Computational de novo design of a self-assembling peptide with predefined structure.

Article IMF

YJMBI-64635; No. of pages: 13; 4C: 2, 4, 7

Computational De Novo Design of a Self-Assembling Peptide with Predefined Structure Sabine Kaltofen 1 , Chenge Li 2 , Po-Ssu Huang 3 , Louise C. Serpell 4 , Andreas Barth 2 and Ingemar André 1 1 2 3 4

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Department of Biochemistry and Structural Biology, Lund University, 221 00 Lund, Sweden Department of Biochemistry and Biophysics, Stockholm University, 114 18 Stockholm, Sweden Department of Biochemistry, University of Washington, Seattle, WA 98195, USA School of Life Sciences, University of Sussex, Brighton BN1 9RH, United Kingdom

Correspondence to Ingemar André: [email protected] http://dx.doi.org/10.1016/j.jmb.2014.12.002 Edited by T. Yeates

Abstract Protein and peptide self-assembly is a powerful design principle for engineering of new biomolecules. More sophisticated biomaterials could be built if both the structure of the overall assembly and that of the self-assembling building block could be controlled. To approach this problem, we developed a computational design protocol to enable de novo design of self-assembling peptides with predefined structure. The protocol was used to design a peptide building block with a βαβ fold that self-assembles into fibrillar structures. The peptide associates into a double β-sheet structure with tightly packed α-helices decorating the exterior of the fibrils. Using circular dichroism, Fourier transform infrared spectroscopy, electron microscopy and X-ray fiber diffraction, we demonstrate that the peptide adopts the designed conformation. The results demonstrate that computational protein design can be used to engineer protein and peptide assemblies with predefined three-dimensional structures, which can serve as scaffolds for the development of functional biomaterials. Rationally designed proteins and peptides could also be used to investigate the subtle energetic and entropic tradeoffs in natural self-assembly processes and the relation between assembly structure and assembly mechanism. We demonstrate that the de novo designed peptide self-assembles with a mechanism that is more complicated than expected, in a process where small changes in solution conditions can lead to significant differences in assembly properties and conformation. These results highlight that formation of structured protein/peptide assemblies is often dependent on the formation of weak but highly precise intermolecular interactions. © 2014 Elsevier Ltd. All rights reserved.

Introduction The process of evolution has given rise to a large number of protein assemblies from a wide variety of structural motifs. This repertoire of structures can be used as a toolbox from which new functional proteins can be developed by modification of preexisting ones. However, synthetic systems that expand the complexity of natural ones can also be built with structures that are beyond nature's repertoire. Recent developments in rational protein design methods have demonstrated that impressive protein assemblies can be built from first principles by redesigning structural building blocks taken from natural systems [1–8]. This includes not only 0022-2836/© 2014 Elsevier Ltd. All rights reserved.

engineering of relatively simple building blocks such as α-helices [2,3,6] but also redesigning globular proteins to trigger formation of protein cages and crystals [4,5,7]. The development of novel proteins building blocks de novo has proven more difficult: a stellar exception being the design of the top7 protein, a synthetic protein with a novel sequence and topology [9]. However, nature seems to have explored only a fraction of all possible folds, suggesting that man-made proteins could expand on nature's structural repertoire [10,11]. Here, we demonstrate that novel building blocks for the creation of self-assembling biomaterials can be rationally designed de novo, as successions to monomeric proteins such as top7 [9] and the ideal J. Mol. Biol. (2014) xx, xxx–xxx

Please cite this article as: Kaltofen Sabine, et al, Computational De Novo Design of a Self-Assembling Peptide with Predefined Structure, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.12.002

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Computational Design of a Self-Assembling Peptide

protein folds designed by Koga et al. [1]. The added complexity in engineering a self-assembling system de novo stems from the fact that interactions for folding of the individual building blocks and those for subunit contacts have to be optimized simultaneously. If such challenges can be overcome, there would be significant benefits because the molecular structure could be precisely controlled. That would open up possibilities for the development of more complex biomaterials with custom-made properties, which would be of considerable interest in biotechnology and synthetic biology. Large research efforts are targeted toward understanding the process of amyloid formation [12,13] due to their central role in disease [13]. The discovery of amyloids has also created significant interest in nanobiotechnology [14]. Because amyloids are very strong, stable and regular, they are excellent scaffolds for functionalization [14]. Amyloid proteins and peptides form fibrils with cross β-spine structure [13]. Short peptide sequences have been demonstrated to form amyloid-like fibrils and crystal structures of such peptides show that they self-assemble into tight steric zipper interfaces that drive assembly formation [15,16]. Here, we design a fibrillar biomaterial by creating a self-assembling building block consisting of two short fibrillizing peptides connected by an intervening α-helical segment. This building block with a novel topology is designed to self-assemble into a fibril with a continuous layer of α-helices on the outside of the fibril (Fig. 1a–c). Using biophysical measurements including X-ray fiber diffraction, electron microscopy, Fourier transform infrared (FTIR) and circular dichroism (CD)

spectroscopy, we demonstrate that the peptide adopts the designed conformation. Protein and peptide self-assembly is extensively used in nature to build larger assemblies. Formation of assemblies with unique three-dimensional structure rather than heterogeneous aggregates is likely dependent on formation of highly specific intermolecular interactions. Rationally designed self-assembled peptides can serve as powerful systems to investigate these features, in particular, small model systems that fold into well-defined three-dimensional structures. Detailed studies of such systems can be used to gain understanding of the relation between assembly structure and self-assembly mechanism. This can be difficult to investigate using natural systems such as amyloidogenic sequences, where the structure of the assembly is typically less well defined. The peptide developed in this study not only can be used as a scaffold for development of functional biomaterials by introducing binding functionality or covalent attachments into the helices or loop segments but also can be used to increase our fundamental understanding of the energetic and structural determinants of protein and peptide self-assembly.

