Article pubs.acs.org/Langmuir
Supramolecular Assembly of Asymmetric Self-Neutralizing Amphiphilic Peptide Wedges Dara Van Gough,† Jill S. Wheeler,† Shengfeng Cheng,‡,§ Mark J. Stevens,‡ and Erik D. Spoerke*,† †
Electronic, Optical, and Nano Materials, and ‡Computational Materials and Data Science, Sandia National Laboratories, Albuquerque, New Mexico 87185-1411, United States S Supporting Information *
ABSTRACT: Mimicking the remarkable dynamic and multifunctional utility of biological nanofibers, such as microtubules, is a challenging and technologically attractive objective in synthetic supramolecular chemistry. Understanding the complex molecular interactions that govern the assembly of synthetic materials, such as peptides, is key to meeting this challenge. Using molecular dynamics simulations to guide molecular design, we explore here the self-assembly of structurally and functionally asymmetric wedge-shaped peptides. Supramolecular assembly into nanofiber gels or multilayered lamellar structures was determined by cooperative influences of hydrogen bonding, amphiphilicity (hydrophilic asymmetry), and the distribution of electrostatic charges on the aqueous self-assembly of asymmetric peptides. Molecular amphiphilicity and β-sheet forming capacity were both identified as necessary, but not independently sufficient, to form supramolecular nanofibers. Imbalances in positive and negative charges prevented nanofiber assembly, while the asymmetric distribution of balanced charges within a peptide is believed to affect peptide conformation and subsequent self-assembly into either nanofibers or lamellar structures. Insights into cooperative molecular interactions and the effects of molecular asymmetry on assembly may aid the development of next-generation supramolecular nanomaterial assemblies.
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INTRODUCTION In biology, dynamic, high aspect ratio protein nanofibers enable a wide range of critical functions within cells. Microtubules (MTs), for example, facilitate chromosome separation during cell division, are a key constituent of the reconfigurable cytoskeleton, and direct the transport of intracellular cargo.1 These protein filaments are noncovalently, reversibly polymerized from heterodimers of α- and β-tubulin whose dynamic assembly is dictated by changes in molecular conformation and cooperative assembly.1 Developing synthetic analogues to these versatile, multifunctional nanofibers is a tantalizing materials chemistry target that would stand to impact a wide range of technical fields. Exactly replicating these complex materials, however, is not only synthetically impractical, but may not even be desirable for certain applications. To make this synthetically daunting challenge more tractable and widely applicable, we looked to molecular dynamics (MD) simulations for a distillation of fundamental design elements critical to nanotube assembly. Cheng et al.2 recently developed an MD model to simulate formation of nanotubes. This work revealed that asymmetry of both molecular shape and the chemistry that governs intermolecular attraction critically affected the morphology of simulated supramolecular assemblies. Figure 1 schematically summarizes the importance of shape asymmetry and the asymmetric distribution of interaction sites on the building © 2014 American Chemical Society
blocks in determining nanotube morphology. These simulated results indicate that a wedge-shaped building block with asymmetrically distributed interaction sites preferentially forms nanotube architectures. Note, however, that within this scheme, the relative strength of “vertical” versus “lateral” interactions also had a significant influence on assembly.2 Inspired by these observations, the present work explores a series of asymmetric, wedge-shaped peptides as building blocks for supramolecular assembly. Peptides are particularly wellsuited for these studies, as the controllable versatility of peptide chemistry facilitates systematic variation of building block composition and properties. In addition, there is precedence for the assembly of peptides, particularly linear amphiphilic peptides, into one-dimensional nanostructures such as nanofibers, nanoribbons, and nanotubes,3−10 engineered to promote a range of cellular, biomolecular, and materials interactions.3,11−19 Limited reports also describe precedence for the assembly of nonlinear, branched peptides, frequently designed to facilitate epitope accessibility or tuning of gelation properties in peptides for biomedical applications.14,20−22 In many of these chemically diverse systems, factors such as amphiphilicity, β-sheet formation, and electrostatic charge are Received: April 26, 2014 Revised: July 3, 2014 Published: July 8, 2014 9201
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blocks. Figure 1 shows a set of different monomers and structures that assemble in molecular dynamics simulations of these monomers. In Figure 1a, there are two pairs of binding sites (faces) on opposite sides of a rhombic triacontahedron (C80 buckyball), which is used to define points on an effectively spherical object. The only attractive interactions occur between sites with the same color (excluding gray). All other interactions are repulsive and yield the overall effective spherical shape. Because of the six-fold symmetry of the binding site (i.e., face), two monomers can bind with six orientations. Consequently, the structure of an assembly is a random, fractallike structure shown on the right of Figure 1a. If the symmetry of the binding region is reduced as in Figure 1b, where again attraction is only between like-colored sites, then the monomers can assemble into flat sheets as shown in the figure. To form tubules, the geometry of the monomer was changed from spherical to a wedge. The monomer’s shape is given by a rigid 3 × 3 × 3 set of gray spheres in the figure. As before, gray spheres interact only by a purely repulsive potential and just give the monomer its shape. The colored spheres in Figure 1c and 1d represent the attractive (binding) sites. For more details of the molecular dynamics simulations used in this model, see Cheng et al.2 In Figure 1d each binding surface has two distinct sites to control the orientation of the two binding monomers. This effect is demonstrated by the difference in assemblies shown in Figure 1c. For the binding sites in Figure 1c, there is a symmetry that allows two orientations of binding. Transforming between the two orientations just involves rotating one monomer by 180°. As shown in the figure, when these two orientations are possible, the system assembles not into tubules but into other geometries, which include a complicated twodimensional surface. When this symmetry is broken and only one orientation is allowed, then the monomers assemble into tubules. Effective tubule formation, then, resulted from the collaboration of several key building block characteristics: (1) an asymmtric wedge shape, (2) asymmetry of interaction sites to orient the wedges for assembly, (3) favorable attraction (as opposed to repulsion) of building blocks, providing both “lateral” and “vertical” attractive interactions needed to form the extended cylindrical structures. Figure 2 shows the chemical structure of the most asymmetric peptide design, Asymm-Wedge, inspired by the asymmetric wedge in Figure 1d. These molecules were designed to comprise hydrophilic, charged branches connected to a relatively hydrophobic, tri-isoleucine (tri-Ile) trunk. The wedge shape of the peptides introduces a structural asymmetry to the building blocks, following the models of wedge-shaped nanotube assemblers described above and, more generally, the conical building blocks described by Isaelachvili.23,24 The isoleucine trunk serves to simultaneously introduce amphiphilicity to the peptide (hydrophilic asymmetry) while the branched side chains of the isoleucine residues are expected to favor β-sheet formation.25 The amphiphilic character is expected to provide the driving force and molecular orientation needed for micelle assembly, while the wedge shape and βsheet formation are expected to direct the formation of these micelles into one-dimensional extended fibers or nanotubes.4−6,8,26−28 Finally, the electrostatic charges in the branches introduce potential attractive or repulsive forces between molecules. The remainder of this paper will focus on exploring how these key molecular characteristics influence nanostructure self-assembly.
Figure 1. Structures of monomers and assembled monomers from molecular dynamics calculations. Within the monomers, gray spheres represent purely repulsive sites that yield the monomer shape. Attraction occurs only between sites with same (nongray) color. Highly symmetric, effectively spherical, building blocks (a) selfassemble into an unordered agglomerate, while monomers with broken symmetry (b) assemble into sheet-like structures. In a similar vein, a wedge-shaped monomer will naturally assemble into hollow tubes, if the binding symmetry is broken (d), but if not, then wavy sheets are formed as two orientations of bound pairs randomly assemble (c).
common elements that strongly influence assembly. In the present work, we evaluate these interactive forces as elements of an asymmetric building block design and explore their influences on molecular self-assembly. Motivated by the results of the MD simulations described above, we focus on asymmetric wedge-shaped peptides designed to incorporate key elements of the MD model to facilitate 1D nanostructure assembly. We specifically investigate the influences of amphiphilicity (hydrophilic asymmetry), hydrogen bonding, and the asymmetric distribution of self-neutralizing positive and negative electrostatic charges within the branches of these wedge-shaped peptide building blocks impact self-assembly.
