Tuning Assembly Size in Peptide-Based Supramolecular Polymers by

Article pubs.acs.org/Biomac

Tuning Assembly Size in Peptide-Based Supramolecular Polymers by Modulation of Subunit Association Affinity Kaylyn M. Oshaben and W. Seth Horne* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States S Supporting Information *

ABSTRACT: Nature uses proteins and nucleic acids to form a wide array of functional architectures, and scientists have found inspiration from these structures in the rational design of synthetic biomaterials. We have recently shown that a modular subunit consisting of two α-helical coiled coil peptides attached at their midpoints by an organic linking group can spontaneously self-assemble in aqueous solution to form a soluble supramolecular polymer. Here we explore the use of coiled-coil association affinity, readily tuned by amino acid sequence, as a means to predictably alter properties of these supramolecular assemblies. A series of dimeric coiled-coil peptide sequences with identical quaternary folded structures but systematically altered folded stability were designed and biophysically characterized. The sequences were cross-linked to generate a series of branched, self-assembling biomacromolecular subunits. A clear relationship is observed between coiled-coil association affinity and apparent hydrodynamic diameter of the supramolecular polymers formed by these subunits. Our results provide a family of soluble supramolecular polymers of tunable size and well-characterized coiled-coil sequences that add to the library of building blocks available for use in the rational design of protein-based supramolecular biomaterials.

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INTRODUCTION

In Nature, proteins and nucleic acids are used to create an array of complex supramolecular architectures from an assorted set of simple building blocks.1,2 In one area of the emerging field of synthetic biology, researchers are applying this toolbox of biomacromolecules to fabricate designer biomaterials. While protein-based assemblies are more common in biological systems, nucleic acids have found wider use in the synthetic realmpresumably because of the very well-defined relationship between DNA sequence and folding.3,4 In contrast to nucleic acids, the interplay between sequence and folding in proteins is more complex, which makes their use as scaffolds for preparation of structurally defined biomaterials more challenging. Despite this challenge, many de novo designed proteinbased supramolecular assemblies have been reported in the literature, including hydrogels,5−7 spheres,8,9 disks,10 and high aspect-ratio nanofibers.11−14 Among protein folding motifs that have found use in supramolecular biomaterials, assemblies of α-helices, called αhelical coiled coils or helix bundles, have a special importance.15,16 The relationship between coiled-coil amino acid sequence and folded structure is very well understood and the quaternary fold can be encoded by peptide chains quite short in length.17 Coiled-coil-forming peptides are defined by a heptad repeat of amino acid residues. Each sequence position in the heptad, designated abcdefg, plays a specific role in influencing folding behavior (Figure 1A). Coiled-coil sequences are typically amphipathic in the folded α-helical state with hydrophobic residues at a/d heptad positions packing to form a © 2014 American Chemical Society

Figure 1. (A) Helical wheel diagram of a dimeric coiled-coil interface with heptad positions labeled. (B) A branched self-assembling “subunit” consisting of two coiled-coil-forming peptide chains connected by an organic small molecule linker. (C) Two different views (side and top) of the supramolecular polymers that can form from self-assembly of the subunit in B.

hydrophobic core upon oligomerization of multiple helices. Residue composition at a/d positions is the primary Received: January 9, 2014 Revised: March 3, 2014 Published: March 5, 2014 1436