Results Computational protein design Our structural understanding of amyloid formation has been greatly aided by the determination of crystal structures of model peptides that self-assemble into

Fig. 1. De novo design of self-assembling βαβ peptides. (a) The computational design is based on the crystal structure of the sup35 heptapeptide with sequence GNNQQNY [15]. A loop–helix–loop segment constructed using flexible backbone design connects two β-strands with backbone conformations taken from the sup35 peptide. (b) Cross-section of the atomic model of the fibril in a view perpendicular to the fiber axis. The interactions between the two layers of sheets are stabilized by a steric zipper interface formed by interdigitating side chains. (c) Structural model for how peptides associate into fibers. (d) Sequences of sup35 heptapeptide, βαβZip and βαβZip2. Green positions are part of the steric zipper interface, red helical residues according to DSSP [20].


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amyloid-like fibrils [15,16]. The first such structure was of a seven-residue peptide (GNNQQNY) from sup35, which is a prion-like protein in yeast that forms amyloid-like fibrils. The peptide self-assembles into a double β-sheet structure with the two parallel sheets facing each other to form a tight interface. β-Sheets interacting in this manner this are referred to as “steric zippers” (Fig. 1b) [15]. The steric zipper interface is formed by interdigitating polar side chains originating from the two sheets. We use this region of the sup35 peptide as a self-assembling scaffold that triggers the formation of fibrils that are decorated with α-helices on the exterior. As shown in Fig. 1c, the helices are running perpendicular to the fibril axis. The design strategy is shown in Fig. 1a. The steric zipper interface is encoded by keeping the three amino acids involved in the interface formation in each sup35 peptide (the underlined letters in GNNQQNY and green in Fig. 1d) while allowing the others to mutate to optimize the desired structure (see below). Two sup35 peptides are connected by a segment with loop–helix–loop secondary structure. The resulting βαβ motif is a common structural motif in protein folds but does not occur on its own [1]. There are several design challenges: the length of secondary structure elements must be optimally chosen, the sequence of the segment between the two parallel strands must be able to fold into a loop–helix–loop secondary structure and the intermolecular interactions between consecutive peptides in the desired assembly must be optimized to facilitate specific association. The overall structure of the fibril can be described as a helix with 180° turn and a pitch of 4.8 Å and the βαβ peptide as the repeating unit. To approach the design problem, we developed a computational procedure for flexible backbone design in the context of helical structural symmetry using the Rosetta macromolecular modeling software [17]. The protocol was developed in a prior version of Rosetta macromolecular modeling suite, but we have recently introduced the same functionality into the RosettaRemodel program [18]. To determine optimal lengths of the two loops and the helix, we built single peptides de novo using sequence-independent fragment assembly simulations. Peptides with different combinations of secondary structures lengths were folded in separate simulations and the optimality of a given combination was estimated from the frequency of models adopting the intended fold. Based on these simulations, two sets of secondary structure lengths were identified as the most optimal, with a (strand)loop: helix:loop(strand) lengths of (6)3:13:3(6) and (6) 3:14:2(6), respectively. These are consistent with the rules for connection of adjacent secondary structure elements in ideal protein structures [1]. Two separate design calculations were carried out using the two secondary structure combinations. In the flexible backbone design, the backbone structures of the two adjacent strands were kept according to the crystal structure of the sup35 heptapeptide [15]. The protein

backbone of the intervening segment was built using a fragment-based loop modeling procedure in a coarse-grained representation of the peptide structure and using helical symmetry to describe the structure and interactions of the fibril. Iterations of sequence optimizations and structure-based energy refinement in an all-atom representation of the fibril were used to arrive at the final sequence. The amino acid identities at positions involved in the steric zipper interface were fixed in the design calculation to the identities found in the sup35 peptides. A large number of independent design calculations were carried out and the final sequences were identified among the best energy models. Further stabilization was attempted by redesign of the surface of the peptide to reduce the net charge and by introducing N-terminal capping residues to stabilize the helices [19]. One sequence for each secondary structure combination was selected for experimental characterization. The peptides based on the 3:14:2 and 3:13:3 secondary structure length are called βαβZip and βαβZip2, respectively. The structural models of βαβZip and βαβZip2 are similar (Fig. S1). However, the longer second loop of βαβZip2 leads to a more tilted helix relative to the sheet with a larger distance to the strands. The sequences of βαβZip and βαβZip2 and the comparison with the sup35 heptapeptide are shown in Fig. 1d. Experimental characterization of designed peptides Assembly structure βαβZip and βαβZip2 were produced by solid-phase synthesis for experimental characterization. At an early stage of our investigation, we found that βαβZip2 does not adopt a significant amount of secondary structure under a wide range of tested conditions. Therefore, detailed investigations of peptide structure and assembly properties were only carried out for βαβZip. All the experiments described in the following section were performed with the peptide dissolved in pure water without a pH buffer. Under these conditions, the pH of the solution is determined by the buffering capacity of the peptide itself and residual amounts of trifluoroacetic acid (TFA) from the peptide synthesis. We typically observed a pH value of ~3.8 in the peptide solution, which is somewhat below the theoretical isoelectric point of 4.4. We found these conditions to be ideal for structure formation and self-assembly. The influence of solvent properties such as pH and ionic strength will be discussed below. The ability of βαβZip to assemble into fibrils was investigated with cryo-transmission electron microscopy (TEM). The peptide spontaneously forms long thin fibrils (Fig. 2a). At higher fibril concentrations, we observe tangling and bundling of individual fibrils in the micrographs. Bundling of fibrils occurs without formation of well-defined fiber structures, in what