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RESULTS AND DISCUSSSION As mentioned above, results from MD simulations of nanotube assembly were used to guide the design of the peptide building 9202
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Within the branches of these peptides there is a mix of positively and negatively charged residues as well as multiple hydrogen bonding groups collectively intended to promote intra- and intermolecular attraction of the peptide branches. One of the advantages of using peptides as building blocks is that it allows for the incorporation of functional peptide sequences to mediate biomolecular or materials interactions. In many cases, the sequences of these mimics include a mix of positive and negative charges, and in the present work, we evaluate the potential impact of where these charges are positioned within a peptide on the subsequent assembly of these structures. Dissolved in water at concentrations as low as 5 mg/mL (0.5 wt %) at near-neutral pH, Asymm-Wedge initially forms a freeflowing liquid solution that spontaneously transforms to become a self-supporting gel, composed of assembled nanofibers and nanofiber bundles, revealed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) in Figures 3, parts b, c, and d, respectively. Figure 3a schematically depicts the molecular assembly and nanofiber bundling. These amphiphilic molecules are believed to assemble into fibers, guided by the sequestration of the relatively hydrophobic isoleucine trunks from the aqueous environment and β-sheet formation as these isoleucines pack together. Subsequent electrostatic, hydrogen bonding, or van der Waals interactions likely promote secondary assembly into nanofiber bundles. Figure 3b shows a scanning electron microscope (SEM) image of a lyophilized nanofiber gel, revealing both individual and bundled nanofibers within an interwoven supramolecular network. The inset in Figure 3b shows the self-supporting nanofiber gel at the top of an inverted vial. The TEM image of negatively stained
Figure 2. Chemical structure of Asymm-Wedge highlighting the charge asymmetry and the tri-Ile trunk. Orange circles highlight negative charges, while purple circles highlight positive charges.
Figure 3. (a) Schematic depicting the assembly of Asymm-Wedge in to nanofiber bundles. (b) Scanning electron microscope image showing bundles of Asymm-Wedge nanofibers. Inset: Self-supporting gel formed from Asymm-Wedge at 5 mg/mL in water at neutral pH. (c) Negatively stained transmission electron microscope (TEM) image of a self-supporting gel. (d) High resolution AFM image of bundled nanofibers. Color scale (right) spans +7.5 nm (white) to −7.5 nm (black). 9203
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Figure 4. (a) FTIR spectrum (black) and deconvolution (gray curves) for Asymm-Wedge. The summation of the deconvoluted peaks is shown as the dashed red overlaying the black spectrum. (b) Circular dichroism spectra for Asymm-Wedge (black) and Asymm-SSS (blue).
combined strong negative peak near 220 nm and the strong positive peak near 190 nm. Formation of β-sheets along the length of supramolecular nanofibers is believed to be important in the formation of the extended nanofiber morphology.4,8,26−28,33 In fact, in analogy to the MD-simulated structures above, these extended β-sheets provide the longrange “vertical” interactions needed to extend the cylindrical micelles into fibers. For Asymm-Wedge, though, the tri-Ile trunk serves double duty, providing both the critical β-sheet forming capacity of the peptides as well as critical amphiphilic character in the peptide. Amphiphilicity is widely recognized as an important driving force for micelle assembly, as the incentive to sequester poorly solvated moieties, such as hydrophobic Ile side chains, from aqueous solvent drives molecular aggregation and packing. The role of peptide amphiphilicity in the present system was explored by replacing the Ile trunk with a tri-serine trunk in to create Asymm-SSS, Figure 5a. Improved solvation of the polar
nanofibers in Figure 3c and the AFM image in Figure 3d both confirm the presence of bundled nanofibers observed in the SEM image in Figure 3b. Nanofiber dimensions, determined from all three images, consistently measure 4.5 ± 0.6 nm in diameter but extend to hundreds of nanometers in length. In a fully extended conformation, the peptides are estimated to be ∼3.8 nm long. Because the nanofibers are only 4.5 nm wide, however, it is clear that the peptides do not assemble end-toend in a fully extended conformation (like spokes on a bicycle.) Although the peptides are believed to assemble in a radial fashion, these data indicate that the peptide branches partially collapse in on one-another, driven by electrostatic attraction between oppositely charged residues. The balance of positive and negative charges present on Asymm-Wedge is critical for the observed spontaneous assembly. In other systems, rich in charged amino acids, electrostatic repulsion from imbalances in charge prevents assembly; only when the charges are neutralized by pH, cationic binding, or secondary molecules does assembly occur.6,21,28,29 Similarly, when Asymm-Wedge is dissolved in strongly acidic (pH 2) or strongly basic (pH 13) solutions, the resulting selective neutralization of either the anionic or cationic residues, respectively, produces a net charge on the peptide and prevents supramolecular assembly. Under neutral conditions, however, because both the positive and negative charges are simultaneously present on the opposing branches of this wedge peptide, Asymm-Wedge spontaneously assembles. This behavior may prove particularly relevant to biomedical applications, for example, in which in situ supramolecular assembly is required, but the addition of secondary chargeneutralizing agents may be undesirable. Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopies were used to characterize the supramolecular character of these nanofibers. The composition of the amide I peak in FTIR provides key information about the secondary structure of peptides and proteins, and Figure 4a shows the deconvoluted amide I peak of Asymm-Wedge. Peaks at 1621 cm−1 and 1633 cm−1 can be assigned as β-sheet and aggregated β-sheet components,30,31 while the peak at 1657 cm−1 is attributed to α-helical components of the peptide.30 The peak at 1673 cm−1 is believed to be due to a small amount of residual trifluoroacetic acid (TFA) present in these synthetic peptides.32 Circular dichroism (Figure 4b) confirms the presence of β-sheet in Asymm-Wedge, evidenced by the
Figure 5. (a) Schematic showing the chemical structure of AsymmSSS. (b) SEM image of structure formed from the assembled wedgeshaped peptide. Inset: At 5 mg/mL in water at neutral pH, AsymmSSS did not form a self-supporting gel. A small defect in the material is visible in the bottom of the micrograph.
hydroxyl side chains of the serines in Asymm-SSS34,35 would be expected to eliminate the amphiphilic driving force for molecular aggregation and self-assembly. In fact, Asymm-SSS did not self-assemble to form a gel, and there was no discernible assembled structure evident by SEM (Figure 5b) or TEM (Supporting Information Figure 1). Deconvolution of the amide I peak in FTIR showed no evidence of β-sheet, revealing 9204
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Figure 6. Amino acid sequence structures for (a) Symm-Vert and (b) Symm-Horiz. SEM images show (c) fibers assembled from Symm-Horiz and (c, d) multilamellar sheets formed from Symm-Vert at high and low magnification. Sheets curled into hollow spheres are visible in part e. SymmHoriz formed a self-supporting gel (inset c), while Symm-Vert did not (inset d) at 5 mg/mL in water at neutral pH.
