This is the accepted version of the journal article: Díaz-Caballero, Marta; Navarro, Susanna; Nuez-Martínez, Miquel; [et al.]. «pH- responsive self-assembly of amyloid fibrils for dual hydrolase-oxidase reactions». ACS catalysis, Vol. 11, Issue 2 (January 2021), p. 595-607. DOI 10.1021/ac- scatal.0c03093 This version is available at https://ddd.uab.cat/record/277745 under the terms of the license Page 1 of 46 ACS Catalysis 1 2 3 4 5 pH-responsive self-assembly of short peptides 6 7 8 9 into multi-catalytic amyloid fibrils 10 11 12 Marta Díaz-Caballero1, Susanna Navarro1, Miquel Nuez-Martínez2, Francesca Peccati3, 13 14 Luis Rodríguez-Santiago4, Mariona Sodupe4,5, Francesc Teixidor2 and Salvador Ventura1,5* 15 16 17 18 19 20 1 Institut de Biotecnologia i de Biomedicina (IBB) and Departament de Bioquímica i Biologia 21 22 Molecular; Universitat Autònoma de Barcelona; 08193 Bellaterra (Barcelona), Spain. 23 24 25 2 Institut de Ciència de Materials de Barcelona (ICMAB-CSIC). Campus UAB, 08193 Bellaterra, 26 27 28 Barcelona (Spain). 29 30 31 3 Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and 32 33 Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio (Spain) 34 35 36 37 4 Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra (Spain) 38 39 40 5 ICREA, Passeig Lluís Companys 23, E-08010 Barcelona (Spain) 41 42 43 44 45 46 *Corresponding author: E-mail: salvador.ventura@uab.es 47 48 49 50 51 52 KEYWORDS: amyloid fibrils, self-assembly, short peptides, artificial enzymes, stimuli-pH- 53 54 responsive materials, hydrogels 55 56 57 58 59 60 ACS Paragon Plus Environment 1 ACS Catalysis Page 2 of 46 1 2 3 ABSTRACT 4 5 6 There is an increasing interest in synthetic systems that can execute bio-inspired chemical 7 8 reactions without requiring the complex structures that characterize enzymes in their components. 9 10 The hierarchical self-assembly of peptides provides a means to create catalytic 11 12 13 microenvironments. Ideally, as it occurs in enzymes, the catalytic activity of peptide 14 15 nanostructures should be reversibly regulated. In a typical enzyme mimetic design, the peptide's 16 17 self-assembling and catalytic activities are segregated into different regions of the sequence. Here, 18 19 20 we aimed to design minimal peptides in which the self-assembly and function were all encoded in 21 22 the same amino acids. Moreover, we wanted to endorse the resulting one-component nanomaterial 23 24 with divergent, chemically unrelated, catalytic activities, a property not observed in natural 25 26 enzymes. We show that short peptides consisting only of histidine and tyrosine residues, arranged 27 28 29 in a binary pattern, form biocompatible amyloid-like fibrils and hydrogels combining hydrolytic 30 31 and electrocatalytic activities. The nanofibers' mesoscopic properties are controlled by pH, the 32 33 34 transition between assembled active b-sheet fibrils, and disassembled inactive random coil species 35 36 occurring in a physiologically relevant pH range. The structure of one of such amyloid-like fibrils, 37 38 as derived from molecular dynamic simulations, provides insights on how they attain this unique 39 40 41 combination of structural and catalytic properties. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 2 Page 3 of 46 ACS Catalysis 1 2 3 INTRODUCTION 4 5 6 Natural organisms use proteins and peptides self-assembly to build up a variety of mesoscopic 7 8 materials, including collagen1 and fibrin2 in humans, spider silk,3 or squid sucker ring teeth.4 There 9 10 is a rising interest in exploiting peptide self-assembly as a route to build up novel nanomaterials5,6 11 12 13 since they might allow incorporating protein-like activity and responsivity to the environment into 14 15 the nanostructure, providing control over the macroscopic and functional properties of the 16 17 assembly. Besides, peptides can assemble under mild, close to physiological conditions, displaying 18 19 20 excellent biocompatibility. 21 22 Amyloid fibrils belong to the group of ordered nanostructures that result from the self-assembly 23 24 of polypeptides.7 They have been traditionally associated with human diseases, but the recent 25 26 27 discovery of amyloids assisting biological functions in a wide range of organisms, from bacteria 28 29 to humans,8,9 has changed this perception. Amyloid fibrils share a common quaternary structure, 30 31 formed by the assembly of monomers into a characteristic “cross-β” structure, in which β-sheets 32 33 are parallel to the fibril axis with their strands perpendicular to this axis.10,11 This architecture is 34 35 36 stabilized by hydrogen bonds between neighboring β-strands, and an array of other non-covalent 37 38 contacts, like π-π stacking, van der Waals, electrostatic and hydrophobic interactions. This renders 39 40 robust supramolecular structures without a need for exogenous cross-linking agents, a property 41 42 43 exploited to design novel materials for biomedical and biotechnological applications.5,12 44 45 The buildup of materials via molecular self-assembly allows to define their ultimate structural 46 47 and functional properties by designing the individual constituent molecules. A long-lasting 48 49 50 objective in catalyst design has been to develop simple synthetic systems that can execute bio- 51 52 inspired chemistry, without requiring defined and complex tertiary structures in the individual 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 3 ACS Catalysis Page 4 of 46 1 2 3 molecules. The self-assembly of peptides provides a means for bringing multiple functional groups 4 5 6 into proximity, in a way that is analogous to the folded conformation of enzymes.13 7 8 Catalytic self-assembled systems are typically modular, incorporating one or more amino acids 9 10 relevant for the catalysis and a block responsible for the assembly, usually separated by a spacer.14– 11 12 16 13 The self-assembly promoting unit may or may not have a pure peptidic nature, like peptide 14 15 amphiphiles, which are constructed by capping peptides with alkyl chains or other aromatic 16 17 molecules. Both peptides and peptide