Self-Assembly of Aβ-Based Peptide Amphiphiles with Double

Two peptide–amphiphiles (PAs), 2C12–Lys–Aβ(12–17) and C12–Aβ(11–17)–C12, were constructed with two alkyl chains attached to a key frag...
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Self-Assembly of Aβ-Based Peptide Amphiphiles with Double Hydrophobic Chains Chengqian He, Yuchun Han, Yaxun Fan, Manli Deng, and Yilin Wang* Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China S Supporting Information *

ABSTRACT: Two peptide−amphiphiles (PAs), 2C12−Lys− Aβ(12−17) and C12−Aβ(11−17)−C12, were constructed with two alkyl chains attached to a key fragment of amyloid βpeptide (Aβ(11−17)) at different positions. The two alkyl chains of 2C12−Lys−Aβ(12−17) were attached to the same terminus of Aβ(12−17), while the two alkyl chains of C12− Aβ(11−17)−C12 were separately attached to each terminus of Aβ(11−17). The self-assembly behavior of both the PAs in aqueous solutions was studied at 25 °C and at pHs 3.0, 4.5, 8.5, and 11.0, focusing on the effects of the attached positions of hydrophobic chains to Aβ(11−17) and the net charge quantity of the Aβ(11−17) headgroup. Cryogenic transmission electron microscopy and atomic force microscopy show that 2C12−Lys−Aβ(12−17) self-assembles into long stable fibrils over the entire pH range, while C12−Aβ(11−17)−C12 forms short twisted ribbons and lamellae by adjusting pHs. The above fibrils, ribbons, and lamellae are generated by the lateral association of nanofibrils. Circular dichroism spectroscopy suggests the formation of β-sheet structure with twist and disorder to different extents in the aggregates of both the PAs. Some of the C12−Aβ(11−17)−C12 molecules adopt turn conformation with the weakly charged peptide sequence, and the Fourier transform infrared spectroscopy indicates that the turn content increases with the pH increase. This work provides additional basis for the manipulations of the PA’s nanostructures and will lead to the development of tunable nanostructure materials.

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INTRODUCTION Peptide amphiphiles (PAs) have gained much interest as molecular building blocks for a new generation of materials because of their ability to adopt structures with precisely defined shapes and spatial distributions of functionality. Prompted by the clear unmet medical need and the emergence of bionanotechnology, PAs have recently been exploited for many technological applications, such as acting as scaffolds for cell culture,1 vectors for gene and drug delivery,2 and templates for nanofabrication3 and biomineralization.4 Normally, a typical PA molecule consists of a structural and functional hydrophilic peptide covalently conjugated to one hydrophobic aliphatic chain. The chemical design versatility of peptides, which can adopt specific secondary structures, provides a unique platform for the design of materials with controllable structural features at the nanoscale. A variety of works demonstrated an amazing level of control over the features in modifying the peptide sequence adjacent to hydrophobic chains,5 adding the number of amino acids of the peptide sequence,6 and incorporation of different peptide epitopes that can be tailored for various purposes.7,8 In the context of peptide choice, specific attention here has been given to the amyloid−β (Aβ) peptide based on two reasons. First, Aβ fibrils self-assembled from Aβ peptides, including Aβ(1−40) and Aβ(1−42), are associated with Alzheimer’s disease. Second, being a kind of insoluble high© 2012 American Chemical Society