only a peak at 1646 cm−1, attributed to unordered peptide, and a minor peak at 1673 cm−1 from residual TFA. These observations were further supported by the change in the shape of CD spectra, visible in the spectrum in Figure 4b; the strong negative peak near 195 nm indicates largely unordered material. These data collectively confirm the lack of β-sheet secondary structure in this material, despite the fact that serine is expected to be a competent (if not as proficient as Ile) βsheet forming amino acid.25 This behavior is particularly telling with respect to the importance of the amphiphilicity in these materials. Not only does the improved solvation of the tri-Ser trunk eliminate the driving force for molecular aggregation, but it also addresses molecular orientation in the present model. In Asymm-Wedge, the relatively poor solvation of the Ile side chains compared with the excellent solvation of the hydrophilic, charged peptide branches creates a hydrophilic asymmetry in the molecule that serves to orient the wedge peptides for assembly as well. The MD-simulated wedge in Figure 1c clearly shows that without asymmetric orientation, tubule formation is not favored. Similarly, when the orienting amphiphilicity of the wedge peptide was removed in Asymm-SSS, undirected intermolecular electrostatic and hydrogen bonding interactions produced a disordered structure. Moreover, in Asymm-Wedge, this orienting influence collaborated with hydrophobic aggregation of the poorly solvated Ile side chains to provide the molecular organization and proximity needed to facilitate the effective βsheet formation so important for nanofiber formation. Simply incorporating a β-sheet-competent trunk (SSS) into the peptide did not produce a sufficient driving force for nanofiber assembly (or measurable β-sheet formation); the cooperative influence of the β-sheet formation and the organizing influences of amphiphilicity (hydrophilic asymmetry) produced effective nanofiber assembly in this system. This cooperative influence was further supported when the hydrogen bonds responsible for β-sheet formation of AsymmWedge were thermally disrupted. When gels were heated to 85
°C, the self-supporting integrity of the gel was disrupted, transforming the gel into a free-flowing liquid. Upon cooling, the self-supporting gel reformed. By thermally disrupting the hydrogen bonds forming the β-sheets within these assemblies, the nanofibers responsible for gel formation became sufficiently disordered that they were unable to support gelation. Only when these hydrogen bonds were allowed to reform was the network of stable nanofiber assemblies reconstituted and the gel reformed. Interestingly, this process was demonstrated through multiple cycles. The reversibility of this assembly represents an interesting parallel between these synthetic assemblies and natural biological nanofibers, like microtubules or actin, though the molecular mechanics of these processes are admittedly very different. In addition to the balance of charges described above, the distribution of electrostatic charges on the peptide branches was varied to investigate how asymmetry of electrostatic interactions might influence molecular assembly. The extreme pH studies described above clearly showed that having both positively charged and negatively charged amino acids present on the peptides is important for assembly, ostensibly because these charges are expected to attract, bind, and neutralize opposite charges both intramolecularly and intermolecularly. Figure 6, parts a and b, shows two peptides with compositions nominally identical to Asymm-Wedge, but in which the charged residues were positioned more symmetrically. In Symm-Vert (Figure 6a), each of the two positive or negative charges were placed on the same branch of the peptide. In Symm-Horiz (Figure 6b), the negative charges were both placed near the crook of the branches, while the positive charges were both placed near the terminal ends of the branches. The scanning electron micrographs in Figure 6c−e show how these variations affected supramolecular assembly. Like Asymm-Wedge, Symm-Horiz also formed a self-supporting nanofiber gel at 5 mg/mL (Figure 6c). FTIR analysis of these materials further showed that nanofibers formed from SymmHoriz contained β-sheet, aggregated β-sheet, and α-helix, 9205
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Figure 7. Illustration of the intramolecular interactions, proposed method of folding, and assembly of the wedge-shaped peptides. (a) Asymm-Wedge possesses the most asymmetry and contains the most intramolecular interactions, collapsing the molecule into a relatively broad cone that favors cylindrical nanofiber assembly. (b) Charge placement on Symm-Horiz promotes branch folding, and a broadly conical shape, which again promotes the formation of nanofibers. (c) Lateral electrostatic interactions between branches in Symm-Vert drive produce a more extended conical conformation that promotes self-assembly into bilayered assemblies. Secondary interactions between assembled nanofibers or bilayers would promote nanofiber bundling or multilamellar stacking shown on the right (a−c).
A recent study by Ting et al.36 used self-consistent field theory (SCFT) to examine a model branched peptide system similar to that described here. In this study, electrostatic charges positioned in the branches of the peptide structures forced either a collapsed or extended branch morphology. Moreover, the more collapsed morphology favored the self-assembly of fibers, while the more extended conformation favored the selfassembly of sheets. On the basis of similarities between this model system and the present peptide structures, we propose that the intramolecular and intermolecular attraction of opposite charges in the peptide branches affects the geometry of the assembling peptides, ultimately influencing the observed self-assembled morphologies. Figure 7 provides a simplified summary of these effects for three self-assembling wedge peptides, nominally identical in composition, but in which the positions of electrostatic charges in the peptide branches vary. Note that although these models are consistent with models produced by SCFT, the structures shown are simplified schematics drawn to aid visualization of the proposed scheme, not simulated molecular structures. With Asymm-Wedge, we propose that electrostatic attraction between the asymmetrically positioned charges both draws the two branches together and collapses the distal ends of the branches toward the central part of the molecule, Figure 7a. The average, collective effects of these two processes creates the moderately conical molecular building blocks needed for cylindrical micelle formation. The 4.5 nm nanofibers, seen in Figure 3, support this model of partial branch collapse and molecular assembly. Following this model, the distribution of charges on Symm-Horiz, which also forms nanofibers, would
evidenced by deconvoluted amide I peaks at 1621, 1633, 1657 cm−1 (Supporting Information Figure S4). In contrast, SymmVert did not form a self-supporting gel, even at double the peptide concentration. Examination of lyophilized Symm-Vert by SEM revealed the predominant formation of sheet-like structures, Figure 6d,e. These sheet-like assemblies are one hundred to several hundreds of nanometers thick, and we propose that these structures are multilamellar stacks of peptide bilayers. Of particular interest are the hollow spherical structures, Figure 6e, ostensibly formed from assembled sheets rolled up to form the observed hollow spheres. Again using FTIR to provide a measure of secondary structure, deconvolution of the amide I peak for Symm-Vert shows that peaks representing β-sheet, aggregated β-sheet, and α-helix are present at 1621, 1633, and 1657 cm−1. (Supporting Information Figure S4). In this case, the β-sheet character of these assemblies did not direct the formation of nanofibers but drove the formation of lamellar sheets. The predominance of these discrete sheet-like structures, seen in Figure 6d,e, accounts for the failure of these assemblies to form selfsupporting gels more commonly formed from interpenetrating networks of one-dimensional nanostructures such as nanofibers. Such bilayers would be expected if the peptide wedges adopted a more extended conformation, as the aspect ratio of assembling amphiphiles has a strong effect on supramolecular structure.23 Severely conical molecules would be expected to produce spherical micelles, moderately conical molecules would produce cylindrical micelles (nanofibers), and slightly conical or rod-shaped molecules would form bilayers.23 9206
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also promote branch collapse toward the central portion of the molecule, Figure 7b. As with Asymm-Wedge, this collapse produces the relatively conical building blocks needed for formation of the observed nanofibers. In contrast, however, we suggest that the distribution of opposite charges in Symm-Vert draws the peptide branches together in a predominantly extended conformation, creating more rod-shaped building blocks. These rod-shaped building blocks would be expected to produce bilayer assemblies, Figure 7c.23 The bilayer structure accommodates the extended molecular conformation, while allowing for sequestration of hydrophobic isoleucine trunks from the aqueous environment and the formation of β-sheets within the layered assemblies. Moreover, the top and bottom of each bilayered structure are decorated with the rich chemistries of the peptide branches. Just as similar chemistries displayed on the exterior of the nanofibers lead to fiber bundling, cooperative electrostatic, hydrogen bonding, and van der Waals forces would be expected to promote secondary assembly of these layered structures into multilamellar sheet-like constructs such as those seen in in Figures 6d, 6e. Although it does not appear that these peptides assume the exact hollow tubular morphology described by the MD simulations, extended nanofibers formed from simulationinspired Asymm-wedge do present a number of interesting parallels to the simulated system. The influences of molecular morphology, amphiphilicity, electrostatic interactions, and hydrogen bonding influence key factors of asymmetric molecular shape, molecular orientation, and molecular attraction identified as important for one-dimensional (nanotube) formation. The conical shape of the collapsed branched peptides influenced the morphology of the assemblies. Molecular amphiphilicity not only provided the driving force for molecular aggregation/assembly but served to orient the asymmetric peptide wedges for micelle formation. Imbalances in electrostatic charges prevented self-assembly through local molecular repulsion. Meanwhile, attractive hydrogen bonding between peptides produced the β-sheet structures (“vertical” interactions) needed for extended nanofiber formation.
