Coassembly in Binary Mixtures of Peptide Amphiphiles Containing

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Coassembly in Binary Mixtures of Peptide Amphiphiles Containing Oppositely Charged Residues I. W. Hamley,* A. Dehsorkhi, and V. Castelletto School of Chemistry, Pharmacy and Food Biosciences, University of Reading, Reading RG6 6AD, U.K. S Supporting Information *

ABSTRACT: The self-assembly in water of designed peptide amphiphile (PA) C16ETTES containing two anionic residues and its mixtures with C16-KTTKS containing two cationic residues has been investigated. Multiple spectroscopy, microscopy, and scattering techniques are used to examine ordering extending from the β-sheet structures up to the fibrillar aggregate structure. The peptide amphiphiles both comprise a hexadecyl alkyl chain and a charged pentapeptide headgroup containing two charged residues. For C16-ETTES, the critical aggregation concentration was determined by fluorescence experiments. FTIR and CD spectroscopy were used to examine β-sheet formation. TEM revealed highly extended tape nanostructures with some striped regions corresponding to bilayer structures viewed edge-on. Small-angle X-ray scattering showed a main 5.3 nm bilayer spacing along with a 3 nm spacing. These spacings are assigned respectively to predominant hydrated bilayers and a fraction of dehydrated bilayers. Signs of cooperative self-assembly are observed in the mixtures, including reduced bundling of peptide amphiphile aggregates (extended tape structures) and enhanced β-sheet formation.

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INTRODUCTION Peptide amphiphiles (PAs) are a fascinating class of selfassembling molecules in which amphiphilicity is combined with the biofunctionality of peptide units.1−7 The association properties of these hybrid molecules are controlled by the hydrophobicity of the lipid chain, along with electrostatic, van der Waals, aromatic, and hydrogen bonding interactions of the amino acids in the peptide chain. In most cases reported to date, peptide amphiphiles have been shown to self-assemble into fibrils with a lipid core and a peptide-functionalized corona. The peptide coating of the fibrils leads to exceptional potential applications in biomedicine, including tissue engineering.8−11 Other self-assembled structures including nanotapes12,13 and vesicles14,15 have been reported, with rather few examples in each case. In fact, the general principles of nanostructure selection in these materials are still poorly understood, especially in comparison to the vast existing literature on conventional lipids. For conventional lipids, self-assembly leads to a variety of nonlayered structures, including hexagonal and cubic structures. We have recently investigated the self-assembly of PA C16KTTKS,13 which is incorporated into antiwrinkle skin-care creams with the trade name Matrixyl. This PA incorporates a collagen-stimulating pentapeptide motif based on a sequence from the carboxyl-terminal propeptide of type I collagen.16 The pentapeptide contains two lysine residues and a carboxyl terminus, leading to a charged headgroup in the PA at sufficiently low pH (below the pKa of lysine, approximately pKa = 10). We showed that this compound self-assembles into nanotapes (also known as nanobelts) in relatively dilute © 2013 American Chemical Society

aqueous solutions. To investigate the role of electrostatic interactions in the self-assembly of the PA, in this Article we investigate the self-assembly of C16-ETTES. This contains glutamic acid (E) residues replacing the K residues in the sequence of the Matrixyl PA. We investigate its self-assembly using a combination of spectroscopy, microscopy, and X-ray and neutron scattering techniques. Transmission electron microscopy (TEM) and cryogenic-TEM (cryo-TEM) reveal the presence of extended tapes that have a striped appearance in some regions. The 5-nm-wide stripes are associated with the bilayer ordering of the PA. This in turn was probed using SAXS, which provided a distinct model of stacked hydrogenbonded β sheets and can account for the widths and intensities of observed SAXS peaks. Here, we also examine self-assembly in mixtures of C16ETTES and C16-KTTKS. Electrostatic interactions between E and K residues are expected to lead to coassembly and the possible modification of self-assembled nanostructures. Several groups have examined the formation of nanofibers by the electrostatic coassembly of two PAs with oppositely charged peptide sequences. Fibril formation was observed by Stupp et al. at neutral pH for the mixed system, whereas the parent PAs formed fibrils only in acidic or basic solution for the anionic or cationic molecules, respectively17 This group also studied the coassembly of PAs with oppositely charged (i.e., free N or free C) termini comprising trilysine and triglutamic acid sequences, Received: January 15, 2013 Revised: March 8, 2013 Published: March 27, 2013 5050

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Table 1. Spacingsa from X-ray Diffraction Patterns, Including a Comparison to Previously Published Data for C16-KTTKS13 sample peaks 40 Å spacing bilayer spacing second order (±0.2) bilayer spacing third order or molecular length (±0.1) 7−14 Å spacing

