucine side chains, and the electrostatic repulsion between β-sheets has little impact due to the symmetric distribution of the two charged K residues along the peptide backbone, here we introduce an organic solvent acetonitrile into the KI4K/water system to regulate the intermolecular hydrophobic interactions and the self-assembled nanostructures. Acetonitrile can decrease the polarity of the solvent and weaken the hydrophobic interactions involved. Previous studies have demonstrated its successful dissolution of hydrophobic amyloid peptides to facilitate further investigations into their selfassembly processes.42−44 Through combining experimental characterizations (cryogenic transmission electron microscopy (cryo-TEM), negative-staining TEM, small angle neutron scattering (SANS), circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR)) with computer simulations, this work aims to outline the main features of the structural and morphological transitions upon introducing acetonitrile in the self-assembly system. The mechanistic insight will also benefit further research to exploit their technological applications.

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EXPERIMENTAL SECTION

Materials. Peptide KI4K was synthesized on a CEM Liberty microwave synthesizer by using the standard Fmoc solid phase synthesis strategy. Its C-terminus was amidated by using the Rink amide resin and its N-terminus was capped with acetic anhydride before the cleavage from resin. The details for the peptide synthesis and purification procedures have been given in our previous work.34−36 The final product was assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF) and reversed-phase high-performance liquid chromatograph (RP-HPLC), indicating its high purity (∼98%). The materials for peptide synthesis were purchased from GL Biochem Ltd. (Shanghai) and used as received. Deuterated acetonitrile (CD3CN), D2O, and other chemicals were purchased from Sigma-Aldrich and used as received. Water used in all experiments was processed with a Millipore Milli-Q system (18.2 MΩ cm). 12976

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Langmuir were from 2.2 to 10.0 Å at 25 Hz. The 64 cm2 detector with 5 mm resolution was placed at a distance of 4.05 m from the samples, producing a wave vector (q) ranging from 0.006 to 0.24 Å−1. The collected data were corrected for the wavelength dependence of the measured sample transmission, the incident spectrum, and relative detector efficiencies before subtracting the solvent background (D2O or the D2O/CD3CN mixtures). Absolute scaling was obtained by comparing the scattering with a partially deuterated polystyrene standard. All data fitting were performed by using SansView 2.1.1 program provided by RAL. In the lamellar model, the scattering intensity I(q) can be approximately expressed as I(q) ≈ 2π(Δρ)2 φδ /q2 ∝ q−2

(1)

where δ is the bilayer thickness, φ is the volume fraction, and Δρ is the difference of scattering length densities of the lamellar layers and the surrounding solvent.45,46 In the hollow cylinder model, P(q) is given in eq 2, where (scale) is a scale factor and H is half of the length of the cylinder. More information about the model is described in ref 47.

P(q) = (scale)Vshell(Δρ)2 2 1/2

(1 − x )

∫0

1

Ψ2[q , R shell(1 − x 2)1/2 , R core

⎡ sin(qHx) ⎤2 ⎥ dx ]⎢ ⎣ qHx ⎦

(2)

Simulation Details. All-atomistic simulations were performed on a KI4K protofibirl in water, acetonitrile/water (molar ratio, 1:1), and acetontirile, by using GROMACS software.48 The peptide and acetonitrile were modeled according to the OPLS force field,49−51 and the water was modeled according to the TIP4P force field.52 The KI4K proto-fibril is composed of four cross-β sheets, each of which contains six β-strands. The cross-β sheet structure was constructed based on the optimal trimer structure in our previous REMD simulation.53 For the system neutralization, the Cl− has been added in the systems containing water. In all the simulations, the systems were first subjected to an energy minimization process by using the steepest decent optimization method. Then, the solvent was relaxed for 0.5 ns in the NPT ensemble with peptide molecules restricted. Finally, 10 ns simulation has been conducted for each system with peptide molecules released at a constant temperature of 300 K and a constant pressure of 1 atm. The van der Waals (VDW) and electrostatic interactions were treated with the cutoff method and the particle-mesh-Ewald method,54 respectively, and the cutoff of VDW interactions and the electrostatic interactions in real space was set to 1.2 nm. The time step in simulation was 1 fs and 1000 instantaneous conformations were evenly sampled for equilibrium runs.