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EXPERIMENTAL SECTION
Materials. N-Fmoc-protected amino acids were purchased from Novabiochem. Diisopropylethylamine (DIEA), trifluoracetic acid (TFA), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), tetrakis(triphenylphosphine)palladium(0), dichloromethane, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N-methylmopholine, acetic acid, triisopropylsilane, acetic anhydride, diethyl ether, piperidine, sodium diethyldithiocarbamate, and N,Ndiisopropylethylamine were purchased from Sigma-Aldrich and used as received. Peptide Synthesis. The peptide trunks and primary branches for Aysmm-Wedge, Asymm-SSS, and Symm-Vert were synthesized C to N from a rink amide resin using standard Fmoc solid-phase peptide synthesis protocols on a Liberty Peptide Synthesizer (CEM Corp., Matthews, NC). Upon completion of the peptide backbone (for Asymm-Wedge, the backbone sequence was IIIKRNSQE, C to N), the sample was rinsed with N-methyl-2-pyrrolidone and dichloromethane. The deprotected amine terminus was then acylated using 10% acetic anhydride in N,N-dimethylformamide for 30 min; reaction completion was verified with a ninhydrin test. The secondary branches were synthesized from the Alloc-protected side group of a lysine residue. The Alloc group, an orthogonal protection group to both base and acid deprotection chemistries, was removed during a 2 h treatment in a solution composed of tetrakis(triphenylphosphine)palladium(0) (0.69 g), chloroform (5.08 mL), acetic acid (0.275 mL), and Nmethylmorpholine (0.138 mL). Excess reagent was removed by rinsing with 0.5% N,N-diisopropylethylamine and then with 0.5% sodium diethyldithiocarbamate (both in N,N-dimethlyformamide). The peptide was then rinsed with both N-methly-2-pyrrolidone and dichloromethane and loaded back onto the Liberty Peptide Synthesizer for branch growth. The peptide was cleaved from the resin during 3 h in a solution containing trifluoroacetic acid (95%), water (2.5%), and triisopropylsilane (2.5%). The peptide was then precipitated from solution with cold diethyl ether (−40 °C) and washed three times with cold diethyl ether using centrifugation. Finally, the peptide product was dried under vacuum overnight. For Symm-Horiz, because the branches are composed of the same amino acid sequences, both branches were simultaneously synthesized from a lysine residue with both amine groups protected with Fmoc groups. Successful peptide syntheses were confirmed by mass spectrometry (University of Minnesota Mass Spectrometry, Minneapolis, MN). Peptide Assembly. Peptides were assembled in aqueous solution concentrations of 5 mg/mL (0.5 wt %) peptide in water. In a typical experiment, 400 μL of deionized water was added to 2 mg of peptide, and the solution pH was adjusted to 7 by adding 0.01 M NaOH. The peptide solutions were vortexed and sonicated to fully dissolve the peptide. The solutions were then heated to 85 °C and allowed to assemble at room temperature overnight. (Assembly will typically occur within tens of minutes, but the extended self-assembly time was used to provide consistent time for potentially kinetically limited systems to assemble.) Sample Characterization. FTIR and SEM analysis were performed on lyophilized peptide assemblies. FTIR spectra were collected on a Varian 2000 FT-IR Scimitar Series equipped with a Pike MIRacle ATR attachment, and SEM analysis was performed using a Zeiss Supra 55VP Field Emission SEM. For TEM analysis, Approximately 3 μL of peptide gel was allowed to dry onto copper holey carbon TEM grids. The samples were then negatively stained with 2% phosphotungstic acid and allowed to air-dry before analysis in a Phillips CM30 TEM. AFM imaging was performed on nanofiber mats created by slowly drying 0.1 mg/mL aqueous peptide onto clean silicon substrates. Images were obtained using peak force tapping mode on a Bruker Dimension Icon AFM, scanning with a 2 nm ScanAsyst tip. Circular dichroism was performed on a Jasco J-815 CD spectrometer using a quartz cuvette with a 0.1 cm path length. Assembled peptides were diluted from 5 mg/mL gel assemblies to ca. 0.1 mg/mL. Molecular Simulations. The dynamics of self-assembly were obtained by performing MD simulations using the LAMMPS
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CONCLUSIONS Here we have presented the simulation-inspired design and synthesis of a series of asymmetric, self-neutralizing, wedgeshaped peptides. Supramolecular assembly of these peptides into nanofiber gels or multilayered lamellar structures was found to depend on cooperation between molecular amphiphilicity, hydrogen bonding, and electrostatic interactions. Molecular amphiphilicity (hydrophilic asymmetry) and βsheet formation, defined by the peptide “trunk”, were both identified as necessary but not independently sufficient drivers for the formation of supramolecular nanofibers. That balance and distribution of electrostatic charges with the peptide “branches” also strongly affected self-assembly. Imbalances in positive and negative charge prevented nanofiber assembly, while the asymmetric distribution of balanced charges within the branches is believed to affect peptide conformation and subsequent self-assembly into either nanofibers or lamellar structures. These insights into the importance of cooperating molecular interactions and the effects of molecular asymmetry on assembly are key to developing the next generation of supramolecular nanomaterial assemblies. 9207
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simulation package.37 In these simulations, each monomer is treated as a rigid body. Two particles in distinct monomers interact purely repulsively through the Lennard−Jones (LJ) potential. The potential is truncated at 1.0 to make the interaction purely repulsive. The attractive interaction is given by a cosine potential
(5) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001, 294, 1684−1688. (6) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Peptide-amphiphile nanofibers: A versatile scaffold for the preparation of self-assembling materials. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5133−5138. (7) Missirlis, D.; Chworos, A.; Fu, C. J.; Khant, H. A.; Krogstad, D. V.; Tirrell, M. Effect of the peptide secondary structure on the peptide amphiphile supramolecular structure and interactions. Langmuir 2011, 27, 6163−6170. (8) Paramonov, S. E.; Jun, H. W.; Hartgerink, J. D. Self-assembly of peptide−amphiphile nanofibers: The roles of hydrogen bonding and amphiphilic packing. J. Am. Chem. Soc. 2006, 128, 7291−7298. (9) Valery, C.; Artzner, F.; Paternostre, M. Peptide nanotubes, molecular organisations, self- assembly mechanisms and applications. Soft Matter 2011, 7, 9583−9594. (10) Vauthey, S.; Santoso, S.; Gong, H. Y.; Watson, N.; Zhang, S. G. Molecular self-assembly of surfactant-like peptides to form nanotubes and nanovesicles. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5355−5360. (11) Arnold, M. S.; Guler, M. O.; Hersam, M. C.; Stupp, S. I. Encapsulation of carbon nanotubes by self-assembling peptide amphiphiles. Langmuir 2005, 21, 4705−4709. (12) Bitton, R.; Schmidt, J.; Biesalski, M.; Tu, R.; Tirrell, M.; BiancoPeled, H. Self-assembly of model DNA-binding peptide amphiphiles. Langmuir 2005, 21, 11888−11895. (13) Bull, S. R.; Guler, M. O.; Bras, R. E.; Meade, T. J.; Stupp, S. I. Self-assembled peptide amphiphile nanofibers conjugated to MRI contrast agents. Nano Lett. 2005, 5, 1−4. (14) Harrington, D. A.; Cheng, E. Y.; Guler, M. O.; Lee, L. K.; Donovan, J. L.; Claussen, R. C.; Stupp, S. I. Branched peptideamphiphiles as self-assembling coatings for tissue engineering scaffolds. J. Biomed Mater. Res., Part A 2006, 78A, 157−167. (15) Reches, M.; Gazit, E. Casting metal nanowires within discrete self-assembled peptide nanotubes. Science 2003, 300, 625−627. (16) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004, 303, 1352−1355. (17) Spoerke, E. D.; Anthony, S. G.; Stupp, S. I. Enzyme directed templating of artificial bone mineral. Adv. Mater. 2009, 21. (18) Tovar, J. D.; Rabatic, B. M.; Stupp, S. I. Conducting polymers confined within bioactive peptide amphiphile nanostructures. Small 2007, 3, 2024−2028. (19) Yuwono, V. M.; Hartgerink, J. D. Peptide amphiphile nanofibers template and catalyze silica nanotube formation. Langmuir 2007, 23, 5033−5038. (20) Koga, T.; Matsui, H.; Matsumoto, T.; Higashi, N. Shape-specific nanofibers via self-assembly of three-branched peptide. J. Colloid Interface Sci. 2011, 358, 81−85. (21) Lin, B. F.; Megley, K. A.; Viswanathan, N.; Krogstad, D. V.; Drews, L. B.; Kade, M. J.; Qian, Y. C.; Tirrell, M. V. pH-responsive branched peptide amphiphile hydrogel designed for applications in regenerative medicine with potential as injectable tissue scaffolds. J. Mater. Chem. 2012, 22, 19447−19454. (22) Liu, Y.; Zhao, X. Presentation of bioactive epitopes with free Ntermini on self-assembling peptide nanofibers. Nano Lett. 2011, 6, 47− 57. (23) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press, Inc.: New York, 1992. (24) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of selfassembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525−1568. (25) Minor, D. L.; Kim, P. S. Measurement of the beta-sheet-forming propensities of amino-acids. Nature 1994, 367, 660−663. (26) Ganesh, S.; Prakash, S.; Jayakumar, R. Spectroscopic investigation on gel-forming beta-sheet assemblage of peptide derivatives. Biopolymers 2003, 70, 346−354.
⎡ ⎛ πr ⎞⎤ U (r ) = − A⎢1 + cos⎜ ⎟⎥ ⎢⎣ ⎝ ra ⎠⎥⎦ where ra is the range and taken to be 1.0 in this work. With this potential, the strength and range of the attraction can be adjusted independently and continuously, which provides more flexibility than the attractive LJ potential. The equations of motion were integrated using a velocity-Verlet algorithm with a time step δt = 0.005τ, where τ = σ(m/ε)1/2 is the unit of time and m is the mass of one particle. The temperature of the system is kept at 1.0ε/kB, where kB is the Boltzmann constant, with a Langevin thermostat of damping rate 1.0τ−1.