5−6 Å

β-strand spacing amino acid/H bonds

a

C16ETTES

1% C16-KTTKS/ 2% C16-ETTES

1% C16-KTTKS/ 1% C16-ETTES

40.39

39.25

1% C16-KTTKS/ 0.5% C16-ETTES

0.5% C16-KTTES/ 1% C16-ETTES

2% C16-KTTKS/ 1% C16-ETTES

28.4

C16KTTKS13 26.4

17.8

18.2

18.4

18.5

18.3

13.88 11.19 8.85 8.02 7.45 6.55 5.99 5.61 5.17 4.72 4.50 4.20 4.09 3.80 3.73

18.22 14.11 11.45 8.91

8.88

14.14 11.38 8.86

12.89 8.87

13.92 11.28 9.18 7.70

6.55 5.66

6.62 5.2

6.69 6.49

4.61

4.71

4.78 4.34 4.14 3.93

5.23

4.75

4.77

4.72 3.25 3.1

In angstroms (Å), ±0.02, unless otherwise stated.

respectively.18 They noted that the coassembled PA nanofibrils exhibited enhanced thermal stability compared to the component PAs. Guler and co-workers have investigated the influence of electrostatic interactions on the self-assembly of lysine- or aspartic acid-based PAs by focusing on the effects of pH or added charged biomolecules rather than studying mixtures of the PAs.19 Yu and co-workers have investigated hydrogelation due to the formation of mixed fibrils from oppositely charged peptides comprising repeat AK or AE motifs.20,21 The ionic strength was found to influence the elastic modulus because of the formation of thinner and stiffer fibrils.21 The influence of the unmatched peptide chain length was also considered.20 The fibrillization of tetrapeptides KFFK, EFFE, KLLE, KAAE, and KVVE has been investigated.22 Individually, peptides KFFK and EFFE form random coil structures; however, fibrils were observed in equimolar mixtures. This highlights the important role of electrostatic interactions between oppositely charged peptides, influencing aggregation. Aggeli and co-workers have recently shown that complementary electrostatic interactions can be used to drive self-assembly into β-sheet hydrogels in mixtures of peptides containing anionic glutamic acid or cationic ornithine.22a In contrast, the unmixed peptides existed in a monomeric form. Complementary electrostatic interactions have also been used to direct the assembly of coiled-coil peptides.23−25 The heterodimeric structures often rely on interactions between K and E residues, as in the present work in which we explore the coassembly of ionic peptide amphiphiles forming β-sheet structures. Complementary interactions involving homopeptide sequences designed to incorporate oppositely charged residues such as K and E have also been exploited in the design of novel bionanomaterials.25,26 We propose that the unique interplay among hydrophobic, electrostatic, and hydrogen bonding forces in peptide amphiphiles can lead to distinct modes of lamellar selfassembly. In these systems, the directed intermolecular (βsheet) hydrogen bonding of the peptide headgroup contributes

to the overall balance of interactions, in contrast to conventional amphiphiles.

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EXPERIMENTAL SECTION

Materials and Sample Preparation. Peptide amphiphile C16ETTES, palmitoyl-Glu-Thr-Thr-Glu-Ser, was custom synthesized by CS Bio (Menlo Park, CA). Two batches were used. For the first, the purity of the sample (supplied as a TFA salt) was 96.51% (by analytical HPLC in a TFA water/acetonitrile gradient). The molecular weight was obtained by electrospray mass spectrometry: found 803.4 Da, expected 803.94 Da. For the second (supplied as a TFA salt), the purity was 96.96% by analytical HPLC with a molecular weight (measured) of 803.47 Da. Peptide amphiphile C16-KTTKS, palmitoyl-Lys-Thr-Thr-Lys-Ser, was purchased from CS Bio (Menlo Park, CA) as the TFA salt. (Previously13,27 we studied the acetate salt.) Three different batches were used. For the first, the purity was 97.6% by analytical HPLC, MS 802.47 Da (expected), and 802.05 Da (measured), and the acetate content was 11% (by HPLC). For the second batch, the purity was 98.61% by analytical HPLC, the molecular weight was 802.2 Da (measured), and the acetate content was 11.61% (by HPLC). For the third batch, the purity was 97.1% by analytical HPLC with MS 802.05 Da (measured). Samples were dissolved directly in ultrapure water from a ThermoFisher Barnstead NanoPure system and were typically allowed to equilibrate for a period of days (at least 12 h). In the following text, all experiments were performed at room temperature unless otherwise stated. The pH (±0.2) of solutions of C16-ETTES was measured to be 5.4 (1 wt % solution) or 6.0 (0.1 wt % solution). The pH of a 1 wt % solution of C16-KTTKS is 3.2.27 Fluorescence Spectroscopy. Thioflavin T and pyrene fluorescence spectra were recorded with a Varian Cary Eclipse fluorescence spectrometer with samples in 4 mm inner width quartz cuvettes. ThT fluorescence assays were conducted using a series of C16-ETTES solutions dissolved in 1.6 × 10−3 wt % ThT. The spectra were recorded from 460 to 600 nm using an excitation wavelength of λex = 440 nm. Pyr fluorescence assays were made using a set of C16ETTES solutions dissolved in 1.2 × 10−5 wt % Pyr. All spectra were measured from 366 to 460 nm using λex = 339 nm. 5051