amphiphiles have been successfully assembled into b-sheet- 18 19 20 rich nanofibers able to hydrolyze activated esters, a typical model reaction for hydrolytic 21 22 enzymes.14,15 Like in many hydrolases, the active residue in the individual subunit is His13,17,18 in 23 24 some cases accompanied by an Arg to facilitate the catalysis.14 Importantly, the architecture of the 25 26 27 assembly provides an adequate enzyme-like microenvironment for the reaction, with the building 28 29 blocks alone displaying low hydrolytic activity.19 The same bottom-up strategy has been employed 30 31 to obtain redox-active nanofibers, exploiting the reduction potentials that Tyr radicals acquire in 32 33 34 assembled structures.20,21 In several of these systems, when the concentration is increased, the 35 36 peptide fibrils entangle to form a fibrillar network, which renders a hydrogel that retains the 37 38 catalytic activity of the individual nanofibers.19,22 The versatile properties of these hydrogels, such 39 40 as high-water content and solid shape, have attracted great attention.23 41 42 43 The inherent sensitivity of polypeptide chains to pH is exploited by nature to control the structure 44 45 of functional amyloids such as PMEL, a protein involved in melanin production, or the storage 46 47 and secretion of pituitary peptide hormones. In both cases, the self-assembly into amyloid fibrils 48 49 50 only occurs under the acidic conditions found in the respective granules.24,25 Mimicking this 51 52 strategy, pH-responsive peptide and peptide amphiphiles have been designed to develop new b- 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 4 Page 5 of 46 ACS Catalysis 1 2 3 sheet-based nanomaterials with tunable properties via a simple self-assembly mechanism without 4 5 6 a need for exogenous cross-linking agents.26,27 7 8 pH-regulated peptide assembly offers an opportunity to mimic the behavior of natural enzymes 9 10 in artificial nanofibrillar catalyzers, which might permit to regulate reactions simply by modulating 11 12 13 the solution pH. Attaining this control is not trivial since it requires the integration of pH- 14 15 responsiveness without interfering with the activity of catalytic residues. This has been achieved 16 17 in a recent study by introducing a His residue at the terminus of a pH-responsive peptide.19 The 18 19 20 peptide exhibits a pH-dependent conformational transition from random coil to b-sheet and 21 22 assembles into fibrils that display esterase activity. In this system, the activity could be reversed 23 24 by pH-induced assembly/disassembly of the fibrils into random coils. This model system, and 25 26 27 indeed most studies,6,23,28,29 exploit the fact that amino acid side chains incorporating -COOH (Asp 28 29 and Glu) or -NH2 (Lys and Arg) groups can be in either a protonated or deprotonated state at pH 30 31 values which are, respectively, below or above their nominal pKa values. Despite the pKa values 32 33 34 in these self-assembling systems can be significantly shifted compared to solution ones,30 the 35 36 transitions necessarily occur far from the physiological pH. 37 38 It has just been reported that the assembly of the Orb2 protein into functional amyloids in 39 40 41 Drosophila melanogaster31 is governed by the protonation state of the imidazole group of His, a 42 43 property shared by suckerin proteins in squids.32,33 This suggests that, under physiological 44 45 conditions, His residues might be used as molecular switches, endorsing synthetic peptides with 46 47 self-assembly reversibility, in addition to catalytic activity, if they are embedded in the proper 48 49 50 microenvironment. 51 52 We have recently designed a collection of synthetic prion-inspired heptapeptides.21 These short 53 54 peptides combine residues enriched in prion-like proteins (Asn, Gln, and Ser) with Tyr, the only 55 56 57 58 59 60 ACS Paragon Plus Environment 5 ACS Catalysis Page 6 of 46 1 2 3 enriched aromatic residue in these polypeptides, forming a binary arrangement (Ac-XYXYXYX- 4 5 6 NH2). Despite their eminent polar nature, these peptides readily assembled into highly ordered 7 8 amyloid fibrils.21 Here we exchanged the polar residues in their sequences by His, obtaining a set 9 10 of short peptides composed only by His and Tyr (hereinafter named as HY-peptides) consisting of 11 12 13 7, 8 and 9 residues, HY7 (Ac-HYHYHYH-NH2), HY8 (Ac-YHYHYHYH-NH2) and HY9 (Ac- 14 15 HYHYHYHYH-NH2), respectively. We rationalized that instead of having separate modules for 16 17 assembly and function, the same residues might account for both activities in these peptides. We 18 19 20 show that, indeed, HY-peptides present unique properties, since they assemble into biocompatible 21 22 amyloid-like fibrils in a pH-dependent manner. These nanofibers act as multi-catalytic artificial 23 24 enzymes because they combine both hydrolase and redox activities, thanks to their His and Tyr 25 26 residues, respectively. The fibrils render active hydrogels at moderate peptide concentrations, 27 28 29 whose assembled state can be controlled by the pH. Atomistic molecular dynamics simulations 30 31 provide a structural explanation for this singular combination of conformational and functional 32 33 features. Our results illustrate how supramolecular assemblies enable the incorporation of 34 35 36 properties not seen in natural enzymes, such as the introduction of two different and unrelated 37 38 catalytic activities into a single pH-regulated nanomolecular structure. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 6 Page 7 of 46 ACS Catalysis 1 2 3 RESULTS AND DISCUSSION 4 5 6 7 HY-peptides self-assemble into amyloid-like structures at pH 8.0 8 9 10 The influence of pH on HY7, HY8 and HY9 peptides self-assembly was assessed by incubating 11 12 each peptide at pH 4.0 or pH 8.0. The assembly was first monitored through synchronous light 13 14 scattering (Figure S1A). HY-peptides incubated at pH 8.0 exhibited a significant increase in the 15 16 17 light scattering signal compared to those incubated at pH 4.0. 