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ASSOCIATED CONTENT
S Supporting Information *
TEM micrograph of Asymm-SSS and deconvoluted FTIR spectra of Asymm-SSS, Symm-Horiz, and Symm-Vert. 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]. Present Address §
Department of Physics, Virginia Polytechnic Institute & State University, Blacksburg, VA. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge Bonnie McKenzie for performing scanning electron microscopy analysis, Ana Trujillo for performing atomic force microscopy, and Dr. Bruce Bunker for insightful discussions. This research was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, Project KC0203010. Sandia National Laboratories is a multiprogram laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Company, for the US Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
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REFERENCES
(1) Lodish, H.; Berk, A.; Zipursky, S.; Matsudaira, P.; Baltimore, D.; Darnell, J. Molecular Cell Biology; W. H. Freeman and Co.: New York, 1999. (2) Cheng, S. F.; Aggarwal, A.; Stevens, M. J. Self-assembly of artificial microtubules. Soft Matter 2012, 8, 5666−5678. (3) Diegelmann, S. R.; Gorham, J. M.; Tovar, J. D. One-dimensional optoelectronic nanostructures derived from the aqueous self-assembly of π-conjugated oligopeptides. J. Am. Chem. Soc. 2008, 130, 13840− 13841. (4) Goeden-Wood, N. L.; Keasling, J. D.; Muller, S. J. Self-assembly of a designed protein polymer into β-sheet fibrils and responsive gels. Macromolecules 2003, 36, 2932−2938. 9208
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(27) Stendahl, J. C.; Rao, M. S.; Guler, M. O.; Stupp, S. I. Intermolecular forces in the self-assembly of peptide amphiphile nanofibers. Adv. Funct. Mater. 2006, 16, 499−508. (28) Niece, K. L.; Hartgerink, J. D.; Donners, J. J. J. M.; Stupp, S. I. Self-assembly combining two bioactive peptide−amphiphile molecules into nanofibers by electrostatic attraction. J. Am. Chem. Soc. 2003, 125, 7146−7147. (29) von Maltzahn, G.; Vauthey, S.; Santoso, S.; Zhang, S. U. Positively charged surfactant-like peptides self-assemble into nanostructures. Langmuir 2003, 19, 4332−4337. (30) Byler, D. M.; Susi, H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers 1986, 25, 469− 487. (31) Gasset, M.; Baldwin, M.; Fletterick, R.; Pruisner, S. Perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 1−5. (32) Haris, P.; Chapman, D. The conformational analysis of peptides using Fourier transform IR spectrscopy. Biopolymers 1995, 37, 251− 263. (33) Jiang, H. Z.; Guler, M. O.; Stupp, S. I. The internal structure of self-assembled peptide amphiphiles nanofibers. Soft Matter 2007, 3, 454−462. (34) Kyte, J.; Doolittle, R. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982, 157, 105−132. (35) Wilmley, W.; White, S. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 1996, 3, 842−848. (36) Ting, C.; Frischknecht, A.; Stevens, M.; Spoerke, E. Electrostatically tuned self-assembly of branched peptide amphiphiles. J. Phys. Chem. B 2014, 118, 8624−8630. (37) Plimpton, S. Fast Parallel Algorithms for Short-Range Molecular-Dynamics. J. Comput. Phys. 1995, 117, 1−19.
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Supramolecular Assembly of Asymmetric Self-Neutralizing Amphiphilic Peptide Wedges Dara Van Gough,† Jill S. Wheeler,† Shengfeng Cheng,‡,§ Mark J. Stevens,‡ and Erik D. Spoerke*,† †
Electronic, Optical, and Nano Materials, and ‡Computational Materials and Data Science, Sandia National Laboratories, Albuquerque, New Mexico 87185-1411, United States S Supporting Information *
ABSTRACT: Mimicking the remarkable dynamic and multifunctional utility of biological nanofibers, such as microtubules, is a challenging and technologically attractive objective in synthetic supramolecular chemistry. Understanding the complex molecular interactions that govern the assembly of synthetic materials, such as peptides, is key to meeting this challenge. Using molecular dynamics simulations to guide molecular design, we explore here the self-assembly of structurally and functionally asymmetric wedge-shaped peptides. Supramolecular assembly into nanofiber gels or multilayered lamellar structures was determined by cooperative influences of hydrogen bonding, amphiphilicity (hydrophilic asymmetry), and the distribution of electrostatic charges on the aqueous self-assembly of asymmetric peptides. Molecular amphiphilicity and β-sheet forming capacity were both identified as necessary, but not independently sufficient, to form supramolecular nanofibers. Imbalances in positive and negative charges prevented nanofiber assembly, while the asymmetric distribution of balanced charges within a peptide is believed to affect peptide conformation and subsequent self-assembly into either nanofibers or lamellar structures. Insights into cooperative molecular interactions and the effects of molecular asymmetry on assembly may aid the development of next-generation supramolecular nanomaterial assemblies.
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INTRODUCTION In biology, dynamic, high aspect ratio protein nanofibers enable a wide range of critical functions within cells. Microtubules (MTs), for example, facilitate chromosome separation during cell division, are a key constituent of the reconfigurable cytoskeleton, and direct the transport of intracellular cargo.1 These protein filaments are noncovalently, reversibly polymerized from heterodimers of α- and β-tubulin whose dynamic assembly is dictated by changes in molecular conformation and cooperative assembly.1 Developing synthetic analogues to these versatile, multifunctional nanofibers is a tantalizing materials chemistry target that would stand to impact a wide range of technical fields. Exactly replicating these complex materials, however, is not only synthetically impractical, but may not even be desirable for certain applications. To make this synthetically daunting challenge more tractable and widely applicable, we looked to molecular dynamics (MD) simulations for a distillation of fundamental design elements critical to nanotube assembly. Cheng et al.2 recently developed an MD model to simulate formation of nanotubes. This work revealed that asymmetry of both molecular shape and the chemistry that governs intermolecular attraction critically affected the morphology of simulated supramolecular assemblies. Figure 1 schematically summarizes the importance of shape asymmetry and the asymmetric distribution of interaction sites on the building © 2014 American Chemical Society
blocks in determining nanotube morphology. These simulated results indicate that a wedge-shaped building block with asymmetrically distributed interaction sites preferentially forms nanotube architectures. Note, however, that within this scheme, the relative strength of “vertical” versus “lateral” interactions also had a significant influence on assembly.2 Inspired by these observations, the present work explores a series of asymmetric, wedge-shaped peptides as building blocks for supramolecular assembly. Peptides are particularly wellsuited for these studies, as the controllable versatility of peptide chemistry facilitates systematic variation of building block composition and properties. In addition, there is precedence for the assembly of peptides, particularly linear amphiphilic peptides, into one-dimensional nanostructures such as nanofibers, nanoribbons, and nanotubes,3−10 engineered to promote a range of cellular, biomolecular, and materials interactions.3,11−19 Limited reports also describe precedence for the assembly of nonlinear, branched peptides, frequently designed to facilitate epitope accessibility or tuning of gelation properties in peptides for biomedical applications.14,20−22 In many of these chemically diverse systems, factors such as amphiphilicity, β-sheet formation, and electrostatic charge are Received: April 26, 2014 Revised: July 3, 2014 Published: July 8, 2014 9201
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blocks. Figure 1 shows a set of different monomers and structures that assemble in molecular dynamics simulations of these monomers. In Figure 1a, there are two pairs of binding sites (faces) on opposite sides of a rhombic triacontahedron (C80 buckyball), which is used to define points on an effectively spherical object. The only attractive interactions occur between sites with the same color (excluding gray). All other interactions are repulsive and yield the overall effective spherical shape. Because of the six-fold symmetry of the binding site (i.e., face), two monomers can bind with six orientations. Consequently, the structure of an assembly is a random, fractallike structure shown on the right of Figure 1a. If the symmetry of the binding region is reduced as in Figure 1b, where again attraction is only between like-colored sites, then the monomers can assemble into flat sheets as shown in the figure. To form tubules, the geometry of the monomer was changed from spherical to a wedge. The monomer’s shape is given by a rigid 3 × 3 × 3 set of gray spheres in the figure. As before, gray spheres interact only by a purely repulsive potential and just give the monomer its shape. The colored spheres in Figure 1c and 1d represent the attractive (binding) sites. For more details of the molecular dynamics simulations used in this model, see Cheng et al.2 In Figure 1d each binding surface has two distinct sites to control the orientation of the two binding monomers. This effect is demonstrated by the difference in assemblies shown in Figure 1c. For the binding sites in Figure 1c, there is a symmetry that allows two orientations of binding. Transforming between the two orientations just involves rotating one monomer by 180°. As shown in the figure, when these two orientations are possible, the system assembles not into tubules but into other geometries, which include a complicated twodimensional surface. When this symmetry is broken and only one orientation is allowed, then the monomers assemble into tubules. Effective tubule formation, then, resulted from the collaboration of several key building block characteristics: (1) an asymmtric wedge shape, (2) asymmetry of interaction sites to orient the wedges for assembly, (3) favorable attraction (as opposed to repulsion) of building blocks, providing both “lateral” and “vertical” attractive interactions needed to form the extended cylindrical structures. Figure 2 shows the chemical structure of the most asymmetric peptide design, Asymm-Wedge, inspired by the asymmetric wedge in Figure 1d. These molecules were designed to comprise hydrophilic, charged branches connected to a relatively hydrophobic, tri-isoleucine (tri-Ile) trunk. The wedge shape of the peptides introduces a structural asymmetry to the building blocks, following the models of wedge-shaped nanotube assemblers described above and, more generally, the conical building blocks described by Isaelachvili.23,24 The isoleucine trunk serves to simultaneously introduce amphiphilicity to the peptide (hydrophilic asymmetry) while the branched side chains of the isoleucine residues are expected to favor β-sheet formation.25 The amphiphilic character is expected to provide the driving force and molecular orientation needed for micelle assembly, while the wedge shape and βsheet formation are expected to direct the formation of these micelles into one-dimensional extended fibers or nanotubes.4−6,8,26−28 Finally, the electrostatic charges in the branches introduce potential attractive or repulsive forces between molecules. The remainder of this paper will focus on exploring how these key molecular characteristics influence nanostructure self-assembly.