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according to the different mixing protocols, heated to 60 °C, and cooled back to room temperature. Additional SAXS measurements were performed at Diamond Light Source (Harwell, U.K.), at SOLEIL (L’Orme des Merisiers, France), and at the ESRF (Grenoble, France). At Diamond, experiments on beamline I22 used X-rays with a wavelength 1.069 Å and a 1.20 m sample-to-detector distance. SAXS data were collected with a gas-filled multiwire RAPID area detector. The data were sector averaged to produce 1D intensity profiles. The q scale was calibrated using silver behenate. On beamline SWING at SOLEIL, the wavenumber q range was set to 0.004−0.5 Å−1, with λ = 1.03 Å (12 keV). The images captured by the AVIEX170170 CCD detector were radially averaged and corrected for transmitted intensity and water background using the software Foxtrot. On beamline ID02 at the ESRF, SAXS/WAXS experiments were performed at a wavelength of λ = 0.995 Å. SAXS data were collected with a FReLoN Kodak CCD with a 1.2 m sample− detector distance, and WAXS data were measured simultaneously with an Aviex CCD. Samples were injected using a syringe into ENKI KIbeam thin-wall (0.05 mm) 1.85-mm-diameter polycarbonate capillaries that optimize background subtraction. Measurements were performed at 25 °C. For the shearing experiments on SWING, 1 mL of a solution containing 3 wt % C16-ETTES was pipetted into a quartz Couette cell, which consisted of concentric cylinders. The sample was performed at steady shear at varying shear rates. The sample gap was 0.5 mm. Shearing experiments were performed at temperatures of 25, 40, and 55 °C.

Circular Dichroism (CD). CD spectra were recorded using a Chirascan spectropolarimeter (Applied Photophysics, U.K.). Solutions of C16-ETTES (concentrations in the range of 0.001−3 wt %) or mixtures of C16-ETTES and C16-KTTKS were loaded in parallel plaque cells (Hellma quartz Suprasil) with a 0.01 mm path length. The CD data were measured using a 1 s acquisition time per point and a 0.5 nm step. The post-acquisition smoothing tool from Chirascan software was used to remove random noise elements from the averaged spectra. A residual plot was generated for each curve in order to verify whether the spectrum had been distorted during the smoothing process. The CD signal from the water was subtracted from the CD data of the peptide solutions. Fourier Transform Infrared (FTIR) Spectroscopy. Spectra were recorded using a Nexus-FTIR spectrometer equipped with a DTGS detector and a multiple reflection attenuated total reflectance (ATR) system. Solutions of C16-ETTES or mixtures with C16-KTTKS in D2O were sandwiched in ring spacers between two CaF2 plate windows (spacer 25 μm). All spectra were scanned 128 times over the range of 4000−950 cm−1. Fiber X-ray Diffraction (XRD). For C16-ETTES, X-ray diffraction was performed on a stalk prepared from a 3 wt % solution in water. A series of mixtures with C16-KTTKS (Table 1) were also used to prepare stalks. Each stalk was mounted (vertically) onto the four-axis goniometer of a RAXIS IV++ X-ray diffractometer (Rigaku) equipped with a rotating anode generator. The XRD data was collected using a Saturn 992 CCD camera. Transmission Electron Microscopy (TEM). To prepare specimens for TEM, a droplet of the PA solution was placed on a Cu grid coated with a carbon film (Agar Scientific, U.K.), stained with uranyl acetate (1 wt %, Agar Scientific, U.K.), and dried. TEM experiments were performed using a JEOL JEM-2010 microscope operated at 200 kV. Cryogenic-Transmission Electron Microscopy (Cryo-TEM). Experiments were performed using a Tecnai 12 -FEI 120 kV BioTwin Spirit transmission electron microscope (TEM). A drop of C16-ETTES solution (0.7 wt %) was placed on a Cu grid coated with a carbon film (Agar Scientific, U.K.) and covered with a perforated carbon film (plasma-treated). The drop was automatically blotted. A FEI Vitrobot Mark IV device was used to plunge the sample into liquid ethane (−183 °C) to form a vitrified specimen. Vitrified specimens were transferred to nitrogen (−196 °C) for storage. The frozen specimens were then analyzed with the microscope using a Gatan cryotransfer specimen holder. Small-Angle X-ray Scattering (SAXS). SAXS was performed using a Bruker Nanostar instrument with Cu Kα radiation from an Incoatec microfocus source. The beam was collimated by a three-slit system. The samples were mounted in capillaries. The sample− detector distance was 67 cm, and a Vantec-2000 photon-counting detector was used to collect SAXS patterns. The wavenumber q = 4π sin θ/λ (where 2θ is the scattering angle) scale was calibrated using silver behenate. Measurements on C16-ETTES were performed for the sample dissolved in ultrapure water. For the mixtures, C16-KTTKS and C16ETTES were mixed as dry powders. Water was then added to make up a solution with a defined concentration. A vial containing the solution was then placed in a sonicator at 30 °C for approximately 1 h in order to dissolve both C16-KTTKS and C16-ETTES in water. For studies on the equilibration of samples at room temperature, two protocols were followed. In the first, a solution (0.5, 1, or 2 wt %) of C16-ETTES was prepared and left to stand for a week (in the refrigerator) to allow for sufficient time for self-assembly to occur. This was then followed by the addition of C16-KTTKS (0.5, 1, or 2 wt %). SAXS was performed immediately after preparing the mixture and again 2 months later to study the effects of aging. In the second procedure, solutions of C16-KTTKS were prepared first and left for a week, followed by the addition of C16-ETTES. To allow for thermal equilibration above the lipid chain melting temperature (previously reported to occur at around 40−45 °C13,28), SAXS measurements were also performed for samples prepared