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diethyl ether. The precipitate was pelleted by centrifugation and the ether decanted. The peptide pellet was dissolved in a mixture of 0.1% TFA in water and 0.1% TFA in acetonitrile prior to purification by high-performance liquid chromatography (HPLC) on a C18 preparative column using gradients between 0.1% TFA in water and 0.1% TFA in acetonitrile. HPLC fractions containing the product were combined, frozen, and lyophilized. Peptide identity was confirmed by mass spectrometry using an Applied Biosystems Voyager DE Pro MALDI-TOF instrument. All peptides were >95% pure by analytical HPLC on a C18 column. Subunit Synthesis. A fresh 1.5 mM solution of piperazinebased bis-bromoacetamide linker 11, synthesized as previously described,14 was prepared in DMF. A stock solution of peptide was prepared in deionized water, and the concentration determined by UV absorption at 276 nm after dilution into aqueous 6 M guanidinium chloride.29 The peptide was diluted to 150 μM in pH 7 phosphate buffer with 1% acetonitrile by volume. The peptide solution was heated to 65 °C on a water bath and the linker stock was added in five equal aliquots to a final concentration of 75 μM. Linker additions were performed at 15 min intervals over the first hour of the reaction. To maintain complete solubility, two aliquots of acetonitrile were added at 30 min intervals (total of ∼3% acetonitrile). After 2 h, the reaction was quenched by addition of 0.1% TFA in 20% acetonitrile. Amicon Ultra filters (MW cutoff 3000 Da) were used to remove the DMF and concentrate the subunit for purification. Equal amounts of reaction solution and water were added to the spin filter, and the mixture was spun at 6000 rpm for 30 min. Additional spins were performed until the reaction mixture had been washed with ∼3X volumes of water. The resulting material was purified was purified by HPLC on a C18 semipreparative column. Identity and purity of the final product was confirmed by analytical HPLC and MALDI-TOF. Circular Dichroism. CD experiments were performed on an Olis DSM17 circular dichroism spectrometer using 0.1 cm quartz cuvettes. Peptide concentration was determined by UV absorbance at 276 nm.29 Samples of 100 μM peptide in 10 mM phosphate buffer pH 7 were prepared and scanned at 20 °C from 200 to 260 nm in 1 nm increments with an integration time of 5 s and a bandwidth of 2 nm. A buffer blank was used to correct each spectrum and baseline molar ellipticity at 260 nm. Variable temperature CD was performed by monitoring molar ellipticity at 222 nm from 20 to 96 °C in 4 °C increments with a 2 min equilibration time between data points and an integration time of 5 s. Thermal melt data was fit to a two-state unfolding model30 to obtain the melting temperature (Tm). Changes in Tm for each mutant relative to wild-type GCN4-p1 (ΔTm) were used to estimate changes to the free energy of folding/association (ΔΔGassoc).31 Uncertainties in the Tm values determined by curve fitting were used to calculate uncertainties in ΔT m and ΔΔG using standard error propagation. Gel Permeation Chromatography. GPC of peptides 1−5 was carried out on a GE Healthcare Superdex 75 10/300 column (10 × 300 mm, 24 mL bed volume, 13 μm average particle size). The column was equilibrated with 0.15 M NaCl in 0.05 M sodium phosphate, pH 7. Peptides were loaded onto the column (100 μL sample at 100 μM concentration in equilibration buffer) and eluted at a flow rate of 0.8 mL/min. A molecular weight calibration curve was obtained by analyzing the elution volumes of 1 mg/mL solutions of BSA, ovalbumin,