Figure 1. (A−C) Negative-staining TEM images and (D−F) cryoTEM images of the self-assembled nanostructures formed by KI4K in the mixture of acetonitrile and water with different acetonitrile contents: (A, D) 0%, (B, E) 20% (v/v), and (C, F) 80% (v/v). Note that the peptide concentration was fixed at 16 mM and peptide solutions were incubated for 7 days prior to experimental characterizations.

ribbons keeps constant while their width increases until the formation of closed nanotubes. In the latter, it is the width of the ribbons that remains constant while the pitch shortens to heal during structural transition.55 In addition to the dominant helical ribbons, some twisted ribbons with a negative Gaussian, saddle-like curvature were also observed (white arrow, Figure 1B). Increasing acetonitrile content to higher ratios led to a more dramatic effect and thin fibrils became dominant. For example, we observed a large population of weakly twisted fibrils with a width of some 10 nm and lengths on the order of micrometers in 80% (v/v) acetonitrile (Figure 1C). Note that the peptide concentration was fixed at 16 mM during the solvent-controlled morphological transition. In order to avoid the possible effects of artifacts caused by sample preparations (e.g., staining and drying) during the negative-staining TEM characterizations, we also performed cryo-TEM imaging. As shown in Figure 1D−F, the same nanostructures still prevailed in this case, suggesting that they did not result from artifacts. The tubular feature of the KI4K assemblies in water was readily identified by the pairing of continuous dark edge lines along the assemblies (Figure 1D), and the widths determined from cryo-TEM were comparable with those from negative-staining TEM. The formation of KI4K

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RESULTS Self-Assembled Morphologies. As shown in Figure 1A, the negative-staining TEM imaging revealed that KI4K underwent self-assembly in water to form nanotubes with diameters mostly in the range of 80−160 nm and lengths on the order of micrometers, consistent with previous observations.35 At relatively low acetonitrile concentrations such as 10% (v/v), the introduction of acetonitrile into water had little effect on the self-assembled nanostructures, and long KI4K nanotubes still dominated (Figure S1). With the acetonitrile content further increasing, we observed a morphological transition from nanotubes to helical ribbons. In water with 20% (v/v) acetonitrile, long helical ribbons with cylindrical curvature were predominant and their widths were mostly in the range of 30−90 nm (Figure 1B). Helical ribbons have been widely perceived as the precursor of nanotubes in peptide selfassembly, and the evolution from helical ribbons to nanotubes was suggested to proceed via the “growth width model” or “closing pitch model”.55−57 In the former, the helical pitch of 12977

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nanotube wall is composed of the KI4K monolayer. As for the sample with 20% (v/v) acetonitrile in water, helical ribbons with heights of some 2.1 nm dominated (Figure 2B), and their widths were also mostly in the range of 30−90 nm (Figure 2B), consistent with the values from TEM. In 80% (v/v) acetonitrile, twisted fibrils were widely observed in the AFM imaging and their heights were some 10 nm, in good agreement with the diameters determined from TEM (Figure 2C). It is evident from the above TEM and AFM characterizations that the introduction of acetonitrile into water significantly affected the KI4K self-assembled nanostructures. With increasing acetonitrile ratio, the self-assembled nanostructures of KI4K underwent morphological transitions between different 1D nanostructures, i.e., from wide nanotubes to helical nanoribbons and then to thin nanofibrils, accompanied by decrease in their diameters or widths. Acetonitrile is a polar aprotic solvent with hydrogen bonding ability (HBA) similar to water but its polarity index is clearly lower.44,58 Methanol has a polarity index similar to that of acetonitrile and its introduction into water also induced a morphological transition similar to acetonitrile, although methanol is a polar protic solvent. As shown in Figure S2, we observed a large population of fibrils with widths of some 10 nm and lengths on the order of micrometers in 80% (v/v) methanol or pure methanol as solvent. Secondary Structures. β-Sheet hydrogen bonding between peptide backbones usually favors the axial growth of peptide assemblies.26,33,34 In spite of different morphologies caused by the addition of acetonitrile, KI4K molecules within these different 1D self-assembled architectures adopt similar secondary structures. As shown in Figure 3A, the CD spectra of 16 mM KI4K in different solvents all exhibited a negative band at about 217 nm and a positive maximum at around 198 nm, indicating the formation of β-sheet secondary structures.59,60 In good agreement with the CD results, FTIR spectra displayed a dominant amide I band around 1620 cm−1 (Figure 3B), also characteristic of β-sheet conformations.61,62 Shen et al. have observed the same degree of β-sheet hydrogen bonding of Aβ(1−39) upon the introduction of 35% (v/v) acetonitrile into water.42 This may be ascribed to similar HBA values of acetonitrile (0.40) and water (0.47),44,58 and the acetonitrile addition would have little influence on the peptide−peptide and peptide−water hydrogen bonds. Layered Nanostructures. In order to gain more structural information on the self-assembled architectures at shorter length scales (several to tens of nanometers), SANS measurements were performed. For all the SANS profiles (Figure 4), their intensity (I) versus wave vector (q) showed an approximate −2 dependence at low q, i.e., I ∝ q−2, suggesting that the fundamental unit of all the KI4K assemblies is the same, i.e., lamellae.6,35,63 The SANS profiles in water and 20% (v/v) acetonitrile were similar over the whole measured q range. Further analysis indicated that a hollow cylinder model plus a lamellar model fitted the profile of KI4K in water well. Because the diameters of the nanotubes were at a relatively larger length scale (∼100 nm) and cannot be determined unambiguously through SANS, we fixed them in the range of 80−160 nm during the fitting, consistent with the TEM measurements. As a result, the wall thicknesses of 1.9−2.4 nm for the hollow cylinders and the lamellar thicknesses of 1.8−2.3 nm were obtained (Figure 4 and Table S1). These values are comparable with those from AFM and all are close to the molecular length of KI4K in the extended conformation, indicating again that the