ordered fibrils, several natural amyloid-like fibrils associated with the extracellular matrix or cell walls in microbial communities are thought to play a functional role9 in the cell adhesion10 and the formation of the bacterial biofilms.11 It has demonstrated that the trifluoroethanol-stabilized monomeric Aβ(1−42) delineates two separated helical domains, but only the destabilization of helix I, comprising residues 11−24, causes a transition from a α-helix to a β-sheet structure and a consequent amyloid formation.12,13 Many truncated shorter fragments of Aβ peptides, including Aβ(10−35),14 Aβ(18− 28),15 and Aβ(16−22),16 also form fibrils in vitro. Especially, the hydrophobic core Aβ(17−20) is essential for β-sheet formation, and the sequence Aβ(16−20) or Aβ(17−21) is critical for fibrillization.17,18 However, Aβ(11−17) is the most stable helical region and significantly contributes to the stability of the α-helical secondary structure of Aβ and the inhibition of peptide fibrillogenesis. Previously, we found that Aβ(11−17) cannot form ordered self-assemblies, but by introducing a long alkyl chain to this sequence, the created C12−Aβ(11−17) displays the prominent biological features of Aβ(1−40) in the morphology of the self-assemblies.19 Received: November 23, 2011 Revised: January 24, 2012 Published: January 24, 2012 3391

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transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy. This study shows that 2C12−Lys−Aβ(12−17) can self-assemble into long stable fibrils in aqueous solutions over the entire pH range studied (pH 3.0−11.0), whereas C12− Aβ(11−17)−C12 displays short twisted ribbons and lamellae, varying with pH condition. This observation highlights the significance of the alkyl chain positions in the stability and structure of the self-assembled nanofibrils. It indicates that with the modification of hydrophobic chain position and peptide charge property, the PA self-assembling nanostructure can be controlled.

The tendency of aliphatic chains to aggregate in aqueous solution provides a driving force for self-assembly of PAs, and the self-assembly is directed by hydrogen-bond forming between peptides.20 The PAs self-assemble in aqueous solution to yield fibrils of which the stability can be tuned by using different alkyl chain lengths21,22 or different hydrophobic components.23 All well-known gemini surfactants consist of two ionic or polar hydrophilic heads with two hydrophobic tails. The addition of two alkyl tails induces a synergistic effect, and the dimeric structure endows them with unique properties and abundant self-assembly behaviors compared to the corresponding monomeric surfactants.24−27 Thus, by introducing two hydrophobic chains, the PA molecules will be expected to possess the features of both gemini surfactants and PAs in self-assembly. Two hydrophobic chains can be connected to a peptide sequence at different positions. These two chains may be connected to the same terminus of a peptide, which brings the alkyl chains into close proximity, thereby may increase the effective concentration of hydrophobic structure and keep the peptide flexible. Another connecting approach is to attach the two hydrophobic chains at each terminus of a peptide separately. Because the hydrophobic chains tend to associate together, the conformation and the secondary structure of the peptide may be affected. Therefore, the positions of two hydrophobic chains relative to a peptide sequence should be an important factor of controlling the ability of PA self-assembly and the assembling structure. Herein, we have designed and studied two Aβ(11−17)-based PAs, i.e., 2C12−Lys−Aβ(12−17) and C12−Aβ(11−17)−C12 as shown in Figure 1. The introduction of Aβ(11−17) endows the