determinant of the stability of the quaternary fold and also influences the number and relative orientation of helices in the assembly.18−22 Residues at e/g positions often form salt bridges between helices, adding to the stability and/or promoting a specific orientation.23 The other three heptad positions are solvent exposed in the folded state and can be used to functionalize the coiled coil for different applications.24 Because of the extensive understanding of sequence−folding relationships in coiled-coil peptides, they are an ideal basis set for creating supramolecular assemblies with tunable properties. Simple linear chains bearing one or more coiled-coil-forming sequences have been designed to form diverse assemblies with a range of structures and properties.12,25−27 Expanding beyond linear chain connectivity, several reports have demonstrated the utility of postsynthetic cross-linking to form branched oligomer topologies capable of self-assembly to form more complex coiled-coil-based materials. An early use of such postsynthetic covalent modification involved branched tetravalent building blocks displaying four peptide chains off a dendrimer core; mixing two such multivalent subunits bearing complementary sequences from a heterodimeric coiled coil produced rope-like fibers.11 A more recent example of postsynthetic covalent modification employed three coiled-coil peptide sequences in two different disulfide-linked subunits to prepare monodisperse protein cages.9 We have recently shown that cross-linking two peptide domains encoding for dimeric coiled coil folds through a Cys residue introduced at the midpoint of each sequence can produce a subunit building block capable of forming soluble supramolecular polymer assemblies in aqueous buffer (Figure 1B,C).14 One advantage of this postsynthetic cross-linking approach to coiled-coil peptide-based materials is the modularity of the system: both peptide sequence and organic linker structure can be altered to rationally tune properties of the noncovalent supramolecular assembly. In our prior work, we demonstrated that altering the length and rigidity of the organic linker between chains in the subunit can control assembly size formed in solution; however, attempts to probe effects of peptide sequence on supramolecular assembly were hampered by poor solubility of the resulting material.14 Here, we designed a series of homodimeric coiled-coil peptide sequences with near-identical folded structure but systematically altered thermodynamic folded stability. These sequences are used to establish the relationship between coiled-coil association affinity and the size of supramolecular assembly formed when such peptides are incorporated into branched self-assembling subunits. The results reported here, coupled with our prior work, demonstrate the potential of using linker structure and peptide sequence in tandem to prepare supramolecular polymer assemblies in aqueous solution of defined chain length, chain diameter, and backbone flexibility.

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MATERIALS AND METHODS Peptide Synthesis. Peptides were synthesized manually by microwave-assisted Fmoc solid-phase methods28 or in automated fashion on a Protein Technologies Tribute Automated Synthesizer. NovaPEG Rink Amide resin was used to generate the C-terminal carboxamide, and all sequences were acetylated at the N-terminus. Peptides were cleaved from resin by treatment with 92.5% trifluoroacetic acid (TFA), 3% water, 3% ethanedithiol and 1.5% triisopropylsilane solution for 3 to 6 h. After the peptide was cleaved from resin, it was precipitated from the filtered cleavage solution by addition of ∼40 mL cold 1437

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RESULTS AND DISCUSSION System Design. Our previous work with supramolecular polymers based on postsynthetically cross-linked peptides14 made use of GCN4-p1 (1), the leucine zipper dimerization domain from the yeast transcription factor GCN4.18 This 33residue sequence forms a dimeric coiled-coil quaternary structure under most experimental conditions34 and has a reported dissociation constant of ∼0.57 μM (corresponding to a ΔG of ∼8.6 kcal mol−1).38 Cross-linking two GCN4-p1 peptides with a small organic linker via a Cys residue introduced at a solvent-exposed f position midway along the helix leads to a “subunit” capable of self-assembly via coiled coil formation to form a supramolecular polymer (Figure 1C).14 Our goal in the present work is to systematically establish the relationship between the self-association affinity of the peptide domains in such a subunit and the properties of the supramolecular assembly the subunit forms in solution. In order to perform the experiments necessary to achieve this goal, we first required a series of peptides based on GCN4-p1 with a range of association affinities but near identical physical properties in the folded state. The relationship between hydrophobic core composition and quaternary structure stability in coiled coils has been well studied.18,19,39,40 Guided by this information, we designed a series of mutants of GCN4-p1 intended to provide a stepwise modulation in dimer thermodynamic stability (Figure 2). In