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RESULTS Self-Assembly of C16-ETTES. We first determined the critical aggregation concentration (cac) of C16-ETTES in water at room temperature using fluorescence assays with dyes pyrene and thioflavin T. The latter is a diagnostic for amyloid formation because this dye is taken up selectively by β-sheet amyloid structures;23,28b,c however, the mechanism of binding to amyloid has not been definitively identified. Although some papers suggest that ThT binds to β-sheet grooves, other papers have provided some evidence that the binding is dependent on electrostatic interactions or diameter-dependent binding to molecular grooves/cavities in β sheets and other fibers.29−31 The fluorescence of pyrene is sensitive to the hydrophobicity of its environment, and as such it is widely used to locate the cac of amphiphilic molecules.31b,c Figure 1 shows that C16-ETTES aggregates into amyloid (β-sheet-based) structures above 0.005 ± 0.001 wt %. This is lower than the cac of C16-KTTKS, determined (by pyrene fluorescence measurements) to be 0.01

Figure 1. Fluorescence assays to determine the critical aggregation concentration of C16-ETTES using dyes thioflavin T (solid circles) and pyrene (open circles). Note that the fluorescence peak for solutions with ThT only is centered at 484 nm. The position of the peak changes from 484 to ∼494 nm upon adding peptide. I1 is the fluorescence intensity of the pyrene 0−0 band at λ ≈ 373 nm. 5052

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It is noteworthy that the fluorescence-based assays using dyes pyrene and thioflavin T are much more sensitive to the aggregation of PA than FTIR spectroscopy in particular, but they also seem to be more sensitive to aggregation than CD spectroscopy. The fact that ThT binding is diagnostic for amyloid (i.e., fibril) formation suggests that β-sheet formation and 1D self-assembly into extended nanostructures (here, tapes) occur concurrently with the sequestration of the hydrophobic lipid chains into the fibril interiors. TEM and cryo-TEM revealed the tapelike structure of selfassemblies of C16-ETTES. A representative cryo-TEM image is shown in Figure 3. This reveals twisted tapes. In some areas,

wt %.28 This is consistent with the different hydrophobicities of the two PAs: The octanol/water partition coefficients are calculated to be log p = −0.527 for C16-ETTES and log p = −0.905 for C16-KTTKS.32 The fact that pyrene shows a break in its fluorescence−concentration curve at the same concentration as the cac determined by ThT fluorescence suggests that the cac corresponds to aggregation into fibrillar structures (probed by ThT) concomitantly with partitioning of the lipid chains into the hydrophobic interior of the fibrils. The secondary structure of C16-ETTES was examined using circular dichroism and FTIR spectroscopies. Figure 2a shows

Figure 3. Cryo-TEM image obtained from a 0.7 wt % solution of C16ETTES with (inset) a higher-magnification image.

Figure 2. (a) Circular dichroism spectra for C16-ETTES measured for concentrations in the range of 0.001−3 wt %. (b) FTIR spectra of C16ETTES measured for 1 and 3 wt % solutions in D2O.