18 19 We investigated the secondary structure content of HY-peptides incubated at pH 8.0 and pH 4.0 20 21 by circular dichroism (CD) spectroscopy (Figure S1B). The far-UV CD spectra of peptides 22 23 24 incubated at pH 8.0 presented a minimum at 217 nm associated with β-sheet structures. In contrast, 25 26 in HY-peptides incubated at pH 4.0, the signal gradually decreased at low wavelengths, a feature 27 28 assigned to random coil conformations, suggesting that HY-peptides remained disordered at this 29 30 pH. At both pHs, the peptide spectra exhibited positive maxima around 230 nm, arising from the 31 32 33 contribution of the peptide aromatic side chains.34 These signals decreased and were redshifted at 34 35 pH 8.0 relative to those at pH 4.0. 36 37 Next, we assessed the secondary structure of the assemblies formed upon incubation at pH 8.0, 38 39 40 by Fourier-Transform Infrared (FTIR) spectroscopy and recording absorbance in the amide I 41 42 region of the spectrum (Figure S1C and Table S1). In addition to a peak at ~1610 cm-1 43 44 corresponding to His and Tyr aromatic side chains, the FTIR spectra were dominated by a signal 45 46 47 comprised between 1615 and 1632 cm-1, accounting for ~ 70 % of the total area of the absorbance 48 49 spectrum for all HY-peptides. This signal can be assigned to intermolecular β-sheet structure. We 50 51 52 could not detect antiparallel β-sheet signals at ~1690 cm-1, suggesting a preferential parallel 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 7 ACS Catalysis Page 8 of 46 1 2 3 disposition of the β-strands in the assemblies. The FTIR and CD data are in good agreement, 4 5 6 indicating the formation of β-sheets when HY-peptides are incubated at pH 8.0. 7 8 We assessed if these β-sheet enriched assemblies exhibited an amyloid-like signature using the 9 10 amyloid-specific dyes Thioflavin-T (Th-T) and Congo Red (CR). Th-T was added to HY-peptides 11 12 13 incubated at pH 4.0 or pH 8.0 for 4 days. As shown in Figure 1A, the three peptides promoted an 14 15 increase in the Th-T fluorescence signal only at pH 8.0, whereas the spectra of samples incubated 16 17 at pH 4.0 resembled that of the buffer with Th-T only. Binding to CR confirmed the differential 18 19 20 behavior of the peptides at the two pHs. As shown in Figure 1B, HY-peptides incubated at pH 8.0 21 22 showed a red-shift of the respective CR absorption spectra, while the peptides incubated at pH 4.0 23 24 presented a spectrum similar to that of the buffer with CR. Thus, both assays indicate that the 25 26 peptides form amyloid-like assemblies only when incubated at alkaline pH. 27 28 29 The morphology of the assemblies was visualized by transmission electron microscopy (TEM). 30 31 Analysis of representative micrographs revealed the presence of small amorphous assemblies in 32 33 HY-peptides incubated at pH 4.0. In contrast, samples incubated at pH 8.0 displayed long and thin 34 35 36 fibrillar structures (Figure 1C), consistent with an amyloid-like nature, although they differed in 37 38 morphology between peptides. We measured the thickness of the imaged HY7, HY8 and HY9 39 40 fibrils at pH 8.0 using high magnification micrographs (Figure 1C) displaying widths 41 42 43 corresponding to 16.5 ± 4.0 nm, 21.0 ± 6.4 nm and 7.2 ± 1.2 nm, respectively, in agreement with 44 45 the dimensions reported for different amyloid structures.35 46 47 The analysis of the peptides incubated at pH 8.0 by dynamic light scattering (DLS) confirmed 48 49 50 the presence of large assemblies (Figure S2). On the contrary, samples incubated at pH 4.0 did 51 52 not show any evident DLS signal since species’ sizes were under the detection range of the 53 54 instrument. The average diameter of HY-peptides assemblies at pH 8.0 was 1031.0 ± 453.8 nm 55 56 57 58 59 60 ACS Paragon Plus Environment 8 Page 9 of 46 ACS Catalysis 1 2 3 (HY7), 3161.4 ± 1004.2 nm (HY8), and 2097.9 ± 940.2 nm (HY9). Although these dimensions 4 5 6 are in the range observed by DLS for different amyloid assemblies,36,37 they should not be taken as 7 8 a proxy of the fibril length since DLS is only accurate in measuring dimensions of spherical 9 10 particles, and they just reflect the relative sizes of the different assemblies. The sizes were not 11 12 13 monotonically increasing with the length of the peptide chain. We hypothesized that this might 14 15 respond to the fact that HY7 and HY9 have a composition of Hn+1Yn, whereas HY8 composition 16 17 formula is HnYn. To explore this possibility, we synthesized an HY6 peptide (Ac-HYHYHY-NH2). 18 19 20 At pH 8.0, HY6 forms assemblies that bind to Th-T (Figure S3A) and CR (Figure S3B), exhibit 21 22 high b-sheet content as measured by FTIR (Figure S3C and Table S1) and appear as highly 23 24 ordered fibrils, 19.0 ± 4.9 nm in width (Figure S3D). As expected, HY6 does not assemble into 25 26 27 amyloid-like structures at pH 4.0 (Figure S3). DLS analysis, indicated that, as hypothesized, the 28 29 diameter of HY6 assemblies at pH 8.0 (765.6 ± 120.5 nm) was smaller than the one of HY8 ones 30 31 (Figure S2). 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 9 ACS Catalysis Page 10 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 10 Page 11 of 46 ACS Catalysis 1 2 3 Figure 1. Characterization of the amyloid-like properties for HY-peptides incubated at pH 4 5 6 4.0 and pH 8.0. The amyloid tinctorial properties of HY7 (red), HY8 (blue) and HY9 (green) 7 8 incubated peptides were assayed using A) Th-T and B) CR. Spectra in the absence (grey line) and 9 10 in the presence of HY7 peptides incubated at pH 4.0 (dashed line) and at pH 8.0 (solid line) are 11 12 13 shown. C) Representative micrographs of HY-peptides samples incubated at pH 4.0 (dashed line, 14 15 upper row) and at pH 8.0 (solid line, middle and bottom rows). Scale bars correspond to 0.2 or 1 16 17 µm. 18 19 20 HY-peptides fibrils are innocuous for human cells 21 22 23 One of the limitations of amyloid-like materials is that they might possess certain cytotoxic 24 25 activity.38 In order to discard this possibility, HY amyloid-like fibrils formed at pH 8.0 were 26 27 isolated by centrifugation and added to SH-SY5Y neuroblastoma cultured cells at concentrations 28 29 30 ranging between 10 and 50 μM (Figure S4). After 72 hours, the cell viability was monitored using 31 32 the PrestoBlue® reagent. None of the fibrillar assemblies display significant cytotoxicity at any of 33 34 the tested concentrations, suggesting that they would have a good biocompatibility. 