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EXPERIMENTAL SECTION

Materials. We designed 2C12−Lys−Aβ(12−17) and C12−Aβ(11− 17)−C12, and the synthesis was carried out by GL Biochem (Shanghai) Ltd. The structures of 2C12−Lys−Aβ(12−17) and C12− Aβ(11−17)−C12 were confirmed by 1H NMR and mass spectra (see Supporting Information). The purities of these two PAs were better than 98% (checked by high-performance liquid chromatography). Inorganic reagents (>99.5%) were purchased from Beijing Chemical Co. Milli-Q water (18 MΩ·cm−1) was used throughout. Sample Preparation. 2C12−Lys−Aβ(12−17) and C12−Aβ(11− 17)−C12, with molecular weight of 1253.72 and 1239.69, were ultrasonically dissolved in water for 2 min at 400 W by a JY92-II N Ultrasonic cell crusher (Ningbo Xinzhi) fitted with a dispersing tool with 2.76 mm outer diameter. The pH was adjusted with concentrated HCl or NaOH solution to achieve the desired values. Cryogenic Transmission Electron Microscopy (Cryo-TEM). The 2C12−Lys−Aβ(12−17) and C12−Aβ(11−17)−C12 samples were embedded in thin layer of vitreous ice on freshly carbon-coated holey TEM grids by blotting the grids with filter paper and then plunging into liquid ethane cooled by liquid nitrogen. Frozen hydrated specimens were imaged by using FEI Tecnai 20 electron microscope (LaB6) operated at 200 kV with low-dose mode (about 2000 e/nm2) and nominal magnification of 50 000. For each specimen area, the defocus was set to 1−2 μm. Images were recorded on Kodak SO163 film and then digitized by Nikon 9000 with the scanning step 2000 dpi corresponding to 2.54 Å/pixel. Atomic Force Microscopy (AFM). A Multimode Nanoscope IIIa AFM (Digital Instruments, CA) was used for AFM imaging. For ambient imaging, 5−7 μL of 2C12−Lys−Aβ(12−17) or C12−Aβ(11− 17)−C12 solution was deposited onto a freshly cleaved piece of mica and left to adhere for 5−10 min. The samples were then briefly rinsed with Milli-Q water and dried with a gentle stream of nitrogen. Probes used were etched silicon probes attached to 125 μm cantilevers with a nominal spring constant of 40 N/m (Digital Instruments, model RTESPW). All the provided morphology images were recorded using a tapping mode at 512 × 512 pixel resolution and a scan speed of 1.0− 1.8 Hz. Topographic data were regularly recorded in both trace and retrace to check on scan artifacts. They were shown in the height mode without any image processing except flattening. Analysis of the images was carried out using the Digital Instruments Nanoscope Software (Version 512r2). Each of the samples was prepared and observed at least five times. Circular Dichroism (CD). The CD spectra were recorded on a JASCO J-815 spectrophotometer at room temperature using a 0.1 mm quartz cell. Scans were obtained in a range between 190 and 260 nm by taking points at 0.5 nm, with an integration time of 0.5 s. Four spectra were averaged to improve the signal-to-noise ratio and smoothed using the noise reducing option in the software supplied by the vendor. Fourier Transform-Infrared Spectroscopy (FT-IR). Samples were prepared by evaporating 10 μL drops of the PA solutions on CaF2 glass slides, then were dried in vacuum, followed by buffer exchanging with D2O and dehydration (repeated twice). Spectra were measured with a Bruker Optics TENSOR-27 FT-IR spectrophotometer. The extracted amide I band contour was subjected to the

Figure 1. Chemical structures and space-filling models of PAs: (A) 2C12−Lys−Aβ(12−17) and (B) C12−Aβ(11−17)−C12.

desired PA molecules with biological features, while the two alkyl chains attached onto the Aβ(11−17) through amide bond promotes the self-assembly of the PA molecules. The overall charges of these two PA molecules are pH dependent. Each molecule approximately carries three positive, two positive, or zero or one negative net charges at pHs 3.0, 4.5, 8.5 or 11.0, respectively. Effects of the pH and alkyl chain positions on the self-assembly of 2C12−Lys−Aβ(12−17) and C12−Aβ(11−17)− C12 in aqueous solutions have been investigated by cryogenic 3392

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Figure 2. Cryo-TEM images and AFM height images of the 2C12−Lys−Aβ(12−17) solutions at 0.2 mM concentration but different pHs. (A1, A2) pH 3.0; (B1, B2) pH 4.5; (C1, C2) pH 8.5; (D1, D2) pH 11.0. All the AFM images are 5 × 5 μm2 in size. second derivative calculation and Gaussian curve-fitting analysis. After decomposition of the amide I band (1700−1600 cm−1), the secondary structure contents were estimated using the criteria described by Pelton and Mclean.28