aprotinin, a 17-residue synthetic peptide (Ac-YEAAAKEAAAKEAAAKA-NH2), and vitamin B12. Dynamic Light Scattering. DLS data were collected on a Malvern Zetasizer Nano ZS90 dynamic light scattering instrument with a 632.8 nm laser at a fixed angle of 90° and a constant temperature of 25 °C. Subunit samples (200 μM peptide in 150 mM NaCl, 50 mM HEPES pH 7) were prepared by adding 10X buffer to concentrated stock solutions of subunit in water. The samples were filtered using 0.22 μm filters to remove any dust, and apparent hydrodynamic diameter was measured periodically over 8 h. Each data points and accompanying uncertainty reported in Figure 6 represent two or three independent samples, each measured in triplicate. Crystallization, Diffraction Data Collection, and Structure Determination. Crystallization was carried out using the hanging drop vapor diffusion method. Drops were prepared by mixing 0.7 or 0.5 μL of peptide stock (10 mg/mL in water) with 0.7 μL of buffer and allowed to equilibrate over a well containing 700 μL of buffer solution. Crystals of peptide 2 were obtained from a well buffer containing 0.05 M sodium acetate, 0.1 M sodium citrate tribasic pH 5.6, 20% w/v PEG 4000. A single crystal was flash frozen in liquid N2 after being soaked in the mother liquor supplemented with 15% glycerol. Crystals of peptide 3 were obtained from a well buffer composed of 0.3 M sodium acetate pH 4.6, 0.1 M sodium citrate tribasic pH 5.6, and 25% w/v PEG 4000. A single crystal was flash frozen in liquid N2 after being soaked in the above buffer supplemented with 25% v/v glycerol. Crystals of peptide 4 were obtained from a well buffer composed of 0.1 M sodium acetate pH 4.6, 0.1 M sodium citrate tribasic pH 5.6, and 25% w/v PEG 4000. A single crystal was flash frozen in liquid N2 after being soaked in the above buffer supplemented with 10% v/v glycerol. Crystals of peptide 5 were obtained from a well buffer composed of 0.15 M sodium citrate tribasic pH 5.6, 20% v/v 2-propanol, and 15% PEG w/v 4000. A single crystal was flash frozen in N2 after being soaked in the above buffer supplemented with 10% v/v glycerol. Diffraction data were collected on Rigaku Saturn 944 CCD or Rigaku Raxis HTC detector using CuKα radiation. d*TREK was utilized to index, integrate, and scale the collected data. Structure refinement was carried out using CCP432 and Phenix.33 The structures were solved by molecular replacement using previously published structures of GCN4-p1 (PDB 2ZTA, 4DMD) as search models.18,34 A combination of refinement programs were used to complete the structure: Refmac35 and Phenix33 for automated refinement, Coot36 for manual model building, ARP/wARP37 for solvent building, and Phenix33 for construction of composite omit maps. Statistics for X-ray data collection and structure refinement can be found in the Supporting Information. Negative Stain Transmission Electron Microscopy. TEM was carried out on an FEI Morgagni 268 electron microscope at an accelerating voltage of 80 kV. Samples of subunit 6s (200 μM) were prepared in 50 mM HEPES buffer pH 7.2. The solution was syringe filtered through a 0.22 μm pore size filter and allowed to equilibrate for 8 h. The equilibrated sample was diluted to 50 μM, dropped onto a carbon Formvar coated 300-mesh grid (Electron Microscopy Science), and allowed to stand. After 10 min, the drop on the grid was diluted with 6 μL of water, excess liquid was wicked off, and the remaining material stained with 2% uranyl acetate for 1 min. The grids were allowed to dry overnight in open air and then stored in a desiccator prior to imaging.

Figure 2. (A) Sequences of GCN4-p1 (1) and hydrophobic core mutants 2−5; structures of valine (Val), 2-aminobutyric acid (Abu), and isoleucine (Ile) residues are shown. (B) Crystal structure of GCN4-p1 (1, PDB 4DMD) with the positions of the mutated residues in the dimeric coiled coil indicated.

peptides 2−5, one or more of the valine residues found in a heptad positions in the hydrophobic core of peptide 1 were mutated to decrease (2) or increase (3−5) folded stability. In peptide 2, Val9 is replaced with the unnatural residue 2aminobutyric acid (Abu), which is slightly less hydrophobic and intended to destabilize the fold. Peptides 3−5 bear mutations of one, two, or three core valine residues to isoleucine, which is known to stabilize dimeric folds in coiled coils.19 The core hydrophilic asparagine at a heptad position 16 was retained across the series; this residue has a destabilizing effect due to its polar nature but helps to specify the dimer oligomerization state.34 1438