4 Computational Design of a Self-Assembling Peptide


Fig. 2. Characterization of βαβZip assembly structure. (a) Cryo-TEM images of fibrils examined after 24 h incubation at room temperature (100 μM). (b) Time dependence of CD signal at 100 μM recorded after 5 min (black), 15 min (red), 45 min (blue) and 90 min (green). (c) Fiber diffraction spectrum of peptide dissolved in water at ~ 20 mg/ml and incubated for 5 days. Fibrils were aligned over night at room temperature. (d) ATR-FTIR absorbance spectrum of 1 mM peptide dried from a D2O solution after 1 h incubation. (e) Second derivative of the ATR-FTIR absorbance spectrum shown in (d). Comparison between samples dried from solutions in D2O and H2O. (f) Simulated fiber diffraction spectrum from the atomic model of fibril, with 5° fiber disorientation. The experimental spectrum has been mapped into reciprocal space and the simulated spectrum is shown in top right quadrant.

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appears to be unspecific interactions. Less bundling is observed in areas of lower fibril concentration, see Fig. S10. Analysis of the width distribution of individual fibrils from the micrographs shows that the most frequent width is around 5 nm (see Fig. S2). This is in excellent agreement with the structural model of the fibril (Fig. 1c), which predicts a width of 4–5 nm. We find no evidence of a significant population of individual fibrils with widths larger than 8 nm. This demonstrates that the peptide largely adopts single fibrils without associating into structured protofilaments. To determine the conformation of individual peptide monomers within the fibrils, we investigated the secondary structure of βαβZip using CD and FTIR spectroscopy. As illustrated in Fig. 2b, the lyophilized peptide adopts a predominantly randomcoil-like state directly after dissolving it in water. Over time, the secondary structure content increases before reaching a steady state after approximately 90 min at a reference concentration of 200 μM. The CD spectra recorded at this state imply a mixed α/β secondary structure as expected from the structural model. FTIR spectroscopy was used to gain further insight into the structure of the peptide within the fibril. Figure 2d shows the ATR (attenuated total reflection)-FTIR absorbance spectrum of a dried peptide film in the amide I region. The spectrum has clear maxima near 1655 cm −1 (α-helix) and 1627 cm − 1 (β-sheet). The spectrum was further analyzed by simultaneous fitting of the absorbance spectrum and of its second derivative. In films dried from solutions in H2O (D2O), the relative band area of bands assigned to α-helices was 25–38% (17–18%) and that assigned to β-sheets was 37–43% (37–45%). The results depended on the relative contribution of the absorbance spectrum on the one hand and its second derivative on the other for judging the deviation between fit and experiment. In D2O but not in H2O, there is a band near 1670 cm −1, which accounts for 4– 14% of the band area. This band is close to the spectral range for α-helices but was not counted as α-helix in this evaluation. This omission can explain the lower α-helix content in D2O. The FTIR results are in good agreement with the structural model, where 11 amides are hydrogen-bonded in an α-helical conformation (35%) and 8 amides form β-strands (26%). The experimental β-content is higher than expected based on the structural model. However, the estimation from the structure is dependent on the precise definition used to assign hydrogen bonds (DSSP [20] in this case). Two additional amides in each strands form weaker polar interactions with neighboring strands that are not assigned as hydrogen bonds by DSSP and their inclusion would increase the β-content to 39%. A careful analysis of band shapes and positions in the FTIR second-derivative spectrum can be used to discriminate between particular types of secondary structure that do not give rise to different

signals in CD spectroscopy. The second-derivative spectrum (Fig. 2e) has bands at 1666, 1653 and 1627 cm −1 (1662 and 1624 cm −1 when dried from D2O). The band position of the latter is indicative of a β-sheet that is composed of many strands [21] and that lacks the twist typically found in sheets from globular proteins. The sharpness of the band suggests a homogeneous structure. There is no evidence for the presence of a significant amount of antiparallel β-sheets because the expected highwavenumber band near 1690 cm −1 is missing. The signal for α-helices is typically found between 1650 and 1660 cm − 1, where a higher wavenumber indicates vibrational coupling between several helices [21]. The parallel alignment and the proximity of helices in the fibril model predict strong coupling between helices. Therefore, it is not surprising to find a band at the upper limit of the range assigned to α-helices at 1666 cm −1. Taken together, the spectroscopic data lend strong support to the structural model. An important property of the system that needs to be verified is the spatial organization of individual, folded monomers within the fibrils. The method of choice to address this question is X-ray fiber diffraction, a scattering method where an aligned fibril specimen is subjected to X-ray radiation that gives rise to a two-dimensional diffraction pattern that arises from the repeating structure along the fibril axis [22,23]. In order to obtain data with high information content, we have to align the fibrils prior to data recording. Typically, fibril diffraction data show some extent of disorientation since perfect alignment cannot be reached. This disorientation results in diffraction intensity that is spread into arcs (Debye-Scherrer arcs) and an approximately linear reduction of intensity in the equatorial direction compared to a perfectly aligned system [23,24]. The experimental fibril diffraction patterns from aligned samples composed of βαβZip fibrils are well oriented with intensity found on the meridian at 4.6 Å and 9.2 Å (Fig. 2c). An intense diffraction signal at around 4.8 Å is typically observed for amyloid structures and is the result of repetition of β-strands that run perpendicular to the fibril axis with a spacing of 4.8 Å [25]. The diffraction data suggest that the distance between strands is slightly shorter in βαβZip, 4.6 Å. An intensity distribution measured in the meridional direction of the diffraction pattern is shown in Fig. S3 and reinforces the identification of the major signals. A number of diffraction rings are present in the diffractogram. In a fiber diffraction experiment, rings are observed for diffracting species without preferred alignment. Because of the sharpness of the rings, they are likely coming from crystalline domains of the sample. A strong equatorial intensity is observed at 9.0 Å that continues on a diffraction ring appearing close to the 9.2-Å signal on the meridian. The intensity is considerably stronger