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RESULTS AND DISCUSSION Both 2C12−Lys−Aβ(12−17) and C12−Aβ(11−17)−C12 are pH−sensitive, so four pHs, 3.0, 4.5, 8.5 and 11.0, were selected to define the protonation states of the two PAs in aqueous solutions. The pKa values of the carboxylic group in Leu and in the side chain of Glu, the imidazolyl group in His, and the pKb value of the amino group in Lys are 2.36, 4.25, 6.00, and 10.53, respectively.29 Thus, each PA molecule carries approximately three positive net charges at pH 3.0, two positive net charges at pH 4.5, zero net charge at pH 8.5, and one negative net charge at pH 11.0. To identify the morphologies of the 2C12−Lys−Aβ(12−17) self-assemblies, Cryo-TEM and AFM were carried out with its aqueous solutions at 0.2 mM concentration but different pHs. The Cryo-TEM images (Figure 2 A1, B1, C1, D1) show that 2C12−Lys−Aβ(12−17) molecules form well-ordered fibrils, which are micrometers in length but 25 nm wide. Meanwhile, the fibrils are observed in the AFM height images (Figure 2 A2, B2, C2, D2) with an average height value of 20 nm that is close to the Cryo-TEM result. It is noteworthy that the morphologies of the fibrils do not change with the pHs, suggesting that the molecule charge condition has no effect on the structures of the self-assemblies of 2C12−Lys−Aβ(12−17). Further insight into the structure of these long fibrils is also provided by the high magnification images of Cryo-TEM (Figure 3 A, B, C, D), which indicate that the fibrils are a bundle of parallel nanofibrils, ∼4.3 nm wide for each. These long but thin nanofibrils tend to laterally bundle together. Figures 4 and 5 present the morphologies of the C12− Aβ(11−17)−C12 assemblies formed at 0.2 mM and at pHs 3.0, 4.5, 8.5, and 11.0, respectively. In the C12−Aβ(11−17)−C12 solutions, numerous short ribbons and lamellae prevail with the pH increase. The pH dependence indicates that the charge property plays an important role in the self-assembly of C12− Aβ(11−17)−C12. Clearly, short assemblies with length of ∼100 nm are presented at the four pHs. At pH 8.5 and 11.0 (Figure 4 C1, D1), the nanofibrils align in nearly lamella arrays with a width larger than 50 nm. The high magnification images (Figure 5 C2, D2) along the direction of the fibril length indicate that the lamellae are bundled by lateral aggregation of

Figure 3. High magnification Cryo-TEM images of the self-assemblies of 2C12−Lys−Aβ(12−17) at 0.2 mM concentration at (A) pH 3.0; (B) pH 4.5; (C) pH 8.5; and (D) pH 11.0.

nanofibrils of 2.4 nm width for each. In contrast to the lamellae formed at pH 8.5 and 11.0, short ribbons consisting of rodlike nanofibrils are observed at pH 3.0 and 4.5 in the Cryo-TEM images (Figure 4 A1, B1), with an average width of 25 nm and a varying length from ∼80 nm to ∼1.8 μm. Meanwhile, the height of the rodlike nanofibrils of C12−Aβ(11−17)−C12 in the AFM images (Figure 4 A2, B2) is ∼18 nm, a little lower than the height of the 2C12−Lys−Aβ(12−17) fibrils (Figure 2 A2, B2, C2, D2). In addition, the ribbons are the bundles of the nanofibrils of ∼2.0 nm width each and are twisted in acidic conditions (as shown by the arrows in Figure 5 A1, B1), which is also proven by the AFM section analyses (Supporting Information). To probe the secondary structures of the above selfassemblies, both CD and FT-IR were applied. CD studies were performed for 0.2 mM 2C12−Lys−Aβ(12− 17) solutions at pHs 3.0, 4.5, 8.5, and 11.0 and for the C12− Aβ(11−17)−C12 solutions at the same conditions (Figure 6). Previous work30 on PAs has shown that β-sheet hydrogen3393

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Figure 4. Cryo-TEM images and AFM height images of the C12−Aβ(11−17)−C12 solutions at 0.2 mM concentration but different pHs. (A1, A2) pH 3.0; (B1, B2) pH 4.5; (C1, C2) pH 8.5; (D1, D2) pH 11.0. All the AFM images are 5 × 5 μm2 in size.