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Structural and Biophysical Characterization of Peptide Building Blocks. Circular dichroism (CD) scans were performed on peptides 1−5 in pH 7 phosphate buffer to assess if the mutations to the hydrophobic core disrupted the α-helical fold of the coiled coil (Figure 3A). Mutants 3-5 all retained

in 2. Gel permeation chromatography (GPC) results indicated the mutations did not change the preferred dimeric oligomerization state in solution. Every peptide in the series 1−5 eluted at the same volume when injected from a 100 μM stock in pH 7 phosphate, and a known trimeric mutant of GCN4-p1 was clearly resolvable (see Supporting Information). In order to probe for any subtle alterations to the coiled coil fold that might result from sequence mutation, we grew diffraction quality crystals of 2−5 by hanging-drop vapor diffusion and solved their structures by X-ray crystallography to 2.2 Å resolution or better (Table S2). All peptides crystallized in the same C2 space group with a dimeric coiled coil as the asymmetric unit. Comparison of the four new mutants with a previously published structure of the wild-type GCN4-p1 dimer show the overall fold of the coiled coil was not altered (Figure 4A).

Figure 3. CD scans at 20 °C (A) and thermal melts (B) of peptides 1−5 at 100 μM concentration in 10 mM pH 7 phosphate buffer. The mutations to the hydrophobic core are minimally disruptive to the overall helicity of the coiled coils but systematically alter thermodynamic stability of the quaternary fold. Figure 4. Comparison of X-ray crystal structures of peptides 1−5. (A) Overlay of residues 2−30 with calculated backbone RMSDs to peptide 1. (B) Comparison of parallel packing at a heptad position 9 in the hydrophobic core when occupied by a Val, Abu, or Ile residue.

helical content similar to that of GCN4-p1 (1). Peptide 2 showed a slightly decreased helicity relative to the rest of the series, which is attributed to partial fraying at the N-terminus in solution due to less ordered hydrophobic core packing around the Abu residue. Results from CD thermal melts bear out design hypotheses about the relationship between hydrophobic core composition and coiled-coil stability in the designed sequences (Figure 3B, Table 1). Each peptide showed a similarly cooperative unfolding transition with thermal unfolding midpoints (Tm) ranging from 53 to 78 °C at 100 μM concentration. The changes in Tm correspond to 0.4−0.7 kcal mol−1 stabilization per Val→Ile mutation among 3-5 and a larger 1.1 kcal mol−1 destabilization for the Val→Abu mutation

A particular aspect of interest in the structural analysis was the packing arrangement in the hydrophobic core. Prior published work on the design of coiled-coil folding motifs discuss how the identity of the residues at the a and d positions affect packing geometry.41 Parallel packing positions accommodate β-branched amino acid side chains (such as Val and Ile) much better than perpendicular packing sites, where the Cα−Cβ bond points directly toward the neighboring helix. In coiled coils based on GCN4-p1, the a position (primarily Val) shows parallel packing, while the d position (primarily Leu) shows perpendicular packing. The design of mutants 2−5 took these packing preferences into account when replacing Val with Abu or Ile at the a positions. Analysis of the crystal structures of 1− 5 shows the mutated residues are all accommodated into the hydrophobic core without changing the side-chain orientation relative to the native structure (Figure 4B). Importantly, the additional hydrophobic CH2 groups in 3−5 are effectively buried in the folded state. This means the Val→Ile mutations should have a minimal effect on the surface physical properties of the folded protein. Analysis of the Asn16 residue packing in 2−5 shows the polar contact has not been disrupted in any of the mutant sequences.