35 36 37 38 39 Amyloid self-assembly of HY-peptides is controlled by pH 40 41 The results presented above converge to indicate that the fibrillation of HY-peptides under mild 42 43 conditions is strongly dependent on pH. We assessed if the degree of peptide self-assembly 44 45 correlates with the solution's pH. HY-peptides were incubated at pHs ranging from 4.0 to 8.5 in 46 47 48 Tris-Citrate buffer (Figure 2). Amyloid formation was monitored using Th-T. Since the peptide 49 50 termini are protected by amidation and acetylation modifications and the tested pH range should 51 52 not affect Tyr's protonation (L-Tyr side chain pKa = 10.5); the behavior of these peptides would 53 54 55 be directly associated to the protonation state of their His residues (L-His side chain pKa = 6.0). 56 57 58 59 60 ACS Paragon Plus Environment 11 ACS Catalysis Page 12 of 46 1 2 3 As shown in Figure 2, the three HY-peptides exhibited a pH-dependent transition with a pH1/2 4 5 6 which is close to the pKa value of 6.88 determined for the imidazole group of histidine in the 7 8 random-coil peptide Ac-Ala-Ala-His-Ala-NH239 (Table S2), indicating that His deprotonation 9 10 facilitates self-assembly, whereas electrostatic repulsion between positively charged His impedes 11 12 13 it. There exist subtle differences in the pH1/2 between the peptides, with HY7 and HY9 pH 14 15 transitioning at slightly higher pH than HY8. This might owe to the different His/Tyr ratio in the 16 17 peptides, the identity of the C-terminal residue, or to a different environment of the His residues 18 19 20 in the different assemblies. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figure 2. HY7, HY8, and HY9 peptides self-assembly as a function of the pH. HY7 peptide 42 43 (red line) was measured at 200 µM final concentration and HY8 (blue line) and HY9 (green line) 44 45 peptides were assayed at 100 µM final concentration in 100 mM Tris HCl 50 mM Citrate buffer 46 47 adjusted at different pHs (4.0 to 8.5 each 0.5 pH units) and the assembled state evidenced by the 48 49 50 addition of Th-T. Th-T maximum emission at 492 nm was recorded, and fluorescence intensity 51 52 normalized. Fitting of the data to a sigmoidal curve was performed using GraphPad Prism 5.0 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 12 Page 13 of 46 ACS Catalysis 1 2 3 software. Data correspond to the mean of three independent experiments and error bars indicate 4 5 6 standard deviation. 7 8 9 pH-responsive HY-peptides amyloids 10 11 12 The properties of HY-peptides suggested that they might experiment bidirectional 13 14 assembly/disassembly in response to changes in the pH, which would allow to control their 15 16 17 supramolecular state. To assess if this was the case, HY-peptides were initially self-assembled at 18 19 pH 8.0 for 4 days and then the solution was acidified to pH 4.0 and allowed to stand for 4 days. 20 21 Finally, the pH was reverted again to pH 8.0 and the peptides solution incubated for 4 additional 22 23 24 days. 25 26 The process was monitored simultaneously by synchronous light scattering, far-UV CD, Th-T 27 28 and CR binding (Figure 3). The results from synchronous light scattering, Th-T, and CR binding 29 30 converged to indicate that the amyloid-like assemblies formed at pH 8.0 were disassembled at pH 31 32 33 4.0 and that upon reversion to pH 8.0 the amyloid properties were at least partially recovered 34 35 (Figure 3A, 3B and 3C). The CD spectra of the HY-peptides revealed that the minimum signal 36 37 detected at 217 nm corresponding to β-sheet present in samples incubated at pH 8.0 disappeared 38 39 40 at pH 4.0, and then was recovered back when the pH turned alkaline (Figure 3D). 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 13 ACS Catalysis Page 14 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 3. HY7, HY8, and HY9 peptides assembly, disassembly and reassembly as a function 41 42 43 of the pH. HY7 (red), HY8 (blue), and HY9 (green) peptides were prepared at 250 µM. An initial 44 45 self-assembling incubation step was performed at pH 8.0 (solid colored line), followed up by a 46 47 disassembling incubation step at pH 4.0 (solid black line), and a re-assembling incubation step 48 49 50 induced at pH 8.0 (dashed colored line). A) The aggregated state of the samples was analyzed by 51 52 synchronous light scattering. Amyloid tinctorial properties were assessed by B) Th-T and C) CR 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 14 Page 15 of 46 ACS Catalysis 1 2 3 binding assays, performed in the absence (grey line) and in the presence (colored and black lines) 4 5 6 of peptide. D) The secondary structure content of the peptides was assessed by far-UV CD. 7 8 9 The peptide solutions were imaged at each step by TEM. As shown in Figure S5, HY-peptides 10 11 incubated at pH 8.0 displayed an amyloid-like fibrillar morphology, whereas upon acidification to 12 13 pH 4.0, fibrils were no longer present and only small and dispersed amorphous aggregates were 14 15 16 observed. In good agreement with the above biophysical data, reversion to pH 8.0 promoted the 17 18 re-organization into fibrillar assemblies, despite their morphology was less regular than the 19 20 original ones. 21 22 23 We used HY8 as a model system to test if the transition between a non-amyloid state at pH 4.0 24 25 and an amyloid-like ensemble at pH 8.0 is sustained after several cycles of acidification- 26 27 alkalinization. Figure S6 shows that this is the case for at least 4 cycles. 28 29 30 The data converge to indicate that the soluble/assembled transitions observed for HY peptides 31 32 depend on the ionizable imidazole side chains, and indeed, a QY7 peptide (Ac-QYQYQYQ-NH2) 33 34 which, as HY-peptides, forms amyloid fibrils at pH 8.021 retains its aggregated state and amyloid- 35 36 37 like properties when the solution is acidified to pH 4.0 (Figure S7). 