bonding plays a crucial role in the formation of nanofibrils. Although supramolecular assemblies of PAs have heterogeneous secondary structures, only β-sheet structure is the possible reason that most PAs form cylindrical nanofibrils rather than spherical micelles.7,30 All the present CD spectra of the present two PAs are predominantly expected for β-sheet, and it is supported by the following FT-IR data (Figure 7). All

Figure 5. High magnification Cryo-TEM images of the self-assemblies of C12−Aβ(11−17)−C12 at 0.2 mM concentration at (A1, A2) pH 3.0; (B1, B2) pH 4.5; (C1, C2) pH 8.5; and (D1, D2) pH 11.0.

Figure 7. FT-IR spectra of 0.2 mM 2C12−Lys−Aβ(12−17) and C12− Aβ(11−17)−C12 solutions at different pHs in the 1800−1450 cm−1 region. (A1, B1) pH 3.0; (A2, B2) pH 4.5; (A3, B3) pH 8.5; (A4, B4) pH 11.0.

the self-assemblies of 2C12−Lys−Aβ(12−17) at the four pHs present a β-sheet secondary structure signaled by the maximum at ∼203 nm and the minimum at ∼224 nm. However, the CD spectra are red-shifted relative to those in typical β-sheets with a maximum at 195 nm and a minimum at 216 nm.31 It has been shown that the red-shifted β-sheet signal is associated with a twisted planar β-sheet.32,30 For the self-assemblies of C12− Aβ(11−17)−C12 at the same pH conditions, the CD spectra are complex and imply more than one type of the secondary

Figure 6. CD spectra of 0.2 mM 2C12−Lys−Aβ(12−17) and C12− Aβ(11−17)−C12 solutions at pHs 3.0, 4.5, 8.5, and 11.0. 3394

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Figure 8. Schematic representation of (A) a model nanofibril formed by 2C12−Lys−Aβ(12−17), (B) a model nanofibril, and (C) a model nanotape formed by C12−Aβ(11−17)−C12 at low and high pHs, respectively.

three main forces controlling the assembly of the PA molecules: hydrophobic interaction, hydrogen bonding, and electrostatic repulsion. The hydrophobic interaction in aqueous solution provides a driving force for self-assembly, which is directed by the hydrogen-bonding between the peptide moieties. While the electrostatic repulsion of the peptide moieties assists the PA molecules assemble in a well-ordered manner. The balance of these interactions dictates the nanostructure and the assembled morphologies. On the basis of the secondary structures and the morphologies of the assemblies described above, a schematic representation of the models for the self-assemblies formed by 2C12−Lys−Aβ(12−17) and C12−Aβ(11−17)−C12 at different pHs is shown in Figure 8. The 2C12−Lys−Aβ(12−17) molecules tend to associate into long fibrils in aqueous solution, the morphology of which would not change with the pHs. Take the molecule charge situation at low pH for an example (Figure 8A): the two positive imidazolyl groups in the middle of the peptide increase the electrostatic repulsion between the neighboring peptide head groups. Hence, the 2C12−Lys−Aβ(12−17) molecules may prefer to organize into a cylinder with a relatively high curvature. The peptide moiety adopts a β-sheet conformation, and the hydrogen bonding is highly aligned down the fibril axis. When the 2C12−Lys−Aβ(12−17) molecules are not charged at high pH, the cylinder assembles are still able to be formed, however, without the regulation of electrostatic repulsion, the 2C12−Lys−Aβ(12−17) molecules will be driven together by strong hydrophobic interaction among the hydrocarbon chains, leading the structure a little more disordered. In contrast, C12−Aβ(11−17)−C12 molecules self-assemble into the short twisted ribbons or lamellae, varying with pH