stripes can be observed, with a stripe spacing of approximately 5 nm. These are observed in some regions along the tapes and not others, and our interpretation of the stripes is that they represent stacks of bilayers viewed side-on, with the presence of bilayers being indicated by SAXS as discussed shortly. The bilayers are extended in the direction of the long tape axis; however, the tape thickness defines the number of bilayer repeats, which is less than 10. Typical TEM images are shown in SI Figure 1. These reveal that most of the grid was covered with a dense network of extended tape structures (SI Figure 1a). However, a few striped tapes were also observed such as that shown in SI Figure 1b, with a spacing of approximately 5 nm, consistent with cryo-TEM. There were typically up to ∼10 stripe repeats. As for C16-KTTKS,13 the tapes are polydisperse in width, a consequence of the primarily 1D self-assembly mechanism. In TEM and cryo-TEM images, the majority of the nanostructures imaged are tapes lying parallel to the surface of the grid, but a fraction are viewed side-on, producing the striped appearance. SAXS reveals that there are in fact two spacings of the stripes corresponding to hydrated (5.3 nm) and dehydrated (4 nm) bilayer structures (vide infra). The fraction of 4 nm striped structures was larger than for C16-KTTKS,27 and we believe that this is due to the reduced solubility of C16-

CD spectra spanning the range above and below the cac, extending up to 3 wt %. At lower concentration, there is a significant contribution from disordered structure, as expected. The spectra look similar to those previously reported by us for C16-KTTKS13 and, at some concentrations, those previously reported for other PA systems.33 At higher concentration, a maximum is observed at 206 nm with a smaller minimum at 227 nm. Our interpretation of these features in the spectra is that they result from red-shifted β-sheet peaks. The spectra for the β-sheet structure are expected to have a maximum at around 195−200 nm and a minimum near 216 nm.34,35 We have discussed elsewhere the influence of red shifting in the CD spectra of small peptides (AAKLVFF) that can result, for instance, from scattering due to the presence of extended fibrillar objects.36 FTIR spectroscopy (Figure 2b) proved to be insensitive to the β-sheet structure at low concentration (1 wt % and below). At 3 wt %, a strong peak is observed in the range of 1607−1615 cm−1 in the amide I′ region that is associated with β-sheet structure.33,37−39 5053

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Figure 5. As previously proposed for C16-KTTKS13 and also observed for conventional lipids, bilayer structures are expected

ETTES compared to that of C16-KTTKS. Also, the two PAs have different hydrophobicities as mentioned above. The nanostructure of the tapes was further probed using SAXS. Figure 4 shows combined SAXS/WAXS intensity

Figure 4. Combined SAXS/WAXS data for 0.8 wt % C16-ETTES measured at room temperature on beamline ID02 at the ESRF. The data is noisy in the high-q part of the SAXS data. Figure 5. Schematic of two configurations of C16-ETTES bilayers. (a) Dehydrated structure and (b) hydrated structure.

profiles (over a large q range) measured at room temperature for a 0.8 wt % solution in water. The SAXS data contain a strong peak assigned to the PA bilayer spacing of 5.3 nm (which is very similar to the value for C16-KTTKS,13 5.25 nm), along with an additional peak corresponding to a spacing of 2.9 nm. We associate the latter with the spacing of the stripes as revealed by TEM. The WAXS data shows peaks at 0.8, 0.46 and 0.39 nm, all of which were also identified in WAXS experiments on dried stalks (vide infra). In addition to measurements at room temperature, SAXS profiles were also obtained on heating 1 and 3 wt % solutions up to 70 °C (in steps of 2 °C at approximately 2 °C/min). No changes in the intensity profiles were observed, in contrast to our previous report on C16KTTKS for which a transition associated with lipid chain melting was observed on heating above 45 °C.13 Flow alignment of a 3 wt % solution of C16-ETTES was observed during and following shear in a Couette cell (data not shown), confirming the alignment of bilayer structures as for C16KTTKS.13 Additional detailed information on the self-assembled structure of C16-ETTES was obtained from the analysis of fiber X-ray diffraction experiments performed on a dried stalk. SI Figure 2 shows an XRD pattern that contains multiple rings (some showing alignment, as expected for a fiber containing aligned β-sheets) pointing to a high degree of ordering within the C16-ETTES assemblies. Corresponding peak positions obtained from the integration of the data to produce 1D intensity profiles are listed in Table 1. Importantly, the presence of several of the primary reflections was confirmed by in situ SAXS on a bulk sample as shown in Figure 4. SI Figure 3 shows a repeat measurement performed at a different synchrotron source (beamline I22 at Diamond). Peaks are present at d/nm = 5.30, 2.85, 1.74, and 0.87, in good agreement with several primary reflections in the XRD pattern (Table 1) and SAXS/WAXS on beamline ID02 (Figure 4). This confirms that drying to produce the stalk used for fiber X-ray diffraction does not disrupt the primary secondary structure. On the basis of our characterization through electron microscopy and X-ray scattering, we suggest a model for the self-assembled structures of C16-ETTES nanotapes illustrated in