38 39 40 41 HY8 peptide forms a pH-responsive hydrogel 42 43 We selected the HY8 peptide as a model and assessed if it can form a mechanically rigid and 44 45 46 reversible hydrogel at higher concentrations (Figure 4). The inverted tube test indicates that when 47 48 the peptide is incubated at 5 mM (0.5 % w/v) and pH 4.0, the solution remains liquid, but upon 49 50 alkalization to pH 8.0 and incubation for 4 days a rigid hydrogel matrix is formed. Hydrogelation 51 52 53 is reversible and lowering the pH to 4.0 causes the gel to fluidize immediately. 54 55 56 57 58 59 60 ACS Paragon Plus Environment 15 ACS Catalysis Page 16 of 46 1 2 3 Other b-sheet-based nanomaterials able to switch between soluble and assembled states as a 4 5 6 function of the pH have been described previously; however, because they either exploit Asp and 7 8 Glu (pKa < 4.5) or Lys and Arg (pKa > 10) residues, the transitions between states occur further 9 10 from the physiological pH than in our case.6,23,28,29 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Figure 4. pH-reversible transition of HY8 from solution to hydrogel. The HY8 peptide was 32 33 initially incubated at pH 4.0 in the presence of phenol red pH indicator, which presents a yellow 34 35 36 colour at this pH (left image). Upon increase of the pH to 8.0, phenol red shifts to red/pink colour 37 38 and a solid hydrogel is formed (middle image). Decreasing the pH to 4.0 disentangles the formed 39 40 hydrogel and turns the pH indicator yellow again (right image). 41 42 43 44 45 HY-peptides amyloid-like fibrils display esterase activity 46 47 The imidazolyl groups of His are present at the active sites of many hydrolytic enzymes, like 48 49 trypsin40 and chymotrypsin.41 Peptides containing His residues have been assembled into 50 51 52 nanofibers that function as mimics of biological hydrolytic enzymes, catalyzing ester 53 54 hydrolysis.14,15 In HY-peptides fibrils, at least 50% of the residues are His, suggesting that they 55 56 57 58 59 60 ACS Paragon Plus Environment 16 Page 17 of 46 ACS Catalysis 1 2 3 might also exhibit esterase activity. To address this possibility, HY-peptides fibrils were incubated 4 5 6 in the presence of the commonly employed substrate p-nitrophenyl acetate (pNPA), which 7 8 hydrolysis produces p-nitrophenol (pNP), a chromogenic molecule, that can be monitored by 9 10 measuring the increase in absorbance at 405 nm. As in biological enzymes, the imidazole is 11 12 13 expected to react with the pNPA to form phenol and acylium ion imidazole complexes. The 14 15 complex would then react with water to form acetic acid and regenerate the imidazole ring. 16 17 Kinetic assays at pH 8.0 revealed that HY-peptides were able to catalyze ester hydrolysis without 18 19 20 any additional co-factor. We calculated the kinetic parameters of the reaction. The initial pNPA 21 22 hydrolysis rates were measured as a function of the substrate concentration, maintaining the 23 24 concentration of peptide fibrils constant (Figure 5A). Double-reciprocal plots were obtained 25 26 (Figure S8), and the apparent kinetic constants calculated according to the Michaelis-Menten 27 28 29 equation. The different peptide fibrils did not present large differences in their kinetic parameters 30 31 (Table S3), with kcat(app)/KM specificity constants of 1.26 ± 0.43 M-1 s-1, 1.36 ± 0.18 M-1 s-1 and 1.64 32 33 ± 0.33 M-1 s-1, for HY7, HY8 and HY9 peptides, respectively. The typical enzymatic saturation 34 35 36 kinetics shown by these scaffolds indicates that they behave as actual enzymes. Kinetic parameters 37 38 were also calculated at pH 7.4 (Figures S9 and S10), as expected, the fibrils were slightly less 39 40 active than at pH 8.0 (Table S3), with kcat(app)/KM values of 0.47 ± 0.49 M-1 s-1, 0.54 ± 0.19 M-1 s-1 41 42 43 and 0.55 ± 0.37 M-1 s-1 for HY7, HY8 and HY9 peptides, respectively. 44 45 No connection between the peptide length and the catalytic activity was evident, as previously 46 47 described for the Zn2+ dependent esterase activity of related amyloid peptides.18 Among the 48 49 50 reported catalytic peptide assemblies for ester hydrolysis, metal-chelated higher-order constructs 51 52 exhibit the highest activities.13,17,42 HY-peptides' activity was in the same range as that of non-metal 53 54 dependent catalytic nanofibrils at the same pH.14 55 56 57 58 59 60 ACS Paragon Plus Environment 17 ACS Catalysis Page 18 of 46 1 2 3 We have shown that HY8 forms a hydrogel and that the phase behavior can be tightly controlled 4 5 6 by the pH. We addressed if the fibrils entangled in the gel state display esterase activity (Figure 7 8 5B). A HY8 gel was prepared at pH 8.0 and 5 mM peptide concentration. A 20 µL aliquot of pNPA 9 10 was then added into the hydrogel. After few minutes the gel acquired the yellow coloration of the 11 12 13 pNP product, a clear indication that the nanostructure was catalytically active. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 5. Esterase activity characterization. A) HY7, HY8 and HY9 esterase activity was 49 50 detected by measuring the absorbance of the yellow-colored pNP product at 405 nm in 25 mM 51 52 Tris-HCl pH 8.0 (E0 concentration 100 µM). Data were fitted to the Michaelis-Menten equation 53 54 55 with GraphPad PRISM 5.0 software. B) HY8 hydrogel esterase activity. HY8 peptide was 56 57 58 59 60 ACS Paragon Plus Environment 18 Page 19 of 46 ACS Catalysis 1 2 3 incubated at 5 mM at pH 8.0. An aliquot of 1 mM pNPA was added and the sample incubated for 4 5 6 30 minutes (right image). The yellow color indicates the production of chromogenic pNP. 7 8 9 HY-peptides amyloid-like fibrils are electrochemically active 10 11 In biological systems such as the photosystem II, Tyr acts as redox-active residue thanks to its 12 13 location in chemical environments that reduce the activation potentials for the generation of tyrosyl 14 15 16 radicals.43 In solution, L-Tyr can generate tyrosyl radicals, but those are rapidly quenched in an 17 18 aqueous solvent. Over the past few years, different studies have created redox-active scaffolds by 19 20 incorporating Tyr residues into peptidic building blocks.20,44 We rationalized that the defined 21 22 23 chemical environment of HY-peptides fibrils might endorse their Tyr radicals with reduction 24 25 potentials high enough to catalyze chemical reactions. 