Figure 1. Structures of monomers and assembled monomers from molecular dynamics calculations. Within the monomers, gray spheres represent purely repulsive sites that yield the monomer shape. Attraction occurs only between sites with same (nongray) color. Highly symmetric, effectively spherical, building blocks (a) selfassemble into an unordered agglomerate, while monomers with broken symmetry (b) assemble into sheet-like structures. In a similar vein, a wedge-shaped monomer will naturally assemble into hollow tubes, if the binding symmetry is broken (d), but if not, then wavy sheets are formed as two orientations of bound pairs randomly assemble (c).
common elements that strongly influence assembly. In the present work, we evaluate these interactive forces as elements of an asymmetric building block design and explore their influences on molecular self-assembly. Motivated by the results of the MD simulations described above, we focus on asymmetric wedge-shaped peptides designed to incorporate key elements of the MD model to facilitate 1D nanostructure assembly. We specifically investigate the influences of amphiphilicity (hydrophilic asymmetry), hydrogen bonding, and the asymmetric distribution of self-neutralizing positive and negative electrostatic charges within the branches of these wedge-shaped peptide building blocks impact self-assembly.
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RESULTS AND DISCUSSSION As mentioned above, results from MD simulations of nanotube assembly were used to guide the design of the peptide building 9202
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Within the branches of these peptides there is a mix of positively and negatively charged residues as well as multiple hydrogen bonding groups collectively intended to promote intra- and intermolecular attraction of the peptide branches. One of the advantages of using peptides as building blocks is that it allows for the incorporation of functional peptide sequences to mediate biomolecular or materials interactions. In many cases, the sequences of these mimics include a mix of positive and negative charges, and in the present work, we evaluate the potential impact of where these charges are positioned within a peptide on the subsequent assembly of these structures. Dissolved in water at concentrations as low as 5 mg/mL (0.5 wt %) at near-neutral pH, Asymm-Wedge initially forms a freeflowing liquid solution that spontaneously transforms to become a self-supporting gel, composed of assembled nanofibers and nanofiber bundles, revealed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) in Figures 3, parts b, c, and d, respectively. Figure 3a schematically depicts the molecular assembly and nanofiber bundling. These amphiphilic molecules are believed to assemble into fibers, guided by the sequestration of the relatively hydrophobic isoleucine trunks from the aqueous environment and β-sheet formation as these isoleucines pack together. Subsequent electrostatic, hydrogen bonding, or van der Waals interactions likely promote secondary assembly into nanofiber bundles. Figure 3b shows a scanning electron microscope (SEM) image of a lyophilized nanofiber gel, revealing both individual and bundled nanofibers within an interwoven supramolecular network. The inset in Figure 3b shows the self-supporting nanofiber gel at the top of an inverted vial. The TEM image of negatively stained
Figure 2. Chemical structure of Asymm-Wedge highlighting the charge asymmetry and the tri-Ile trunk. Orange circles highlight negative charges, while purple circles highlight positive charges.
Figure 3. (a) Schematic depicting the assembly of Asymm-Wedge in to nanofiber bundles. (b) Scanning electron microscope image showing bundles of Asymm-Wedge nanofibers. Inset: Self-supporting gel formed from Asymm-Wedge at 5 mg/mL in water at neutral pH. (c) Negatively stained transmission electron microscope (TEM) image of a self-supporting gel. (d) High resolution AFM image of bundled nanofibers. Color scale (right) spans +7.5 nm (white) to −7.5 nm (black). 9203
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Figure 4. (a) FTIR spectrum (black) and deconvolution (gray curves) for Asymm-Wedge. The summation of the deconvoluted peaks is shown as the dashed red overlaying the black spectrum. (b) Circular dichroism spectra for Asymm-Wedge (black) and Asymm-SSS (blue).
combined strong negative peak near 220 nm and the strong positive peak near 190 nm. Formation of β-sheets along the length of supramolecular nanofibers is believed to be important in the formation of the extended nanofiber morphology.4,8,26−28,33 In fact, in analogy to the MD-simulated structures above, these extended β-sheets provide the longrange “vertical” interactions needed to extend the cylindrical micelles into fibers. For Asymm-Wedge, though, the tri-Ile trunk serves double duty, providing both the critical β-sheet forming capacity of the peptides as well as critical amphiphilic character in the peptide. Amphiphilicity is widely recognized as an important driving force for micelle assembly, as the incentive to sequester poorly solvated moieties, such as hydrophobic Ile side chains, from aqueous solvent drives molecular aggregation and packing. The role of peptide amphiphilicity in the present system was explored by replacing the Ile trunk with a tri-serine trunk in to create Asymm-SSS, Figure 5a. Improved solvation of the polar
nanofibers in Figure 3c and the AFM image in Figure 3d both confirm the presence of bundled nanofibers observed in the SEM image in Figure 3b. Nanofiber dimensions, determined from all three images, consistently measure 4.5 ± 0.6 nm in diameter but extend to hundreds of nanometers in length. In a fully extended conformation, the peptides are estimated to be ∼3.8 nm long. Because the nanofibers are only 4.5 nm wide, however, it is clear that the peptides do not assemble end-toend in a fully extended conformation (like spokes on a bicycle.) Although the peptides are believed to assemble in a radial fashion, these data indicate that the peptide branches partially collapse in on one-another, driven by electrostatic attraction between oppositely charged residues. The balance of positive and negative charges present on Asymm-Wedge is critical for the observed spontaneous assembly. In other systems, rich in charged amino acids, electrostatic repulsion from imbalances in charge prevents assembly; only when the charges are neutralized by pH, cationic binding, or secondary molecules does assembly occur.6,21,28,29 Similarly, when Asymm-Wedge is dissolved in strongly acidic (pH 2) or strongly basic (pH 13) solutions, the resulting selective neutralization of either the anionic or cationic residues, respectively, produces a net charge on the peptide and prevents supramolecular assembly. Under neutral conditions, however, because both the positive and negative charges are simultaneously present on the opposing branches of this wedge peptide, Asymm-Wedge spontaneously assembles. This behavior may prove particularly relevant to biomedical applications, for example, in which in situ supramolecular assembly is required, but the addition of secondary chargeneutralizing agents may be undesirable. Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopies were used to characterize the supramolecular character of these nanofibers. The composition of the amide I peak in FTIR provides key information about the secondary structure of peptides and proteins, and Figure 4a shows the deconvoluted amide I peak of Asymm-Wedge. Peaks at 1621 cm−1 and 1633 cm−1 can be assigned as β-sheet and aggregated β-sheet components,30,31 while the peak at 1657 cm−1 is attributed to α-helical components of the peptide.30 The peak at 1673 cm−1 is believed to be due to a small amount of residual trifluoroacetic acid (TFA) present in these synthetic peptides.32 Circular dichroism (Figure 4b) confirms the presence of β-sheet in Asymm-Wedge, evidenced by the
Figure 5. (a) Schematic showing the chemical structure of AsymmSSS. (b) SEM image of structure formed from the assembled wedgeshaped peptide. Inset: At 5 mg/mL in water at neutral pH, AsymmSSS did not form a self-supporting gel. A small defect in the material is visible in the bottom of the micrograph.