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in the equatorial direction, which suggests that the diffracting species is partially aligned. A strong equatorial intensity corresponding to intersheet separation is often observed for cross β-structure [26]. Fibrils formed by the GNNQQNY peptide have a strong meridional reflection at 4.7 Å and an equatorial reflection at around 10 Å [27]. A diffraction pattern was calculated from the structural model (Fig. 1c) and shows excellent agreement with the experimental data (Fig. 2f). A helical pitch of 4.6 Å was used for the simulation. The calculated pattern predicts a strong intensity signal at 4.6 Å and considerably weaker signals at 9.2 Å and on the equator. A parallel β-sheet structure with a repeating unit of 4.6 Å would not give rise to intensity at 9.2 Å because of systematic absences; thus, it must be due to another element repeating every 9.2 Å along the fibril axis. This matches the distance between the α-helices. From the biophysical characterization, we conclude that the assembly structure of βαβZip is in close agreement with the structural model. Parallel β-sheets could also form with an extended peptide in βαβ conformation (Fig. S8). The alternative arrangements can be ruled out based on the geometry of β-strands and α-helices, together with the results of the electron microscopy, FTIR and fiber diffraction experiments, see Fig. S8 for more information. We conclude that the available data support the intended structure for βαβZip shown in Fig. 1c. Assembly mechanism and assembly conditions Acquiring a deeper understanding of the solution conditions that lead up to successful self-assembly of βαβZip into structured fibrils and the mechanism of assembly formation will be important for the development of applications. βαβZip can also serve as an excellent model system for studies of the relationship between assembly structure and assembly mechanism in fibrillation reactions. To get further insight into the assembly properties of βαβZip, we carried out a number of additional experiments. βαβZip can adopt three different structural states (α/β, all-β and unstructured) or mixtures thereof, depending on the solution conditions. The presence of a structural state can readily be diagnosed by CD spectroscopy. The α/β conformation is observed only when the concentration exceeds a certain threshold. A clear difference is seen between 10 μM and 40 μM peptide concentration (Fig. 3a). The concentration dependence of structure formation could be explained by equilibrium between free unfolded monomers and peptides associated into fibrils, where an increase in peptide concentration shifts the equilibrium to the assembled state. However, it does not necessarily prove that unfolded monomers can spontaneously assemble into fibrils. Amyloid fibril formation typically happens with a nucleation-dependent mechanism

[28], where the nucleation event can be the ratedetermining step. Although the CD spectra of a freshly dissolved sample suggest an unstructured peptide, they do not rule out the presence of smaller amounts of nucleating material in the lyophilized peptide powder, which may be crucial for formation of fibrils. The FTIR spectra of lyophilized βαβZip show a significant amount of β/β secondary structure and the presence of extended β-sheets, which demonstrate that fibrils are likely to be present in this preparation (Fig. S4). To determine whether fibril formation can happen spontaneously from unstructured peptides or if nucleating material is required, we denatured fibrils by heat and followed the potential reassembly of fibrils with CD. Thermal denaturation of preformed fibrils was followed by CD to ensure that all secondary structure is lost. The transition from α/β conformation to an unstructured state has a midpoint at 53 °C (Fig. 3c) and is not reversible upon cooling. Sedimentation equilibrium experiments with analytical ultracentrifugation confirm that peptides stay monomeric (average molecular weight of 3065 ± 36 Da, close to the theoretical value for the monomer) 3 days after denaturation (Fig. S5). These data show that spontaneous oligomerization does not occur at a significant rate even at concentrations well above the critical concentration for structure formation (~ 40 μM). However, fibrillation of denatured peptides can be triggered by adding a small amount of preformed fibrils as seeds (Fig. 3b). This shows that nucleating species are present in the lyophilized peptide, which enables efficient self-assembly in the timeframe of our experiments, typically minutes to several hours. TEM images of the material after the seeding experiment confirm that fibrils have formed. The ability of the peptide to fold and spontaneously assemble can also be rescued by lyophilizing the denatured material, see Fig. S9. The formation of βαβZip in the α/β conformation is strongly influenced by pH. The emergence of folded peptide at different pH values was monitored by following the position of the minimum in the CD signal of the peptide, where spectra of pure random coil and α-helix have minima at 196 and 208 nm, respectively (Fig. 3d). When the pH value is raised above a threshold of ~ 4.2, the structure formation is completely abolished. This coincides with the theoretical isoelectric point of the peptide (Fig. 3d) and the titration of aspartic and glutamic acid (assuming model pKa values), two of which are located in close proximity (positions E9 and D11) at the N-terminal end of the α-helix. The deprotonation of these residues and subsequent charge–charge repulsion might be involved in shifting the conformational equilibrium toward the unfolded population. The all-β conformation can be triggered by an increase in ionic strength. In CD spectroscopy (see Fig. 3e), we observe a rapid loss in α-helical content upon addition of, for example, sodium chloride to preformed fibrils. This transition occurs regardless of