26 27 We explored if HY-peptides fibrils can act as bioelectrocatalysts by assessing the oxidative 28 29 30 polymerization of pyrrole as a model reaction.45 Polypyrrole (PPy) can be synthesized 31 32 electrochemically by applying a potential greater than 0.8 V vs. Ag/AgCl or chemically by using 33 34 an oxidant agent. Although electropolymerization can be initiated at 0.8 V vs. Ag/AgCl, the rate 35 36 37 at this potential is too slow to attain macroscopic PPy deposition. Our results showed that at 0.8 V 38 39 vs. Ag/AgCl, a negligible amount of PPy was produced in the presence of L-Tyr (10 μA at 500 40 41 seconds) (Figure 6A and Figure S11). In contrast, when this voltage was applied to HY7, HY8, 42 43 and HY9 fibrils, the chronoamperometry curves indicated that pyrrole polymerized rapidly, in the 44 45 46 range of seconds. Intensities of 850 μA were attained for HY8 and HY9 fibrils and 400 μA for 47 48 HY7 ones. Continuous deposition of PPy was observed in all cases (Figure 6A). The lower 49 50 bioelectrocatalytic activity of the HY7 peptide might respond to its inferior self-assembly 51 52 53 propensity or to the fact that it contains only 3 Tyr residues instead of the 4 Tyr in HY8 and HY9. 54 55 56 57 58 59 60 ACS Paragon Plus Environment 19 ACS Catalysis Page 20 of 46 1 2 3 We investigated whether HY-peptide fibrils would allow Cu(II) ions to oxidize pyrrole and thus 4 5 6 promote PPy formation through tyrosyl mediated electron transfer, without applying any 7 8 additional potential. The fibrils were dissolved in a 4 mM CuCl2 solution and exposed to pyrrole 9 10 vapor (Figure 6B). Solutions containing L-Tyr and CuCl2 or CuCl2 only were used as controls. In 11 12 13 agreement with the electrochemical studies, after incubation for 16 hours, only the peptide 14 15 containing tubes exhibited a black material, corresponding to PPy that precipitated together with 16 17 the fibrils (Figure 6C and Figure S11). Consistent with their bioelectrocatalytic activities, the 18 19 20 quantification of the generated PPy for each peptide by absorbance showed a higher capacity to 21 22 generate PPy for HY9 and HY8 respect to HY7 chemically (Figure 6C). 23 24 When the above described experiments were performed with HY7, HY8 and HY9 peptides 25 26 incubated at pH 4.0, no electrochemical or Cu(II)-mediated pyrrole oxidizing activity was evident 27 28 29 (Figure S12). 30 31 The results in this section indicate that, as in the case of His, the chemical environment of Tyr 32 33 in the HY-peptides amyloid fibrils allows them to contribute to biocatalysis. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 20 Page 21 of 46 ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Figure 6. HY7, HY8, and HY9 fibrils act as bioelectrocatalyst. A) Chronoamperometry (CA) 32 33 curves of the electropolymerization of polypyrrole (PPy) using HY7 (red line), HY8 (blue line), 34 35 36 HY9 (green line) fibers and monomeric L-Tyr (grey line). Potential applied at 0.8 V vs Ag/AgCl 37 38 in 0.1 M NaCl containing 50 mM pyrrole and 250 μM peptide fibrils or 250 μM L-Tyr. B) Image 39 40 of the centrifuged tubes corresponding to the chemical polymerization after 16 hours exposition to 41 42 43 distilled pyrrole vapor. C) Graphs corresponding to the UV-Vis absorbance spectra of the 44 45 generated PPy. Spectra were acquired from 300 to 800 nm. Control tubes contain CuCl2 (black 46 47 line) and L-Tyr (grey line). 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Catalysis Page 22 of 46 1 2 3 Atomistic simulations of HY8 peptide amyloid-like structures. 4 5 6 7 To gain structural insight on HY-peptides' pH-responsiveness, we selected HY8 as a model and 8 9 performed atomistic molecular dynamics simulations (see Supplementary Information for 10 11 computational details). As FTIR results suggest that self-assembly leads to the formation of 12 13 parallel β-sheets, we explored the six possible parallel steric zippers arising from HY8 14 15 16 aggregation,11 probing the whole range of side chain distributions at the interface between two 17 18 parallel b-sheets (Figure S13). These models can be classified into three main families: i) those 19 20 21 that pack only histidine residues at the steric zipper, leaving tyrosines exposed to the solvent, ii) 22 23 those that pack only tyrosines at the interface, leaving out histidines, and iii) mixed systems where 24 25 both tyrosines and histidines coexist at the steric zipper. Since our simulations target fibrillar 26 27 structures at an atomistic level, we had to account for the two possible tautomers - δ-nitrogen 28 29 30 protonated and ε-nitrogen protonated - of histidine at neutral pH. Each parallel steric zipper 31 32 architecture was built with all histidines in either one of the two tautomeric states, leading to fully 33 34 δ-nitrogen protonated (HY8d) and fully ε-nitrogen protonated (HY8e) models. We then compared 35 36 37 the relative stability of all models for each tautomeric state (see Supplementary Information). 38 39 Results show that the preferred arrangement for both HY8e and HY8d fibrils involves packing of 40 41 histidines at the steric zipper (P-FF1-UD), leaving tyrosines exposed to the solvent (Figure 7A). 42 43 44 Analysis of intersheet non-covalent interaction integrals46 indicates that this preferred architecture 45 46 for HY8e is the one that maximizes side chain packing at the interface (Table S4 and S5). 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 22 Page 23 of 46 ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 7. A) Structural model of the preferred ε-protonated steric zipper organization of HY8 34 35 amyloid-like fibrils (HY8e-P-FF1-UD), B) steric zipper histidine-histidine interactions, and C) 36 37 tyrosine side chains exposed to the solvent. 