helical ribbons in water containing 20% (v/v) acetonitrile can be confirmed by the occurrence of discontinuous dark lines (red arrows, Figure 1E), which correspond to the folded edges of helical ribbons. Similarly, twisted ribbons were also observed in this case (white arrows, Figure 1E). Note that the helical ribbons were wider than the twisted ones (Figure 1B and E). For the sample in 80% (v/v) acetonitrile, cryo-TEM imaging showed a network of thin and flexible fibrils (Figure 1F). As a complementary technique, AFM is capable of providing more detailed structural information. In addition to the dominant nanotubes formed by KI4K in water, nanotubes with helical markings, fragmented nanotubes with clear openings, and short ribbons were also observed from AFM imaging (Figure 2A). Some of these structural features could be

Figure 2. AFM height images of the assembled nanostructures by KI4K in the mixtures of acetonitrile and water with different acetonitrile contents: (A) 0, (B) 20% (v/v), and (C) 80% (v/v). Note that the peptide concentration was fixed at 16 mM and peptide solutions were incubated for 7 days before further experimental characterization.

caused by either the interference of the substrate (mica) or the sample preparation procedures (water rinsing and N2 drying) during the AFM characterization, or a combination of both. Furthermore, the confinement of the nanotubes to the mica surface could cause the nanotubes to deform, leading to their apparent widening. The height measurement from AFM could be useful. The wall thickness of the KI4K nanotubes was found to be 2.1 nm (inset in Figure 2A), close to the molecular length of KI4K in the extended conformation, suggesting that the 12978

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Figure 3. (A) CD and (B) FTIR spectra of KI4K in the mixtures of acetonitrile/water with different acetonitrile contents (0%, 20%, and 80%, vol/ vol). To avoid interference from H2O to the amide peak region, D2O was used instead in the FTIR measurements. Note that the peptide concentration was fixed at 16 mM and peptide solutions were incubated for 7 days before the measurements.