Fig. 3. Characterization of βαβZip assembly mechanism and assembly conditions. (a) Comparison of CD spectra of peptides dissolved at 10 μM (blue line) and 40 μM (red line). (b) Effect of thermal denaturation and seeding. A CD spectrum of fibrils assembled at 100 μM is shown in black; red, the same sample after denaturation at 85 °C; blue, after seeding with preformed fibrils. (c) Thermal denaturation of fibers assembled at 100 μM followed by CD at 208 nm (blue) and 218 nm (red). The lines are only present as visual aids and were generated by fitting a two-state model to the data. (d) The effect of pH on the secondary structure. The position of the lower-wavelength peak in CD is shown as a function of pH (black circles, see the left axis). The red curve follows the net charge of the peptide as a function of pH calculated using model pKa values (right axis). (e) Effect of salt on the secondary structure. CD spectra were recorded before (black) and after (red) addition of 25 mM NaCl. (f) Comparison of the second-derivative FTIR spectra of fibrils formed in water before (black) and after (red) incubation with NaCl.

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the size, valence or type (chaotropic or kosmotropic) of the salt; only the rate of transformation varies, without an obvious correlation with physiochemical properties of the ions. The corresponding changes in the FTIR spectra of dried peptide are more subtle (see Fig. 3f) but are in line with a decrease in the content of α-helices and a slight increase in the absorption on the low-wavenumber side of the β-sheet band at 1624 cm −1. A difference spectrum (Fig. S7) between the two spectra with and without NaCl confirms this observation. It shows a negative band at 1651 cm −1 and a positive band at 1612 cm −1, as well as a positive shoulder at 1631 cm −1. In line with the more subtle changes in the FTIR spectra, the cryo-TEM images shown in Fig. S6 demonstrate that the fibrils neither dissociate nor change their morphology or dimensions with increased ionic strength. A more thorough investigation of this intriguing phenomenon is necessary, but we hypothesize that the α-helical portion of the peptide undergoes a locally restricted conformational change that quenches the CD signal but results in a state with vibrations at similar wavenumbers to that of an α-helix (1656 cm −1). While the addition of salt to preformed fibrils does not dissolve them, it prohibits the formation of fibrils starting from the unfolded state. Even very low concentrations of salt lead to completely inhibited fibril formation.

Discussion One of the hallmarks of biological self-assembly is that the process is typically driven by the formation of a multitude of weak but specific interactions that direct the building blocks into unique structural states. In order to mimic the properties of self-assembling proteins and peptides in designed systems, we therefore need to develop capabilities to precisely engineer highly specific interactions, such as hydrogen bonding and tight atomic packing. Steric zipper sequences are very useful scaffolds for protein/peptide self-assembly because they can trigger specific association of proteins and peptides but with a structural motif that rely on highly specific interactions. The steric zipper formation can be encoded in a small number of polar residues whose side chains interdigitate to form an extremely packed interface (see Fig. 1b). Elongation of a fibril is driven by hydrogen bonding between consecutive β-strands. In our design, only three positions per strand are used to encode the steric zipper formation. However, the presence of these residues in a designed peptide is not sufficient for efficient self-assembly. The second peptide, βαβZip2, did not assembly into a fibril despite significant similarities in sequence of the core positions and modeled structure to βαβZip. Neither was it possible to use seeds from βαβZip to trigger assembly of βαβZip2. Furthermore, the assembly of βαβZip into fibrils happens only under fairly narrow solvent

conditions. This is in stark contrast to many natural amyloidogenic sequences, which provide a very strong driving force for aggregation when fused to proteins [29]. Aggregation-prone sequences are problematic to use as the basis for biomaterial design because they form relatively heterogeneous assemblies in a process that is difficult to control. The assembly of βαβZip, on the other hand, utilizes features of natural self-assembling processes; it is driven by the formation of weak but specific interactions that result in controllable assembly that can be reversed and modified. The design of βαβZip-like fibrils is constrained by a number of geometrical considerations. The βαβ motif is a common supersecondary structure element in proteins that almost always has a right-handed twist. Certain combinations of secondary structure lengths are highly preferred when connecting secondary structure elements in proteins [1]. In addition to these general considerations, the assembly of the βαβ motifs into a fibril adds further geometrical constraints. The loops and the helix must have a conformation that avoids overlaps with consecutive peptide units in the fibril. Thus, for a given combination of secondary structure lengths, the sequence space is quite limited in the core and for residues that encode helix–helix interactions. Variants of βαβZip could be designed with different lengths and conformation of loop segments. Further stabilization of the fibrils could potentially be achieved by introducing more hydrophobicity in the core. This requires an extension of loops to increase the distance between helix and the sheet. For example, the lengthening of one of the loops in βαβZip2 leads to a larger core with more room for bulkier hydrophobic side chains (Fig. S1). Amyloid fibers are typically composed of several protofilaments. βαβZip was designed to form single fibrils and the cryo-TEM data suggest that, while the fibrils can occasionally associate laterally, they do not form protofilaments with well-defined structures as is found for the sup35 heptapeptide. Formation of structured protofilaments would lead to greater thermal stability and formation of kinetically trapped assemblies. The βαβZip fibrils are stabilized only by relatively few interactions within the steric zipper interface and longitudinal interactions between consecutive subunits in the fibril. With the consideration of this and the fact that the peptide also has to fold to assemble into fibrils, a midpoint in unfolding of peptide of 53 °C is quite respectable. A full understanding of the assembly pathway of βαβZip calls for further experimental investigations of the assembly kinetics. However, the data presented here suggest a model for how fibrillation may occur. Fibril formation occurs only above a critical concentration, below which the peptide predominantly forms a random-coil-like state. This concentration dependence can be explained by equilibrium between unstructured monomers and a structured