to predominate. The presence of β sheets within the peptide headgroup layers is expected on the basis of spectroscopic analyses discussed above. We propose that two layer structures coexist in the system (Figure 5): a hydrated bilayer structure with a 5.3 nm periodicity and a 3 nm spacing corresponding to dehydrated bilayers. The hydrated bilayer spacing is consistent with interdigitated bilayers of the C16-ETTES molecules, estimated to be approximately 3.4 nm, comprising 1.8 nm for the C16 chain in an extended conformation plus 1.6 nm for the ETTES headgroup, assuming a parallel β-sheet structure. The 3 nm spacing is associated with a dehydrated monolayer. It may be noted that this model of coexisting hydrated and dehydrated peptide amphiphile bilayer structures is consistent with our recent report on C16-βAH.40 However, it differs from the previous model suggested for C16-KTTKS with SDS,27 in which we proposed a 4 nm stripe spacing perpendicular to the main 5.3 nm bilayer spacing. In fact, we cannot find a physical mechanism to explain a 3 to 4 nm spacing of isolated β sheets because the typical spacing of β sheets is 1 to 1.2 nm23,41 and furthermore SDS is expected to change the solubility of the PA. (An increase in fraction of the 4 nm spacing structure was observed with increasing SDS concentration.) For selfassembled PAs, the coexistence of hydrated and dehydrated bilayer structures is more physically realistic, especially considering the solubility as discussed shortly. In SAXS experiments, information on the relative intensity and width of the 5.3 nm spacing (bilayer spacing for C16ETTES) compared to the 3 nm spacing provides data on the proportion of the two structures present and on the correlation length of the two periodic structures, respectively. In the case of C16-ETTES, it is evident (Figure 4) that the 5.3 nm peak is both significantly more intense and sharper than the 3 nm spacing, pointing to a larger proportion of hydrated bilayers. As suggested by TEM images such as Figure 3, the correlation length of the 3 nm spacing over the width of the tapes extends only up to 10 repeats, leading to a correspondingly broad peak in the SAXS profile. 5054

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Mixtures of C16-ETTES and C16-KTTKS. These PAs each contain two ionizable residues as well as the carboxyl terminus. In C16-KTTKS, 97% of species are predicted to be doubly protonated (at one lysine and the carboxyl terminus), leading to a net charge of +1,42 as also calculated using the Henderson−Hasselbach equation.43 We have performed a similar calculation for C16-ETTES using HySS44 software with pKa(Glu1) = pKa(Glu2) = 4.445 and pKa(α-carboxyl) = 3.0.46 We note that pKa values may be shifted upon aggregation, as observed for other amphiphilic peptides,47,48 which indicates that 91% of species are singly protonated at the measured pH 5.4, leading to a net charge of z = 2.89. On the basis of these calculations, we therefore expect charge neutrality for a concentration ratio of approximately 3:1 C16-KTTKS/C16ETTES. In fact, the images of the samples shown in Figure 6 indicate that the solubility of the mixed system is greatest for mixtures

in part be due to differences between measured and calculated pKa values for the charged residues in the peptide,47,48 although a full study of this for our mixed PA system is beyond the scope of the current article. Also, the two PAs have different hydrophobicities, as discussed above. In addition, electroneutrality in the mixed system does not necessarily correlate to reduced PA aggregation and decreased turbidity. The images in Figure 6 were obtained at 65 °C, under conditions used for the thermal equilibration studies. At room temperature, all samples formed cloudy gels. The secondary structure of mixtures of the two PAs (C16ETTES and C16-KTTKS) in aqueous solution were probed using FTIR and CD spectroscopy for a series of selected mixture compositions. Figure 7 shows FTIR spectra covering

Figure 7. FTIR spectra in the amide I′ region for the mixtures indicated (C16-KTTKS/C16-ETTES wt %/wt %).

the amide I′ region. The spectra show a peak at 1609 cm−1 associated with β-sheet ordering. As previously reported,13 C16KTTKS also shows this feature, as does C16-ETTES (Figure 2). Each spectrum in Figure 7 also contains a strong peak at 1672 cm−1 associated with the binding of the TFA counterion to the lysine residues on C16-KTTKS.49−51 This feature was not observed in our previous measurements13 using the acetate salt of this PA and is not present in the spectrum for C16-ETTES (Figure 2) because the TFA does not bind to the acidic Glu residues. Another feature in the amide I′ region is the shoulder peak observed in all spectra at around 1585 cm−1, which is ascribed to the asymmetrical stretching of ionized carboxylate groups (at the C terminus and in the Glu residues in C16ETTES).38,48,52 There are no large qualitative differences in these spectra except that the intensity of the β-sheet peak at 1609 cm−1 relative to the TFA peak appears noticeably stronger for the 2:1 C16-KTTKS/C16-ETTES mixture; however, the baseline is also higher for this sample at low wavenumbers. Outside the amide I′ region, the FTIR spectra contain features at 1438−1439 and 1465 cm−1. The former is probably due to the CO stretching vibration in the CH3CO linkage between the lipid chain and the peptide headgroup or the C−CH3 asymmetrical stretch, and the latter is associated with the CH2/ CH3 scissoring mode vibration in the palmitoyl chain.27,39,52 The shoulder peak at around 1419 cm−1 is probably due to the symmetric CO2− stretch in the Glu residues of C16-ETTES and the C terminus of the ionized molecule.38,39,52 Circular dichroism spectroscopy (SI Figure 4) confirms the presence of β-sheet structures for all of the mixture compositions studied because all spectra exhibit a pronounced minimum at around 222 nm. This is slightly red-shifted