the values measured from TEM and AFM, but the latter was difficult to determine by using TEM and AFM, possibly due to their resolution limit. Note also that both sizes were very sensitive in the data fitting, similar to the cases in water or 20% (v/v) acetonitrile. In spite of different morphologies and diameters/widths of the KI4K assemblies in different solvents, the SANS measurements revealed the same fundamental unit, i.e., the KI4K monolayer under different solvent compositions. Computer Simulations. In order to qualitatively reveal the acetonitrile effect on the KI4K self-assembly at much shorter length scales, we conducted all atomistic simulations with 10 ns trajectory of a constructed KI 4K proto-fibril in three representative systems, i.e., water, acetonitrile, and acetonitrile/water (1:1, molar ratio). The proto-fibril is composed of four cross-β sheets, each of which contains six β-strands (Figure 5), and can be regarded as a simple prototype of KI4K monolayer assemblies. Because the time scale is too large to study the formation process of the proto-fibril with 24 peptide molecules from a random disorder state by using all atomistic simulation, we mainly considered the solvent effect on the proto-fibril’s stability. Figure 5 shows the snapshots of the proto-fibril with time in different solvent environments. In spite of the twisting of each cross-β sheet of the proto-fibrils observed in different solvents, the β-sheet structuring between strands remained stable with time in all cases, indicating that acetonitrile does not interrupt the hydrogen bonding between neighboring strands in these cross-β sheets. The twisting is expected as a result of the inherent chirality of amino acids. However, we can see that there existed different dynamics for the lateral stacking between β-sheets in different solvents. By calculating the distance of the mass centers of neighboring sheets, we found that the distances between sheet1 and sheet2, sheet2 and sheet3, and sheet3 and sheet4 in water decreased from 1.332, 1.350, and 1.360 nm at the initial configuration to 1.267, 1.246, and 1.258 nm at 10 ns configuration, respectively. Note that the sheet order was labeled from the top to bottom of the proto-fibril (Figure 5). These variations in the mass center distance indicated that the lateral stacking structure of βsheets in water became slightly tighter with time. In contrast, the proto-fibril in acetonitrile underwent a large deformation and there existed obvious separation between sheet1 and sheet2 and between sheet3 and sheet4 with time. The mass center distance between sheet1 and sheet2 increased from 1.350 nm at the initial configuration to 1.854 nm at 10 ns configuration, and

Figure 4. SANS profiles measured from 16 mM KI4K in the mixtures of water (D2O) and acetonitrile (CD3CN) with different acetonitrile ratios after 7 days of incubation. The solid lines are the best fits with models and parameters described in the text and Table S1. The measured profiles for the samples with different acetonitrile contents of 0%, 20% (v/v), and 80% (v/v) were represented by circle (○), square (□), and inverted triangle (▽), respectively.

nanotube wall is composed of the KI4K monolayer. Based on the TEM and AFM results, a lamellar model with a thickness of 2.0 ± 0.3 nm was found to fit the profile in 20% (v/v) acetonitrile (Figure 4 and Table S1), indicating that the helical ribbons are also composed of the KI4K monolayer. Note that during the above fitting, the wall and ribbon thicknesses were very sensitive and a slight variation (more than 0.3 nm) in them would lead to an obvious deviation of the fitting curves from the measured ones. These SANS results, together with the above TEM and AFM results, reveal that the helical ribbons are the precursor of the nanotubes, and during the structural transitions from nanotubes to helical ribbons or vice versa, there is little change in their layered structure. However, when the acetonitrile content was further increased to 80% (v/v), the SANS profile of KI4K changed significantly and an obvious convex shape appeared at high q values (Figure 4). In this case, a single hollow cylinder model rather than a lamellar model fitted the profile well, producing an outer radius of 4.2 ± 0.2 nm and a core radius of 2.1 ± 0.3 nm (Figure 4 and Table S1), suggesting that the observed fibrils are thin tubular ones with a wall thickness of some 2.1 nm. The former is in agreement with 12979

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Figure 6. Total number of hydrophobic contacts for the KI4K protofibril in different solvents (ACN denotes acetonitrile). The hydrophobic contact number was calculated by counting the contact number of isoleucine side chains.

water and acetonitrile, and acetonitrile, respectively. These results are consistent with the above snapshots, because the sheet separation in the mixture of water and acetonitrile or acetonitrile will decrease the number of hydrophobic contacts. The variation in the hydrophobic contact number is helpful in understanding the microscopic origin of the above weakening effect. Relative to water, acetonitrile has a nonpolar methyl group, and as a result, it tends to interact with the isoleucine side chain and forces acetonitrile to go into the hydrophobic core formed between two sheets. The solvation in the hydrophobic cores drives the neighboring sheets to separate.

Figure 5. Snapshots of the KI4K proto-fibril and the interfacial distributions of water and acetonitrile (abbreviated as ACN in simulations) molecules with time under different solution conditions. The proto-fibril is composed of four cross-β sheets, each of which contains six β-strands. In water, little sheet separation was observed and water molecules tented to escape from the hydrophobic core between two sheets. In ACN and ACN/water mixture (1:1, molar ratio), obvious sheet separation was observed and solvent molecules tended to approach the hydrophobic cores.