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form of the peptide. The oligomeric forms of the peptide serve as nuclei that are critical for the rapid association of peptides into fibrils. This hypothesis is supported by the thermal denaturation experiment, which demonstrates that peptide concentrations above the critical value is not sufficient for efficient self-assembly. In the absence of nucleating species, the intrinsic folding rate of the monomer may be too slow to yield a sufficient concentration of assemblycompetent peptides. Addition of small amounts of preformed fibrils can serve as templates for fibril extension or serve as catalysts for folding of peptides monomers. Nuclei can also form during lyophilization of unstructured peptide. The drastic increase in peptide concentration in the drying promotes fibril formation, probably by increasing the concentration of association-competent species. Alternatively, ice surfaces formed during the lyophilization process may catalyze oligomerization. From the ATR-FTIR spectra measured for the lyophilized powder, we know that α/β structure is present in the dried state. Nuclei can be reproducible regenerated by lyophilization as demonstrated by the successful fibrillation of thermally denatured peptide. The presence of salt inhibits the fibrillation process but does not lead to dissociation of fibrils already formed. Rather it triggers the local unfolding of α-helical portion of the peptide. This suggests that ions influence the folding equilibrium and increase the population of unfolded peptides. βαβZip has many favorable properties for a designed biomaterial: the assembly formation is highly reproducible and produces homogenous fibrils with specific geometry, it forms single fibrils which maximizes the access to the surface and it folds into a unique structural state that can be switched by the addition of salt. However, for many applications, it may be beneficial to extend the narrow solution conditions at which the assembly can occur. The considerable effect on the peptide assembly from changes in pH and ionic strength suggests that interactions of charged residues on the surface of the peptide are important. This suggests that redesign of the surface-exposed positions could be used to modify the assembly conditions. The pH-dependent assembly and salt-dependent conformational change can be a useful property in some application. However, it also highlights a fundamental challenge in the rational design of selfassembling proteins and peptides. For some systems, it may be necessary to design not only the molecular structure of assemblies but also the assembly pathway. The observed assembly mechanism of βαβZip was not the result of a rational design process. While the assembly pathway is also encoded in the amino acid sequence, it cannot be readily predicted based on the structural model of the fibril. Several applications can be anticipated for peptides based on the βαβZip scaffold. The surface residues can be designed for specific binding to other biomolecules. The α-helix can be used as an anchor for

covalent attachment of functional molecules, for example, by covalent modification of surface-exposed cysteine residues. The loop regions can be replaced to graft metal binding site into them, potentially making the fibrils into electrically conducting peptide wires. The peptide sequence can also be fused to other proteins to introduce them into fibrils. This can be used to create catalytic wires or scaffolds for multiprotein binding. In a recent study, short amyloid-forming peptides were demonstrated to self-assemble into amyloids with catalytic function [30]. The peptides can also be used as model systems to understand the relation between sequence, structure and assembly pathway in amyloid proteins.

Methods Computational design The design was based on the crystal structure of the seven-residue amyloid-like peptide from the yeast protein sup35 [15]. This peptide, with the sequence GNNQQNY, self-assembles into double β-sheet with side chains protruding from the sheets forming a tight steric zipper interface. The design objective was to connect two consecutive peptides along the sheets into one chain with a βαβ motif. The segment between the two strands must have a loop– helix–loop secondary structure. The optimal length of the secondary structure elements in the peptide was determined by de novo folding simulations using Rosetta [17] with the use of a similar strategy used by Koga et al. to design ideal protein structures [1]. Monomeric peptides were folded using Rosetta abinitio [31] with different lengths of loops, strands and helices using a sequence-independent backbone model. The fraction of models that folded into the desired topology was used as a measure to select the optimal lengths of secondary structure elements. The simulations indicated that the most optimal way to connect the 2 seven-residue strands is with loop:helix:loop motifs with lengths 3:13:3 or 3:14:2. These two options were explored in flexible backbone design. The design task can be described as a loop-insertion problem with symmetry constraints. To carry out the calculations, we developed a symmetrical version of the RosettaRemodel protocol, which is a generalized framework for flexible backbone design [18]. The protocol was developed in the framework of the rosetta++ program [31], which is the predecessor of the publically available Rosetta program [17]. However, we have now re-implemented the RosettaRemodel protocol in the Rosetta macromolecular modeling suite with all the functionality necessary to repeat this work. Command lines and input files for the rosetta++ design calculations are shown in the supplementary information. The flexible backbone design was carried out as follows. All simulations were performed using perfect helical symmetry to describe the overall structure of the fibril and the interaction between monomers. The backbone structure of six residues of the two connecting strands was kept fixed to the conformation from the crystal structure. The loop–helix–loop motif connecting the strands was built using fragment insertion with a sequence-independent