Figure 6. Image of vials containing mixtures of C16-KTTKS/C16ETTES at the ratios shown (wt %/wt %). Top (left, right): (i) 1:0.33 and (ii) 3:1. Bottom (left to right): (i) 1:1, (ii) 1:0.5, (iii) 1:2, (iv) 0.5:1, and (v) 2: 1. Pictures were taken for samples immediately following heating to 65 °C.

with 1:0.5 C16-KTTKS/C16-ETTES rather than 3:1 mixtures because the solution with 1:0.5 (wt%) C16-KTTKS/C16-ETTES is clear and transparent, and the next clearest solution is the 2:1 one. The others are cloudy to varying extents because of the formation of extended fibrillar structures. (As previously noted for C16-KTTKS,13 the aggregates have lengths extending up to tens of micrometers, which causes light scattering.) As shown shortly by spectroscopic and microscopic methods, β-sheet fibrils are present for all of these mixtures; therefore, the clarity of the 1:0.5 ratio (C16-KTTKS/C16-ETTES) mixture is not due to the break-up of fibrillar structures but must instead be due to the formation of less extended aggregate structures. These images show the modulation of solubility possible by mixing PAs containing oppositely charged residues. The reason that the 3:1 ratio mixtures does not show the greatest solubility may 5055

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Figure 8. TEM images for samples with C16-KTTKS/C16-ETTES mixtures. (a) 1:0.5, (b) 1:1, and (c) 2:1 . The scale bar represents 1 μm.

compared to the canonical position of this minimum for βsheets at 216 nm, probably because of light-scattering effects arising from the extended aggregates.36 The β-sheet minimum is most pronounced for the clear sample, consistent with reduced light scattering in this case. The spectra are notably different from those for the individual PAs. The spectra for C16ETTES in Figure 2a show strong maxima at 206 nm, except at the lower concentrations. Our previously published spectra for C16-KTTKS (acetate salt) show maxima at 210 nm at low concentration and the 217 nm β-sheet minimum develops only at high concentration (5 wt % and above).13 The spectra in SI Figure 4 therefore suggest a co-operative coassembly mechanism in the mixtures. Furthermore, compared to C16ETTES on its own there seems to be a reduction in the red shifting of the β-sheet minimum, consistent with reduced light scattering effects due to aggregation.23,41 To eludicate the β-sheet structure, X-ray diffraction was performed on dried stalks. Typical cross-β patterns were observed (similar to those in SI Figure 2), consistent with βsheet structures.41 From these patterns, sector integration produced intensity profiles from which d spacings were obtained. These are listed in Table 1 for the mixtures and the two PAs. Characteristic β-sheet features include the 4.7 to 4.8 Å spacing of the β strands and the 11 to 12 Å spacing of the β sheets. The nanostructure of self-assemblies in mixtures of C16KTTKS and C16-ETTES was examined by electron microscopy and SAXS. Figure 8 shows representative TEM images. The clear sample (1:0.5 C16-KTTKS/C16-ETTES) shows relatively straight twisted tapes (Figure 8a); however, most other samples (cloudy, Figure 6) showed well-defined tapes coexisting with clusters of more diffuse/branched aggregates with a lower persistence length. (Figure 8b is a representative image.) The sample with 2:1 C16-KTTKS/C16-ETTES (translucent, Figure 6) showed long, wide tapes with a large persistence length (Figure 8c). The tape and fibril aggregates are highly extended and can be observed by optical microscopy (Figure 9). The aggregates for the 2:1 C16-KTTKS/C16-ETTES sample (Figure 9c) seem straighter and wider than for the other two mixtures shown in Figure 9a,b, consistent with TEM. The exceptional length of these nanotapes is noteworthy and may be relevant to applications where highly extended nanostructures are desired. SAXS confirmed the presence of bilayer structures, through the observation of a Bragg peak for all samples (Figure 10), with a spacing of 5.3 to 5.6 nm. On the basis of our analysis for the individual PAs, this is proposed to correspond to a hydrated bilayer structure. A state of mixed fibrils was achieved through thermal treatment, heating to 60 °C above the lipid chain melting transition, and cooling back to 20 °C. The Bragg peak

Figure 9. Optical microscope images for samples with C16-KTTKS/ C16-ETTES mixtures. (a) 1:0.5. (b) 1:1, and (c) 2:1.