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DISCUSSION Self-assembly of peptides is usually a hierarchical process. For β-sheet self-assembling peptides, hydrogen bonding between peptide backbones drives them to pack longitudinally into βsheets, and the attractions among amino acid side chains promote the lateral stacking (association) of β-sheets.6,7,26,33,35 The degree of their lateral stacking is predominantly dictated by the attractive and repulsive interactions among amino acid side chains and the inherent twisting of β-sheets. Different extents of stacking result in different self-assembled morphologies, and a limited stacking is usually related to the formation of thin fibrils and a significant stacking leads to wide ribbons and tubes. KI4K tends to form β-sheet secondary structures, as indicted by CD characterizations (Figure 3). The strong hydrophobic interactions among isoleucine side chains promote the lateral stacking of KI4K sheets in water. At the same time, the electrostatic repulsion among lysine side chain amino groups produces little effect on the lateral stacking because the two lysine residues of each molecule are symmetrically distributed along the backbone. The combination of these factors leads to a significant stacking of KI4K β-sheets and subsequently the formation of wide ribbons, which readily curl into wide tubes. Upon introduction of acetonitrile into water, the peptide remained β-sheet secondary structures (Figure 3), suggesting that acetonitrile has little effect on the peptide−peptide hydrogen bonding and that there are still extensive hydrogenbonded β-sheets in the mixture of acetonitrile and water. Such a phenomenon can be ascribed to the similar HBA ability of acetonitrile with water.44,58 In comparison with water, however, acetonitrile has a lower polarity index due to the presence of the nonpolar methyl group. As a result, the addition of

the one between sheet3 and sheet4 increased to 1.470 nm from the initial 1.387 nm. In acetonitrile/water, the lateral stacking structure of β-sheets showed a broadly similar separation to that in acetonitrile. Accompanied with the different sheet separations, the dynamics of solvent molecules around the proto-fibril was also different. It appeared that water molecules escaped from the hydrophobic cores between the neighboring sheets with time in water while acetonitrile molecules came into the hydrophobic cores in acetonitrile. In the latter, the internalization of acetonitrile molecules would force the neighboring sheets to separate. The microscopic phenomenon that water molecules escape from the hydrophobic cores of peptide nanostructures has been extensively studied as dewetting.64,65 The above results indicate that acetonitrile has an opposite effect, i.e., wetting the hydrophobic cores. It is interesting that in the mixture of acetonitrile and water, both solvent molecules came to the hydrophobic cores with time and made the neighboring sheets separate. Thus, acetonitrile can not only wet the hydrophobic cores formed between the neighboring sheets, but also assist water to wet the hydrophobic cores. To quantify the wetting (weakening) effect on the hydrophobic interactions, we calculated the total number of hydrophobic contacts in the proto-fibril as shown in Figure 6. Two isoleucine side chains were considered to be in contact if the distance of their two CG2 atoms was less than 6 Å apart. The average numbers over time for the hydrophobic contacts were found to be 223, 204, and 195 in water, the mixture of 12980