10 fragment library. The secondary structure of the inserted fragments was selected to match the optimal secondary structure found in the folding simulation (3:13:3 or 3:14:2). The loop modeling was carried out in a coarse-grained representation of the protein with a knowledge-based energy function [31]. The intervening loop, with loop– helix–loop secondary structure, was built from the Nterminal strand (from the backbone coordinates of Y7 in the original peptide). Models where the loops are successfully closed in the folding simulation (connecting the final loop to the second strand with reasonable bond lengths and geometry) are further optimized in the all-atom part of the remodel protocol. After moving into the all-atom representation, we optimized the loop conformation using CCD closure refinement [32] with the use of poly-alanine sequence for the peptide. The final part of the computational procedure is the sequence design. All residues except the ones involved in the steric zipper interface were allowed to change. The surface-exposed positions were kept to alanine until the final design step. The sequence was optimized by iteratively carrying out fixed-backbone simulated annealing optimization of the sequence followed by CCD closure refinement of the loop conformation. This approach allows for simultaneous optimization of backbone, side-chain conformations and sequence. Five rounds of iterative refinement were carried out followed by a final design calculation where also the sequence of solvent-exposed positions was allowed to change. The sequences of solvent-exposed and buried positions were restricted to polar and apolar amino acids, respectively. Solvent exposure was determined through a calculation of the solvent-accessible surface area. Around 1000 models for each combination of secondary structure lengths were calculated. The best energy models were manually optimized by redesigning the surface residues to reduce electrostatic repulsion between subunits in the fibril, to introduce aromatic residues in order to help with experimental characterization and to introduce an N-terminal capping residue to stabilize the helix [19]. One peptide with 3:14:2 and one with 3:13:3 secondary structure lengths were selected for experimental characterization. Peptide synthesis and sample preparation Peptide manufactured by standard solid-phase synthesis and purified by HPLC to a purity of N 95% was purchased from Genscript Inc. Residual TFA from the manufacturing process was removed by an additional chromatography step. Hereby, the mobile phase was a water/acetonitrile mixture either without any acid or with acetic acid as replacement for TFA. During our study, we observed that the presence of acetic acid impairs structure formation of the peptide. A rechromatography step with an acid-free solvent (H2O/ acetonitrile) fully restored the observed features. Samples were prepared from the lyophilised peptide powder by dissolution into pure H2O. The peptide concentrations were determined by UV absorbance at 205 and 280 nm, following the protocol of Scopes [33]. Adjustment of the pH was achieved by addition of TFA or NaOH, where appropriate. The pH titration experiment was performed by adjusting the pH of separate samples to a given value, followed by incubation recording of CD spectra (see below). The position of the lower-wavelength peak (b 200 nm for random-coil

Computational Design of a Self-Assembling Peptide structures and 208 nm for α-helices) was used as indicator for the amount of secondary structure since the peak intensity at, for example, 208 nm is also influenced by minor differences in concentrations and incubation times. For experiments investigating the influence of ionic strength, sodium chloride was added to a final concentration of 50 mM. Denaturation of material was performed by incubating preformed fibrils at 85 °C for 20 min. For nucleation experiments, intact fibrils were added to such denatured material in a molar ratio of 1:100. CD spectroscopy Spectra were recorded in a quartz cuvette (Hellma) with 1 mm path length using a J-815 spectropolarimeter (Jasco) equipped with a Peltier temperature control. The temperature was set to 20 °C except for the thermal denaturation experiments. Here, spectra were recorded from 20 to 95 °C with a ramp of 1 °C/min. Typically, peptide concentrations were 200 μM unless states otherwise in the results section. FTIR spectroscopy Fibril samples were prepared by dissolving peptide batches that had undergone additional purification to remove TFA in H2O, as well as in D2O. Infrared spectra were recorded at room temperature (20 °C) and with 4 cm − 1 resolution using a Bruker Vertex 70 spectrometer equipped with a SensIR 9-reflection diamond ATR accessory. We averaged 100 interferometer scans for each spectrum. The integrity of the samples prepared for FTIR measurements was confirmed by CD spectroscopy. We added 4 μl of 1 mM peptide in H2O or D2O onto the ATR crystal. Consecutive spectra were measured while the sample was dried under a gentle stream of N2, whereupon a peptide film was formed on the surface of the crystal. Spectra of the lyophilized peptide powder were recorded using the Bruker Platinum ATR accessory. The powder was gently pressed onto the diamond ATR crystal during measurement. The ATR infrared spectra were fitted using the program Kinetics (kindly provided by Erik Goormaghtigh, Université Libre de Bruxelles). The program simultaneously minimizes the error between the fit and the experimental spectrum, as well as between the second derivative of the fit and the second derivative of the experimental spectrum. The second derivative is weighted by a factor in order to account for its smaller amplitude. The weighting factor used was between 50 and 2000. Electron microscopy Carbon-coated copper grids (Electron Microscopy Science) were placed upside-down onto the sample for 30 s, blotted and negatively stained with a ready-to-use vanadin solution (Nanoprobes) After blotting, we air-dried the samples for 24 h prior to inspection on a Philips CM120 BioTWIN cryo-electron microscope. For cryo-TEM, samples were prepared in a vitrification system. We applied 5 μl of a fibril solution to lacey Formvar/carbon-coated copper grids (Ted Pella Inc.) as a thin film and flash-frozen in liquid ethane to avoid water crystallization and perturbance of the sample.