Table 1. Circular Dichroism Data for Peptides 1−5 peptide

[θ]222 (deg cm2 dmol−1 res−1) × 103

1 2 3 4 5

−35.5 −23.7 −33.1 −35.9 −33.7

Tm (°C)a

ΔΔGfold (kcal mol−1)b

± ± ± ± ±

-+1.1 ± 0.4 −0.7 ± 0.04 −1.1 ± 0.05 −1.7 ± 0.07

62.3 52.7 69.4 73.0 77.9

0.3 0.1 0.2 0.3 0.2

Midpoint of the thermal melt of 100 μM peptide in 10 mM phosphate buffer, pH 7. bChange in folding free energy with respect to 1.

a

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Synthesis of Cross-Linked Subunits and Characterization of Assembly Behavior. The above experiments provided a series of sequences (1−5) encoding for dimeric coiled coils of identical folded structure but systematically altered thermodynamic stability. We next incorporated these sequences into branched subunits (Figure 1B) to test the hypothesis that subunit association affinity could be used to tune to properties of the supramolecular polymer formed by self-assembly of that subunit (Figure 1C). Guided by our prior work with GCN4-p1 (1),14 a single Cys residue was introduced in place of solvent-exposed Ser14 in each peptide to produce mutants 6−10. The resulting thiol was use as a handle for postsynthetic peptide cross-linking via alkylation of a piperazine-based linker (11) functionalized with two bromoacetyl groups. Each cross-linked subunit 6s−10s was obtained by mixing 2 equiv of the corresponding peptide (6−10) with 1 equiv of linker 11 in a mixture of DMF and pH 7 phosphate at 65 °C (Figure 5). Subunits were purified by semipreparative

Figure 6. (A) Time-dependent DLS data monitoring the self-assembly of subunits 6s−10s. (B) Plot of apparent assembly size by DLS at 8 h against free energy of association of the coiled coil domain (ΔΔGassoc vs 1) estimated by biophysical analysis of 1−5 (see Table 1).

actual nature of the assemblies formed by 6s−10s (long, flexible chains); however, the DLS data still bear on the qualitative trend among the series. Comparison of the apparent hydrodynamic diameter obtained by DLS for subunits in the series 6s−10s supports our underlying hypothesis that tuning coiled-coil stability by rational alteration to the hydrophobic core can be a useful means to control size of the supramolecular polymers. In previous work, we showed that the thermodynamic stability of the GCN4-p1 (1) fold is not altered upon mutation of Ser14 to Cys (6) or by linking two chains via linker 11 (as in subunit 6s).14 Thus, we plotted apparent hydrodynamic diameter of the supramolecular assembly formed by 6s−10s versus the relative association free energy of the corresponding isolated coiled coil domain 1−5 found in each subunit (Figure 6B). In the series of subunits 6s−8s, apparent assembly size changes by ∼10 nm with each stabilizing core mutation. The difference in apparent hydrodynamic diameter among assemblies formed by 8s−10s shows a >100 nm change per core substitution. We interpret the nonlinear relationship as resulting from the increasing sized linear assemblies formed from the most stable coiled coil domains having a greater tendency to associate laterally to form larger aggregates. In order to visualize the assemblies directly and test for possible interchain association, we carried out negative-stain transmission electron microscopy (TEM) on the materials formed by subunit 6s, based on wild-type GCN4-p1 peptide 1. TEM results showed high aspect-ratio fibers, consistent with the putative mechanism for self-assembly (Figure 1C). The diameter of the fibers (∼6 nm) is in good agreement with the predicted diameter based on the crystal structure of the isolated coiled coil. Instances of interchain association are apparent from bundling of two or more fibers (Figure 7, arrows). We

Figure 5. Peptides 6−10 (A), Ser14→Cys point mutants of 1−5, were used to synthesize cross-linked subunits 6s−10s by reaction with small molecule linker 11 (B).