DOI: 10.1021/acs.langmuir.5b02303 Langmuir 2015, 31, 12975−12983

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Langmuir acetonitrile can decrease the polarity of the solvent system and weaken hydrophobic interactions.42 In this case, the hydrophobic interactions among isoleucine side chains are expected to decrease with the acetonitrile addition, and MD simulations indicated that acetonitrile molecules tended to enter into the hydrophobic cores between sheets and force the neighboring sheets to separate (Figures 5 and 6). Experimental observations revealed morphological transitions from wide nanotubes to helical ribbons with decreased widths and then to thin fibrils with addition of acetonitrile (Figures 1 and 2), indicating the weakening of the hydrophobic interactions between KI4K βsheets and the consequent reduction of their lateral associations. Furthermore, the decreasing degree was proportional to the acetonitrile content in the solvent. These experimental results are consistent with MD simulation results. It should be noted that addition of acetonitrile into water lowers not only the polarity of the solvent system, but also the dielectric constant.66 Therefore, the electrostatic repulsions among lysine side chain amino groups should be enhanced upon the addition of acetonitrile into water. The enhanced electrostatic repulsion between the two terminal lysine residues may make KI4K monomer adopt more extended conformations, which appears to favor their docking and locking into βsheets through hydrogen bonding. However, the enhanced intermolecular electrostatic repulsions within and between βsheets have little effect on their longitudinal extension (predominantly driven by hydrogen bonding) and lateral association (predominantly driven by hydrophobic interactions), because of the symmetric molecular geometry of KI4K.35,53 Overall, due to its specific molecular geometry, the selfassembling KI4K provides a good model for exploring how the extent of hydrophobic interactions affects the morphological transitions. With the decrease of the hydrophobic interactions between the isoleucine side chains via the introduction of acetonitrile into water, the lateral stacking of KI4K sheets decreases. Significant stacking leads to wide ribbons that readily curl into wide helical tubes; intermediate stacking gives rise to helical and twisted ribbons with decreased widths; limited stacking results in thin twisted fibrils. During the entire morphological transition processes, the fundamental unit remained the same, that is, the KI4K monolayer held a constant thickness of about 2 nm, close to the full length of the peptide molecule. Lynn et al. have argued that the width of peptide aggregates had a direct bearing on their morphologies.57 They observed that helical ribbons or nanotubes were dominant when the width was much larger. On the other hand, twisted fibrils became dominant when the ribbons had much smaller widths. Also consistent with our results, Shen et al. have observed that addition of acetonitrile into aqueous solution inhibited the self-assembly of Aβ(1−39) sheets into the multimers for fibril initiation.42 Huang et al. have observed a structural transition of FF assemblies from microtubes in water to nanofibers with acetonitrile addition.44 These studies together thus depict a consistent picture of weakening of hydrophobic interaction by addition of acetonitrile resulting in altered peptide nanostructures.

bonding drove the formation of β-sheets, the strong hydrophobic interactions among isoleucine side chains play a vital role in the lateral stacking of KI4K β-sheets and the extent of twisting. Addition of acetonitrile was found to induce morphological transitions from nanotubes to helical/twisted ribbons and then to thin twisted fibrils while imposing little influence on the β-sheet hydrogen bonding between peptide backbones. In 20% and 80% (v/v) acetonitrile, helical ribbons with widths mostly in the range of 30−90 nm and thin twisted fibrils with a width of some 10 nm prevailed, respectively. These morphological transitions were ascribed to the weakening of the hydrophobic interactions and the reduction in the lateral stacking of KI4K β-sheets. Due to the symmetric molecular geometry of KI4K, the increased intermolecular electrostatic repulsions among lysine side chain amino groups appeared to produce little effect on the lateral stacking of KI4K β-sheets. Complementary MD simulations also indicated that acetonitrile molecules tended to enter into the hydrophobic cores between sheets and force the neighboring sheets to separate. These results clearly demonstrate the role of the hydrophobic interactions among the side chains and the significance of the molecular geometry in mediating the selfassembled structures of short peptides.

CONCLUSIONS We have examined morphological transitions of a symmetrical peptide KI4K through varying solvent. In aqueous solution, KI4K self-assembled into nanotubes with diameters mostly in the range of 80−160 nm. We explained that while hydrogen

(1) Ghosh, S.; Singh, S. K.; Verma, S. Self-Assembly and Potassium Ion Triggered Disruption of Peptide-Based Soft Structures. Chem. Commun. 2007, 2296−2298. (2) Yoon, Y.-R.; Lim, Y.-B.; Lee, E. J.; Lee, M. Self-Assembly of a Peptide Rod-Coil: a Polyproline Rod and a Cell-Penetrating Peptide Tat Coil. Chem. Commun. 2008, 1892−1894.

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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.langmuir.5b02303. TEM image of the KI4K self-assembled nanostructures in 10% (v/v) acetonitrile, AFM images of the KI4K selfassembled nanostructures in 80% (v/v) methanol and methanol, and the best fitted parameters for the SANS profiles (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21503275 and 21373270), the National Science Foundation for Postdoctoral Scientists of China (2012M511555), the Fundamental Research Funds for the Central Universities (15CX02026A), and the Special Funds for Postdoctoral Innovative Projects of Shandong Province of China (201203110). J.R.L. thanks UK Engineering and Physical Sciences Research Council (EPSRC) and the Royal Society (London) for support.

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DOI: 10.1021/acs.langmuir.5b02303 Langmuir 2015, 31, 12975−12983

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