38 39 40 41 Main inter-b-sheet contacts between histidine side chains include dispersion and N-H/C-H···π 42 43 interactions (Figure 7B). This is likely to produce a network of cooperative hydrogen bonds that 44 45 contribute to fibril stability. The pH-dependent behavior of HY8 self-assembly can be explained 46 47 48 in terms of the availability of singly protonated histidine residues for establishing stabilizing 49 50 contacts at the steric zipper. Lowering the pH, an increasing fraction of histidine residues will be 51 52 doubly protonated, bearing a positive charge, and hindering fibril formation due to electrostatic 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 23 ACS Catalysis Page 24 of 46 1 2 3 repulsion. Increasing the pH, histidines lose the positive charge and can be engaged in the zipper 4 5 6 fibril formation, which would explain the pH-responsiveness HY-peptide amyloid. 7 8 9 While our simulations predict the preferred arrangement of the bilayer – the basic unit of the 10 11 aggregate – they cannot predict the global morphology of the fibril; indeed, both imperfections in 12 13 the growth mechanism and the supramolecular assembly of individual steric zippers can fine-tune 14 15 16 fibrillar architectures through subtle energy changes. Thus, while we predict that Tyr are the more 17 18 likely residues to be exposed to the solvent, and consequently the more available for catalysis, we 19 20 cannot exclude that also His residues can be exposed, either through lateral offsets between steric 21 22 23 zipper layers or hybrid bilayer organization. Indeed, it has already been shown that fibril 24 25 polymorphism can occur even at the level of short peptides with repeating units.47 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 24 Page 25 of 46 ACS Catalysis 1 2 3 CONCLUSIONS 4 5 6 7 In this study, we designed de novo short peptides containing only His and Tyr residues in a 8 9 binary pattern to form biocompatible amyloid-like nanofibers that act as enzyme mimetics and 10 11 exhibit two different and unrelated catalytic activities. His residues provide the contacts that 12 13 14 sustain the parallel b-sheet amyloid zipper and endorse the fibrils with hydrolytic activity, whereas 15 16 Tyr residues stabilize the exterior of the bilayer by a ladder of aromatic ring packing, allowing to 17 18 perform electrocatalytic reactions. This structure/function duality is in stark contrast with most 19 20 21 peptide-based catalytic materials designed to date, which exploit a modular architecture with 22 23 different blocks accounting for the assembly and the activity, making them necessarily more 24 25 complex. The fact that the imidazolyl groups of His contribute the interactions sustaining the inner 26 27 steric zipper makes them pH-responsive, their reversible conformational transition being regulated 28 29 30 by pH changes. In contrast to most pH-responsive b-sheet fibrils reported to date, the mechanical 31 32 properties transition occurs in a physiological pH range, as it happens in pH-responsive natural 33 34 35 amyloids.31–33 Analogously to enzymes catalytic sites, it is the three-dimensional fibril 36 37 microenvironment that permits the catalytic activity. This allows forming catalytically active stiff 38 39 hydrogels at moderate peptide concentrations, where the product can be potentially separated by 40 41 42 inducing a gel-to-solution transition. Thus, in HY-peptides, form follows function. 43 44 Overall, we show how a short peptide with a very low sequence complexity can assemble in a 45 46 pH-dependent manner and in the absence of cofactors into an enzyme mimetic nanomaterial 47 48 displaying two divergent catalytic activities. To the best of our knowledge, this combination of 49 50 51 structural and catalytic properties has not been previously described in designed or natural 52 53 enzymes. This bottom-up strategy lays the foundations to generate "smart" nanostructured 54 55 materials, with a la carte combinations of catalytic functions for practical applications. In order to 56 57 58 59 60 ACS Paragon Plus Environment 25 ACS Catalysis Page 26 of 46 1 2 3 scale up the production of this kind of novel bio-catalyzers, their preparation from small 4 5 6 unmodified peptides is advantageous because they can be straightforwardly prepared via solid- 7 8 phase synthesis with precise control over the sequence and molecular dimensions, low cost, and 9 10 high biocompatibility. 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 26 Page 27 of 46 ACS Catalysis 1 2 3 MATERIALS AND METHODS 4 5 6 7 Peptides preparation. Synthetic peptides, HY6 (Ac-YHYHYH-NH2), HY7 (Ac-HYHYHYH- 8 9 NH2), HY8 (Ac-YHYHYHYH-NH2) and HY9 (Ac-HYHYHYHYH-NH2) were purchased from 10 11 RoyoBiotech Co. ltd. (Shanghai, China). The peptides were N-terminal acetylated and C-terminal 12 13 amidated, to avoid the effect of the terminal charges, mimicking a protein sequential context. 14 15 16 Lyophilized peptides were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to obtain a 10 17 18 mM stock solution, aliquoted and frozen at -80°C. For hydrogel formation, HY8 peptide stock 19 20 solution was prepared as described above at a final concentration of 20 mM, aliquoted and frozen 21 22 23 at -80°C. 24 25 Dependence of the peptides self-assembly on the pH. HY7 peptide was diluted from 10 mM 26 27 stock to 200 µM and HY8 and HY9 peptides were diluted from 10 mM stock to 100 µM in 100 28 29 30 mM Tris, 50 mM Citrate buffer at different pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5). 31 32 Independent triplicates were prepared. For each peptide, the ten solutions were incubated at room 33 34 temperature and quiescent conditions for 24 hours and then analyzed by fluorescence 35 36 37 spectrophotometry diluting to 25 µM final concentration and adding Th-T amyloid binding dye at 38 39 25 µM in MilliQ H2O. Th-T emission fluorescence was detected on a Jasco FP-8200 fluorescence 40 41 spectrophotometer (Jasco Corporation, Japan) in the range 460-600 nm, using an excitation 42 43 wavelength of 445 nm, with excitation and emission bandwidths of 5 nm. The signal at 492 nm 44 45 46 was recorded to obtain the pH dependence curves. Data were normalized and curves were fitted 47 48 using a non-linear fitting to the Henderson-Hasselbalch equation in GraphPad Prism 5.0, obtaining 49 50 the pH1/2 values for each peptide. Data correspond to the mean of three independent experiments 51 52 53 and error bars correspond to standard deviation. 