is well-defined for the samples studied, except 2:1 C16-KTTKS/ C16-ETTES in which case the peak is very broad. It is noted that this sample also showed differences in the morphology by TEM compared to the other samples (i.e., very broad tapes 5056

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presumed to be an electrostatic interaction between the peptide headgroups, leads to enhanced β-sheet formation compared to single-component solutions of either PA at the same concentration, as characterized by CD spectroscopy, and FTIR spectroscopy. Our findings are in accord with previous research that shows that E-K electrostatic interactions in mixtures of peptides or PAs with oppositely charged residues can stabilize β-sheet fibrils.17,20−22a,b This in turn can lead to hydrogelation or other changes in macroscopic properties. In a similar vein, interactions between lysine and glutamic acid residues are widely used to stabilize coiled coil structures in designed peptides25,53 and polymer−peptide conjugates.54 Here the electrostatic interactions between C16-KTTKS and C16ETTES lead to the formation of large fibrillar aggregates within cloudy hydrogels. In PA systems, the influence of bilayer hydration forces needs further examination, and thermal dehydration effects will be the subject of ongoing research.

Figure 10. SAXS profiles (shifted vertically by arbitrary scale factors for ease of comparison) for mixtures of C16-KTTKS and C16-ETTES (compositions shown) after thermal treatment.

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that, according to SAXS, have an accompanying dispersity in bilayer spacing). To eludicate the possible effect of cofibrillization versus the self-sorting of fibrils of each component, the mixing protocol was varied. SI Figure 5 shows the SAXS profiles corresponding to the 2:1 mixture. In some cases, the profiles exhibit two peaks before the thermal treatment procedure, pointing to the formation of two populations of bilayer structures, suggesting possible self-sorting. This effect was eliminated by thermal treatment that produced the SAXS profiles containing a single bilayer peak as shown in Figure 10. It is also possible that dehydration plays a role at high temperature because the peaks after heating generally show a shoulder at high q, indicating the presence of a structure with a smaller spacing such as the dehydrated bilayer structure. However, pronounced Bragg peaks corresponding to 3 nm dehydrated bilayer structures, proposed for C16-ETTES (Figure 5), were not observed in most cases (Figure 10 and SI Figure 5). Further studies are planned on possible thermally induced bilayer dehydration processes.

ASSOCIATED CONTENT

S Supporting Information *

TEM images obtained from a 1 wt% solution of C16-ETTES. Fiber X-ray diffraction pattern obtained from a stalk dried from a 3 wt % solution of C16-ETTES. Synchrotron SAXS data for a 1 wt % solution of C16-ETTES at 20 °C, measured on I22 at Diamond. CD spectra for the mixtures indicated. SAXS profiles from experiments on the 2:1 C16-KTTKS/C16-ETTES solution subjected to different mixing protocols. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by EPSRC grants EP/F048114/1 and EP/G026203/1 to I.W.H. Use of the Chemical Analysis Facility at the University of Reading is acknowledged. The assistance of Laura Felisari at the Centre for Advanced Microscopy at the University of Reading is acknowledged. Cryo-TEM was performed in the Wolfson Bioimaging Facility at the University of Bristol, and we thank Judith Mantell for assistance. Some of the synchrotron SAXS was performed at Diamond Light Source (beam time reference 6401-1). We thank Marc Malfois for assistance with the experiments. We are grateful to Javier Perez for assistance with SAXS at SOLEIL (beam time reference 20110562). Beam time at the ESRF on beamline ID02 was awarded under reference SC3468, and we thank Dr. Theyencheri Narayanan for assistance. We thank Dr. Juan Miravet (Universitat Jaume I) for helpful discussions.

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CONCLUSIONS A designed PA, C16-ETTES self-assembles into extended nanotapes, a small fraction of which exhibit a stripe pattern. On the basis of TEM images and the analysis of SAXS profiles, a model is proposed for the nanostructure that comprises bilayers with hydrogen-bonded peptide amphiphile headgroup arrays. The majority of the PA self-assembles into a hydrated bilayer structure. The two bilayer spacings within lamellar structures depend on the solubility that can be adjusted by mixing C16-ETTES with C16-KTTKS. The two bilayer spacings of 3 and 5.3 nm are assigned to dehydrated and hydrated bilayer structures, respectively. These spacings can be imaged when tapes are presented edge-on in TEM and cryo-TEM images. Differences in the self-assembly of C16-ETTES, compared to that of C16-KTTKS, include the reduced cac and the increased fraction of dehydrated bilayer structures. Both of these features are due to the reduced solubility of C16-ETTES. Indeed, this PA is only partially soluble at room temperature. Mixing C16-ETTES with the PA C16-KTTKS containing lysine residues complementary to the glutamic acid residues in C16-ETTES enables the aggregation of tapes to be tuned at an appropriate mixture composition. The cloudiness is ascribed to the formation of partially soluble extended (>micrometersized) fibrillar aggregates. The interaction between the PAs,

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