HPLC, and the identity and purity of the isolated material were confirmed by mass spectrometry and analytical HPLC prior to biophysical characterization. Dynamic light scattering (DLS) was used to measure the apparent hydrodynamic diameter of the supramolecular assemblies formed by cross-linked subunits 6s−10s in solution. Each subunit was analyzed at 200 μM concentration in 50 mM HEPES buffer pH 7 with 150 mM NaCl. After filtration and addition of buffer from a concentrated stock solution to initiate self-assembly, each sample was monitored by DLS over time (Figure 6A). Subunit 6s based on wild-type GCN4-p1 and 7s based on the slightly destabilized Val→Abu mutant reached a stable equilibrium after about 3 h. Subunits with one (8s) or two (9s) stabilizing Val→Ile core mutations per peptide chain equilibrated on a slightly longer time scale but reached a stable size distribution by 8 h. Subunit 10s, based on the most stable dimeric coiled coil in the series showed signs of turbidity after 6 h and substantial aggregation when left at room temperature for 25 h. It is important to point out that the model used in the analysis of the raw DLS data (diameter of a hard sphere with equivalent scattering behavior) is not a good match for the 1440

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ASSOCIATED CONTENT

S Supporting Information *

Characterization data for peptides 1−10 and subunits 6s−10s, gel permeation chromatography data for peptides 1−5, and crystallography data collection and refinement statistics for peptides 2−5. This material is available free of charge via the Internet at http://pubs.acs.org. Coordinates and structure factors for peptides 2−5 have been deposited in the PDB under accession codes 4NIZ, 4NJ0, 4NJ1, and 4NJ2.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Jill Millstone for advice on TEM experiments and Tom Harper for assistance with TEM instrumentation. We also thank Patrick Straney and Ryan Ruenroeng for advice on TEM grid preparation. Funding for this work was provided by the University of Pittsburgh and a CAREER award from the National Science Foundation (DMR1149067 to W.S.H).

Figure 7. Negative-stain transmission electron microscopy image of the fibrous assemblies formed by subunit 6s. Arrows indicate examples of apparent interchain association.

reason this interchain association, a form of nonideal behavior in the supramolecular polymerization, would become more pronounced with increasing chain length. Thus, it may be responsible for the uncontrolled aggregation observed for subunit 10s.

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ABBREVIATIONS CD, circular dichroism; GPC, gel permeation chromatography; PEG, polyethylene glycol; DLS, dynamic light scatter; TEM, transmission electron microscopy; DMF, dimethylformamide

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CONCLUSIONS

In summary, we have shown here that coiled-coil association affinity can be used to tune assembly size in supramolecular polymers formed through self-assembly of subunits composed of postsynthetically cross-linked peptides. A series of hydrophobic core mutants of the dimeric coiled coil GCN4-p1 were designed incorporating destabilizing Val→Abu or stabilizing Val→Ile mutations at a heptad positions in the sequence. Solution-phase biophysical measurements confirm the dimeric oligomerization state of the wild-type sequence is maintained in each mutant; however, association free energies vary over a range of 3 kcal mol−1 in ∼0.6 kcal mol−1 increments. Highresolution crystal structures show the parallel chain topology and tight packing of the hydrophobic core remain intact in all cases. The above set of coiled-coil-forming peptides were used to prepare a family of self-assembling subunits through postsynthetic cross-linking at a solvent-exposed Cys introduced into each sequence, as described in our prior work.14 Solution analysis of self-assembly in the resulting subunits by DLS indicates a direct correlation between peptide association affinity and supramolecular polymer size. Transmission electron microscopy experiments confirm the fibrous morphology of the materials formed. The family of soluble supramolecular polymers of tunable size will find use in our ongoing work to prepare functional assemblies based on this system. Moreover, our results suggest that maintaining ideal behavior in larger linear assemblies may be possible through introduction of capping groups in the coiled-coil domains that disfavor interchain association in the supramolecular polymers through electrostatic or steric effects. The series of well-characterized peptides with tunable association affinity adds to the library of building blocks that can be used in the rational design of peptide-based supramolecular biomaterials.

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dx.doi.org/10.1021/bm5000423 | Biomacromolecules 2014, 15, 1436−1442