hydroxyl side chains of the serines in Asymm-SSS34,35 would be expected to eliminate the amphiphilic driving force for molecular aggregation and self-assembly. In fact, Asymm-SSS did not self-assemble to form a gel, and there was no discernible assembled structure evident by SEM (Figure 5b) or TEM (Supporting Information Figure 1). Deconvolution of the amide I peak in FTIR showed no evidence of β-sheet, revealing 9204
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Figure 6. Amino acid sequence structures for (a) Symm-Vert and (b) Symm-Horiz. SEM images show (c) fibers assembled from Symm-Horiz and (c, d) multilamellar sheets formed from Symm-Vert at high and low magnification. Sheets curled into hollow spheres are visible in part e. SymmHoriz formed a self-supporting gel (inset c), while Symm-Vert did not (inset d) at 5 mg/mL in water at neutral pH.
only a peak at 1646 cm−1, attributed to unordered peptide, and a minor peak at 1673 cm−1 from residual TFA. These observations were further supported by the change in the shape of CD spectra, visible in the spectrum in Figure 4b; the strong negative peak near 195 nm indicates largely unordered material. These data collectively confirm the lack of β-sheet secondary structure in this material, despite the fact that serine is expected to be a competent (if not as proficient as Ile) βsheet forming amino acid.25 This behavior is particularly telling with respect to the importance of the amphiphilicity in these materials. Not only does the improved solvation of the tri-Ser trunk eliminate the driving force for molecular aggregation, but it also addresses molecular orientation in the present model. In Asymm-Wedge, the relatively poor solvation of the Ile side chains compared with the excellent solvation of the hydrophilic, charged peptide branches creates a hydrophilic asymmetry in the molecule that serves to orient the wedge peptides for assembly as well. The MD-simulated wedge in Figure 1c clearly shows that without asymmetric orientation, tubule formation is not favored. Similarly, when the orienting amphiphilicity of the wedge peptide was removed in Asymm-SSS, undirected intermolecular electrostatic and hydrogen bonding interactions produced a disordered structure. Moreover, in Asymm-Wedge, this orienting influence collaborated with hydrophobic aggregation of the poorly solvated Ile side chains to provide the molecular organization and proximity needed to facilitate the effective βsheet formation so important for nanofiber formation. Simply incorporating a β-sheet-competent trunk (SSS) into the peptide did not produce a sufficient driving force for nanofiber assembly (or measurable β-sheet formation); the cooperative influence of the β-sheet formation and the organizing influences of amphiphilicity (hydrophilic asymmetry) produced effective nanofiber assembly in this system. This cooperative influence was further supported when the hydrogen bonds responsible for β-sheet formation of AsymmWedge were thermally disrupted. When gels were heated to 85
°C, the self-supporting integrity of the gel was disrupted, transforming the gel into a free-flowing liquid. Upon cooling, the self-supporting gel reformed. By thermally disrupting the hydrogen bonds forming the β-sheets within these assemblies, the nanofibers responsible for gel formation became sufficiently disordered that they were unable to support gelation. Only when these hydrogen bonds were allowed to reform was the network of stable nanofiber assemblies reconstituted and the gel reformed. Interestingly, this process was demonstrated through multiple cycles. The reversibility of this assembly represents an interesting parallel between these synthetic assemblies and natural biological nanofibers, like microtubules or actin, though the molecular mechanics of these processes are admittedly very different. In addition to the balance of charges described above, the distribution of electrostatic charges on the peptide branches was varied to investigate how asymmetry of electrostatic interactions might influence molecular assembly. The extreme pH studies described above clearly showed that having both positively charged and negatively charged amino acids present on the peptides is important for assembly, ostensibly because these charges are expected to attract, bind, and neutralize opposite charges both intramolecularly and intermolecularly. Figure 6, parts a and b, shows two peptides with compositions nominally identical to Asymm-Wedge, but in which the charged residues were positioned more symmetrically. In Symm-Vert (Figure 6a), each of the two positive or negative charges were placed on the same branch of the peptide. In Symm-Horiz (Figure 6b), the negative charges were both placed near the crook of the branches, while the positive charges were both placed near the terminal ends of the branches. The scanning electron micrographs in Figure 6c−e show how these variations affected supramolecular assembly. Like Asymm-Wedge, Symm-Horiz also formed a self-supporting nanofiber gel at 5 mg/mL (Figure 6c). FTIR analysis of these materials further showed that nanofibers formed from SymmHoriz contained β-sheet, aggregated β-sheet, and α-helix, 9205
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Figure 7. Illustration of the intramolecular interactions, proposed method of folding, and assembly of the wedge-shaped peptides. (a) Asymm-Wedge possesses the most asymmetry and contains the most intramolecular interactions, collapsing the molecule into a relatively broad cone that favors cylindrical nanofiber assembly. (b) Charge placement on Symm-Horiz promotes branch folding, and a broadly conical shape, which again promotes the formation of nanofibers. (c) Lateral electrostatic interactions between branches in Symm-Vert drive produce a more extended conical conformation that promotes self-assembly into bilayered assemblies. Secondary interactions between assembled nanofibers or bilayers would promote nanofiber bundling or multilamellar stacking shown on the right (a−c).
A recent study by Ting et al.36 used self-consistent field theory (SCFT) to examine a model branched peptide system similar to that described here. In this study, electrostatic charges positioned in the branches of the peptide structures forced either a collapsed or extended branch morphology. Moreover, the more collapsed morphology favored the self-assembly of fibers, while the more extended conformation favored the selfassembly of sheets. On the basis of similarities between this model system and the present peptide structures, we propose that the intramolecular and intermolecular attraction of opposite charges in the peptide branches affects the geometry of the assembling peptides, ultimately influencing the observed self-assembled morphologies. Figure 7 provides a simplified summary of these effects for three self-assembling wedge peptides, nominally identical in composition, but in which the positions of electrostatic charges in the peptide branches vary. Note that although these models are consistent with models produced by SCFT, the structures shown are simplified schematics drawn to aid visualization of the proposed scheme, not simulated molecular structures. With Asymm-Wedge, we propose that electrostatic attraction between the asymmetrically positioned charges both draws the two branches together and collapses the distal ends of the branches toward the central part of the molecule, Figure 7a. The average, collective effects of these two processes creates the moderately conical molecular building blocks needed for cylindrical micelle formation. The 4.5 nm nanofibers, seen in Figure 3, support this model of partial branch collapse and molecular assembly. Following this model, the distribution of charges on Symm-Horiz, which also forms nanofibers, would
evidenced by deconvoluted amide I peaks at 1621, 1633, 1657 cm−1 (Supporting Information Figure S4). In contrast, SymmVert did not form a self-supporting gel, even at double the peptide concentration. Examination of lyophilized Symm-Vert by SEM revealed the predominant formation of sheet-like structures, Figure 6d,e. These sheet-like assemblies are one hundred to several hundreds of nanometers thick, and we propose that these structures are multilamellar stacks of peptide bilayers. Of particular interest are the hollow spherical structures, Figure 6e, ostensibly formed from assembled sheets rolled up to form the observed hollow spheres. Again using FTIR to provide a measure of secondary structure, deconvolution of the amide I peak for Symm-Vert shows that peaks representing β-sheet, aggregated β-sheet, and α-helix are present at 1621, 1633, and 1657 cm−1. (Supporting Information Figure S4). In this case, the β-sheet character of these assemblies did not direct the formation of nanofibers but drove the formation of lamellar sheets. The predominance of these discrete sheet-like structures, seen in Figure 6d,e, accounts for the failure of these assemblies to form selfsupporting gels more commonly formed from interpenetrating networks of one-dimensional nanostructures such as nanofibers. Such bilayers would be expected if the peptide wedges adopted a more extended conformation, as the aspect ratio of assembling amphiphiles has a strong effect on supramolecular structure.23 Severely conical molecules would be expected to produce spherical micelles, moderately conical molecules would produce cylindrical micelles (nanofibers), and slightly conical or rod-shaped molecules would form bilayers.23 9206
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also promote branch collapse toward the central portion of the molecule, Figure 7b. As with Asymm-Wedge, this collapse produces the relatively conical building blocks needed for formation of the observed nanofibers. In contrast, however, we suggest that the distribution of opposite charges in Symm-Vert draws the peptide branches together in a predominantly extended conformation, creating more rod-shaped building blocks. These rod-shaped building blocks would be expected to produce bilayer assemblies, Figure 7c.23 The bilayer structure accommodates the extended molecular conformation, while allowing for sequestration of hydrophobic isoleucine trunks from the aqueous environment and the formation of β-sheets within the layered assemblies. Moreover, the top and bottom of each bilayered structure are decorated with the rich chemistries of the peptide branches. Just as similar chemistries displayed on the exterior of the nanofibers lead to fiber bundling, cooperative electrostatic, hydrogen bonding, and van der Waals forces would be expected to promote secondary assembly of these layered structures into multilamellar sheet-like constructs such as those seen in in Figures 6d, 6e. Although it does not appear that these peptides assume the exact hollow tubular morphology described by the MD simulations, extended nanofibers formed from simulationinspired Asymm-wedge do present a number of interesting parallels to the simulated system. The influences of molecular morphology, amphiphilicity, electrostatic interactions, and hydrogen bonding influence key factors of asymmetric molecular shape, molecular orientation, and molecular attraction identified as important for one-dimensional (nanotube) formation. The conical shape of the collapsed branched peptides influenced the morphology of the assemblies. Molecular amphiphilicity not only provided the driving force for molecular aggregation/assembly but served to orient the asymmetric peptide wedges for micelle formation. Imbalances in electrostatic charges prevented self-assembly through local molecular repulsion. Meanwhile, attractive hydrogen bonding between peptides produced the β-sheet structures (“vertical” interactions) needed for extended nanofiber formation.