structure. They display the characteristics of two negative peaks at ∼196 and ∼227 nm and one positive peak at ∼206 nm. Because a typical β-turn CD spectrum has two minima at 196 and 230 nm plus a maximum at 215 nm,17 the observed spectra may represent a superposition of turn and a red-shifted β-sheet. It is noteworthy that the CD signal intensity of the selfassemblies of C12−Aβ(11−17)−C12 is significantly weakened with the increase of the pH value. The reduction of the CD signal is probably related to the twist of the planar β-sheet structure, which may lead to an increase in the hydrogenbonding scope but weakens the strength.32 To support the above conformational findings, the FT-IR spectra of the self-assemblies of 2C12−Lys−Aβ(12−17) and C12−Aβ(11−17)−C12 were performed, quantifying the transition of the secondary structures (Figure 7). After decomposition of the amide I band (1700−1600 cm−1), the secondary structure contents were estimated. The band at 1625 cm−1 indicates the β-sheet structure, while the peaks at ∼1680 cm−1 may reflect turn structure. The contents of the β-sheet and turn structures remain nearly unchanged (∼70% for β-sheet structure and ∼30% for turn structure) with the increase of pH for the self-assemblies of 2C12−Lys−Aβ(12−17). On the contrary, the β-sheet content decreases from about 80% to 50% accompanying with the increase of the turn content in the selfassemblies of C12−Aβ(11−17)−C12 when the pH changes from 3.0 to 11.0, i.e., the self-assemblies of C12−Aβ(11−17)−C12 transfer from ordered β-sheet to lose-turn conformation. The variation in the fibril morphologies of 2C12−Lys− Aβ(12−17) and C12−Aβ(11−17)−C 12 at different pH conditions should arise from the different manners of the intermolecular interactions within the self-assemblies. There are 3395

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condition, as illustrated in Figure 8B,C. At low pH, hydrogen bonding attracts the C12−Aβ(11−17)−C12 molecules to align side by side, while the strong electrostatic repulsion between the neighboring peptide head groups will favor a highly curved structure. Moreover, hydrophobic interactions among the hydrocarbon chains could lead to a further aggregation of the fibrils through crimping inward. These factors may lead to the twisted structure in the ribbons so that the molecules can pack in a low energy approach. Differently, at high pH, the relatively weak electrostatic repulsion between the peptides in the C12− Aβ(11−17)−C12 molecules may favor the lamellar structure (Figure 8C). Especially, because the hydrophobic chains attached at each terminus of the peptide tend to associate together, the conformation of the peptide may be transferred into a turn structure. The decrease in electrostatic repulsion between the molecules allows stronger lateral interactions, resulting in the formation of the flat lamellae, primarily comprised of the C12−Aβ(11−17)−C12 bilayers. Thus, the low curvature of the self-assembled lamellar structures limits the well-defined width.

CONCLUSIONS The present work studied the effects of the attached positions of the hydrophobic chains and the charge situation of the peptide moiety on the structures and molecular interaction in the PA self-assemblies. The results show that both 2C12−Lys− Aβ(12−17) and C12−Aβ(11−17)−C12 can self-assemble into ordered aggregates but display different situations in the structures and morphologies. Therein, 2C12−Lys−Aβ(12−17) tends to assemble into long stable fibrils in aqueous solution over the entire pH range. In contrast, C12−Aβ(11−17)−C12 presents short twisted ribbons and lamellae, varying with pH condition. All the fibrils, ribbons, and lamellae are generated by the lateral association of nanofibrils. This observation highlights the significance of the alkyl chain positions in the stability and structure of the self-assembled nanofibrils. It reveals that with the modification of the hydrophobic chain positions and the peptide charge properties, the PA self-assembling nanostructures can be controlled. This work provides additional basis for the manipulations of the PA’s nanostructures and will lead to the development of tunable nanostructure biomaterials by inscribing biological signals in the self-assemblies. ASSOCIATED CONTENT

S Supporting Information *

Additional AFM images of the self-assemblies as well as the 1H NMR, HPLC, and mass spectra of the PAs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (21025313, 21003137, 20973181, and 21021003). 3396

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