11


Grids were stored under liquid nitrogen until inspection. An Oxford CT3500 cryoholder was used to transfer the samples into the microscope. Images were recorded with a CCD camera using low-electron-dose conditions. For the measurement of the fibril dimensions, individual fibrils were selected from the images using the script e2helixboxer.py from the EMAN2 image analysis package [8]. The fibril width was measured manually using the software DigitalMicrograph 3.4 (Gatan Inc). X-ray fiber diffraction A peptide solution at 10 mg/ml was incubated at room temperature for 24 h. For fiber alignment, 10-μl drops were placed between two capillaries, sealed with wax and kept at room temperature until dry. The dry fiber sample was placed on a goniometer head and data were collected using a CuKα rotating anode (Rigaku) and a CCD + detector, with a specimen-to-detector distance of 100 mm. For inspection of the diffractograms, the software Clearer 2.0 was used [34]. The intensity profile along the meridian was calculated using Clearer by taking the radial average of the intensity in a sector of 20° in the direction that is not obstructed by the beam stop. Experimental diffractograms were transformed into reciprocal space using the software WCEN v2.6.5 [35] with a bin size of 0.01 Å − 1.

To simulate the fiber diffraction spectra, we need to consider that experimental layer lines are not infinitely thin and to take the effect of fiber disorientation into consideration. The distribution of intensity d(lz) across a layer line can be approximated by a Gaussian distribution [39] d ðl z Þ ¼ exp −p 2 π ðl z −Z 0 Þ2 where lz is a reciprocal space coordinate along Z, p is the coherence length of the fiber and Z0 is the value of the reciprocal Z value at the center of the line. p was set to 100 Å in the simulation. The effect of fiber disorientation was simulated with a Gaussian probability disorientation function according to Holmes and Barrington-Leigh [24] N ðα Þ ¼

2 2 α exp − 2 α0 α0

where N(α)dΩ/4π is the probability of finding the fibers at an angle α to the common fiber axis in an element of solid angle dΩ. α0 is a parameter that defines the degree of angular disorientation. We simulate with a α0 = 5° disorientation. The disoriented intensity Idis can now be calculated as I dis ðR; Z Þ ¼

Analytical ultracentrifugation Sedimentation equilibrium experiments were performed with an Optima XL-A ultracentrifuge (Beckman Coulter) in two-sector Epon centerpieces providing 12 mm optical path length. Centrifugation was carried out at 20 °C with rotor speeds of 40,000, 50,000 and 60,000 rpm, respectively. The data were fitted to a single-species model using the software Sedfit 14.1 [36]. The partial specific volume of the peptide was estimated from the amino acid composition with SEDNTERP [37]. Fiber diffraction simulation The fiber diffraction spectrum was simulated from the atomic model of the design by first calculating the intensity along layer lines without disorientation according to Franklin and Klug [38] as follows: I ðR; Z ¼ l=c Þ XX 2πl ¼ f i f j J n ð2πRr i Þ J n 2πRr j cos n φi −φ j − z i −z j c i; j n

where I(R, Z = l/c) is the intensity along layer line l at reciprocal coordinates (R,Z) in cylindrical coordinates, (r,φ,z) are positions of atoms in cylindrical coordinates, f are solvent-corrected atomic scattering factors, Jn is the Bessel function of order n and c is the helical repeat distance. The Bessel orders that contribute to a layer line are determined by the helical selection rule l ¼ um þ v n where u is the number of residues in v turns of the fiber helix and m is an integer.

1 4π

Z

2π Z π

φ¼0 γ¼0

I ðs Þl d ðl z ÞN ðα Þ sinγ dγdφ

where γ and φ are angles defined in a coordinate system described by Holmes and Barrington-Leigh [24] with l z ¼ v cos γ s ¼ v sinγ v¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 R2 þ Z 2

cos α ¼ Z l z þ Rs cos φ=v 2 The double integral was evaluated numerically with MATLAB R2013b, MathWorks Inc., Natick, MA, USA.

Acknowledgment This work was supported by the Swedish Research Council and Olle Engkvist Foundation (I.A.). We would like to thank Erik Goormaghtigh (Université Libre de Bruxelles) for introducing co-fitting of second-derivative spectra into his spectroscopy software Kinetics and for sharing his program with us. We thank Gunnel Karlsson for excellent technical assistance with the collection of electron microscopy images, Nobu Koga (University of Washington) for help with folding simulations, Wojciech Potrzebowski for help with fibril diffraction


12


simulations and comments on the manuscript and David Baker (University of Washington) for discussions in the early stage of this project.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2014.12. 002. Received 15 September 2014; Received in revised form 26 November 2014; Accepted 2 December 2014 Available online xxxx Keywords: self-assembly; computational protein design; protein structure; Fourier transform infrared spectroscopy; de novo design

Abbreviations used: FTIR, Fourier transform infrared; TFA, trifluoroacetic acid; TEM, transmission electron microscopy.

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