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EXPERIMENTAL SECTION
Materials. N-Fmoc-protected amino acids were purchased from Novabiochem. Diisopropylethylamine (DIEA), trifluoracetic acid (TFA), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), tetrakis(triphenylphosphine)palladium(0), dichloromethane, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N-methylmopholine, acetic acid, triisopropylsilane, acetic anhydride, diethyl ether, piperidine, sodium diethyldithiocarbamate, and N,Ndiisopropylethylamine were purchased from Sigma-Aldrich and used as received. Peptide Synthesis. The peptide trunks and primary branches for Aysmm-Wedge, Asymm-SSS, and Symm-Vert were synthesized C to N from a rink amide resin using standard Fmoc solid-phase peptide synthesis protocols on a Liberty Peptide Synthesizer (CEM Corp., Matthews, NC). Upon completion of the peptide backbone (for Asymm-Wedge, the backbone sequence was IIIKRNSQE, C to N), the sample was rinsed with N-methyl-2-pyrrolidone and dichloromethane. The deprotected amine terminus was then acylated using 10% acetic anhydride in N,N-dimethylformamide for 30 min; reaction completion was verified with a ninhydrin test. The secondary branches were synthesized from the Alloc-protected side group of a lysine residue. The Alloc group, an orthogonal protection group to both base and acid deprotection chemistries, was removed during a 2 h treatment in a solution composed of tetrakis(triphenylphosphine)palladium(0) (0.69 g), chloroform (5.08 mL), acetic acid (0.275 mL), and Nmethylmorpholine (0.138 mL). Excess reagent was removed by rinsing with 0.5% N,N-diisopropylethylamine and then with 0.5% sodium diethyldithiocarbamate (both in N,N-dimethlyformamide). The peptide was then rinsed with both N-methly-2-pyrrolidone and dichloromethane and loaded back onto the Liberty Peptide Synthesizer for branch growth. The peptide was cleaved from the resin during 3 h in a solution containing trifluoroacetic acid (95%), water (2.5%), and triisopropylsilane (2.5%). The peptide was then precipitated from solution with cold diethyl ether (−40 °C) and washed three times with cold diethyl ether using centrifugation. Finally, the peptide product was dried under vacuum overnight. For Symm-Horiz, because the branches are composed of the same amino acid sequences, both branches were simultaneously synthesized from a lysine residue with both amine groups protected with Fmoc groups. Successful peptide syntheses were confirmed by mass spectrometry (University of Minnesota Mass Spectrometry, Minneapolis, MN). Peptide Assembly. Peptides were assembled in aqueous solution concentrations of 5 mg/mL (0.5 wt %) peptide in water. In a typical experiment, 400 μL of deionized water was added to 2 mg of peptide, and the solution pH was adjusted to 7 by adding 0.01 M NaOH. The peptide solutions were vortexed and sonicated to fully dissolve the peptide. The solutions were then heated to 85 °C and allowed to assemble at room temperature overnight. (Assembly will typically occur within tens of minutes, but the extended self-assembly time was used to provide consistent time for potentially kinetically limited systems to assemble.) Sample Characterization. FTIR and SEM analysis were performed on lyophilized peptide assemblies. FTIR spectra were collected on a Varian 2000 FT-IR Scimitar Series equipped with a Pike MIRacle ATR attachment, and SEM analysis was performed using a Zeiss Supra 55VP Field Emission SEM. For TEM analysis, Approximately 3 μL of peptide gel was allowed to dry onto copper holey carbon TEM grids. The samples were then negatively stained with 2% phosphotungstic acid and allowed to air-dry before analysis in a Phillips CM30 TEM. AFM imaging was performed on nanofiber mats created by slowly drying 0.1 mg/mL aqueous peptide onto clean silicon substrates. Images were obtained using peak force tapping mode on a Bruker Dimension Icon AFM, scanning with a 2 nm ScanAsyst tip. Circular dichroism was performed on a Jasco J-815 CD spectrometer using a quartz cuvette with a 0.1 cm path length. Assembled peptides were diluted from 5 mg/mL gel assemblies to ca. 0.1 mg/mL. Molecular Simulations. The dynamics of self-assembly were obtained by performing MD simulations using the LAMMPS
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CONCLUSIONS Here we have presented the simulation-inspired design and synthesis of a series of asymmetric, self-neutralizing, wedgeshaped peptides. Supramolecular assembly of these peptides into nanofiber gels or multilayered lamellar structures was found to depend on cooperation between molecular amphiphilicity, hydrogen bonding, and electrostatic interactions. Molecular amphiphilicity (hydrophilic asymmetry) and βsheet formation, defined by the peptide “trunk”, were both identified as necessary but not independently sufficient drivers for the formation of supramolecular nanofibers. That balance and distribution of electrostatic charges with the peptide “branches” also strongly affected self-assembly. Imbalances in positive and negative charge prevented nanofiber assembly, while the asymmetric distribution of balanced charges within the branches is believed to affect peptide conformation and subsequent self-assembly into either nanofibers or lamellar structures. These insights into the importance of cooperating molecular interactions and the effects of molecular asymmetry on assembly are key to developing the next generation of supramolecular nanomaterial assemblies. 9207
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simulation package.37 In these simulations, each monomer is treated as a rigid body. Two particles in distinct monomers interact purely repulsively through the Lennard−Jones (LJ) potential. The potential is truncated at 1.0 to make the interaction purely repulsive. The attractive interaction is given by a cosine potential
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⎡ ⎛ πr ⎞⎤ U (r ) = − A⎢1 + cos⎜ ⎟⎥ ⎢⎣ ⎝ ra ⎠⎥⎦ where ra is the range and taken to be 1.0 in this work. With this potential, the strength and range of the attraction can be adjusted independently and continuously, which provides more flexibility than the attractive LJ potential. The equations of motion were integrated using a velocity-Verlet algorithm with a time step δt = 0.005τ, where τ = σ(m/ε)1/2 is the unit of time and m is the mass of one particle. The temperature of the system is kept at 1.0ε/kB, where kB is the Boltzmann constant, with a Langevin thermostat of damping rate 1.0τ−1.
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ASSOCIATED CONTENT
S Supporting Information *
TEM micrograph of Asymm-SSS and deconvoluted FTIR spectra of Asymm-SSS, Symm-Horiz, and Symm-Vert. 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]. Present Address §
Department of Physics, Virginia Polytechnic Institute & State University, Blacksburg, VA. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge Bonnie McKenzie for performing scanning electron microscopy analysis, Ana Trujillo for performing atomic force microscopy, and Dr. Bruce Bunker for insightful discussions. This research was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, Project KC0203010. Sandia National Laboratories is a multiprogram laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Company, for the US Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
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