54 55 56 57 58 59 60 ACS Paragon Plus Environment 27 ACS Catalysis Page 28 of 46 1 2 3 Peptides aggregation. HY6, HY7, HY8, and HY9 peptides were diluted from 10 mM stock to 4 5 6 250 µM final concentration in 100 mM Tris HCl pH 8.0 buffer and in 100 mM Tris HCl, 50 mM 7 8 Citrate pH 4.0 buffer, and reactions were incubated 4 days at room temperature in quiescent 9 10 conditions. 11 12 13 Peptide assembly, disassembly and reassembly reactions. HY7, HY8, and HY9 peptides were 14 15 diluted from 10 mM stock to a final concentration of 250 µM in 100 mM Tris, 50 mM Citrate pH 16 17 8.0 and incubated for 4 days at room temperature in quiescent conditions for peptide aggregation. 18 19 20 After the initial aggregation reaction incubation, pH was modified to 4.0 with the addition of a 21 22 small amount (approximately 5 µL) of concentrated 37% HCl solution (Sigma Aldrich, Germany) 23 24 and samples were incubated for 4 days at room temperature and quiescent conditions for 25 26 disaggregation. To promote reaggregation, a small amount of 5 M NaOH solution was added to 27 28 29 the reactions to increase the pH to 8.0 (approximately 5 μL). Samples were then incubated for 4 30 31 days at room temperature and quiescent conditions for the reassembling of the fibrils. 32 33 34 HY8 peptide was exposed to a total of 4 assembling-disassembling cycles by pH modulation 35 36 between pH 8.0 and pH 4.0. Acidification was promoted by the addition of 5 to 10 μL of 37 38 hydrochloric acid and pH was checked with a pH-meter. Alkalinization was promoted by the 39 40 41 addition of 5 to 10 μL of 10 M NaOH solution and pH was checked with a pH-meter. Samples 42 43 were incubated in each cycle for 4 days to promote the assembly/disassembly of the fibrils and 44 45 analyzed as previously described. 46 47 48 QY7 assembly and disassembly reaction. QY7 peptide was previously demonstrated to form 49 50 highly stable fibrils under physiological conditions.21 QY7 peptide was incubated in 100 mM PO4 51 52 buffer at pH 8.0 for 7 days. Then, the sample was split in two tubes and centrifuged at 12,000 x g 53 54 55 for 30 minutes. Pelleted fibrils were resuspended in the same volume of 100 mM PO4 buffer at pH 56 57 58 59 60 ACS Paragon Plus Environment 28 Page 29 of 46 ACS Catalysis 1 2 3 8.0 or in 100 mM Tris HCl 50 mM Citrate buffer at pH 4.0. The reactions were incubated for 2 4 5 6 days. The samples were analyzed by measuring the Th-T, CR and light scattering spectra as 7 8 described above. 9 10 Amyloid dyes binding. Thioflavin T (Th-T) and Congo Red (CR) dyes were used to determine 11 12 13 amyloid fibers formation. For Th-T binding assay, incubated peptides were diluted to a final 14 15 concentration of 25 µM in MilliQ H2O and Th-T was added to a final concentration of 25 µM. Th- 16 17 T emission fluorescence was detected on a Jasco FP-8200 fluorescence spectrophotometer (Jasco 18 19 20 Corporation, Japan) in the range 460-600 nm, using an excitation wavelength of 445 nm and with 21 22 excitation and emission bandwidths of 5 nm. For CR binding assay, incubated peptides were 23 24 diluted 1:10 in MilliQ H2O and CR was added to a final concentration of 10 µM. Optical absorption 25 26 spectra were recorded from 375 to 700 nm in a Specord200 Plus spectrophotometer (Analytik Jena, 27 28 29 Germany). Spectra of protein alone were acquired to subtract protein scattering. 30 31 Light scattering. HY-peptides were diluted to a final concentration of 25 µM and light 32 33 scattering was measured in a Jasco FP-8200 fluorescence spectrophotometer (Jasco Corporation, 34 35 36 Japan), exciting at 330 nm, and recording in the range 320-340 nm using an excitation and emission 37 38 bandwidth of 5 nm. 39 40 Circular dichroism (CD). CD spectra were acquired in a Jasco J-715 Spectropolarimeter 41 42 43 (Jasco, Japan). HY7, HY8, and HY9 peptides were diluted from the incubated reaction to 50 µM 44 45 in MilliQ H2O. CD spectra measurements were performed in the far-UV range (260-200 nm) in a 46 47 1 mm path-length polarized quartz cuvette, with standard sensitivity, 1 nm bandwidth, 1 second 48 49 50 response, 0.5 nm data pitch and 100 nm/min speed. Spectra were recorded at 25°C. The final 51 52 spectrum is the result of 10 spectrums average and subtraction of buffer spectrum. 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 29 ACS Catalysis Page 30 of 46 1 2 3 Fourier Transform Infrared (FTIR) spectroscopy. Secondary structure conformation was 4 5 6 assessed by means of Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) 7 8 spectroscopy. Assembled HY7, HY8, and HY9 fibers were centrifuged at maximum speed 12,000 9 10 x g for 30 minutes and resuspended in MilliQ H2O. Samples were placed on the ATR diamond 11 12 13 crystal and dried out with nitrogen flow. The experiments were carried out in a Bruker Tensor 27 14 15 FTIR (Bruker Optics, United States) supplied with a Specac® Golden Gate MKII ATR accessory. 16 17 Each spectrum consists of 32 acquisitions measured at a resolution of 1 cm-1. Data were acquired 18 19 20 and normalized using the OPUS MIR Tensor 27 software (Bruker Optics, United States). FTIR 21 22 spectra were fitted employing a nonlinear peak-fitting equation using PeakFit package v4.12 23 24 (Systat Software, United States). The area for each Gaussian curve was calculated in the amide I 25 26 region from 1700 to 1600 cm-1 using the second derivative deconvolution method in PeakFit 27 28 29 package v4.12 (Systat Software, United States). 30 31 Transmission Electron Microscopy (TEM). HY6, HY7, HY8, and HY9 incubated samples 32 33 were deposited over carbon-coated copper grids for 10 minutes. Sample excess was dried with a 34 35 36 filter paper and negative staining was applied over the samples incubating 5 µL of 2 % (w/v) uranyl 37 38 acetate for 1 minute and excess was dried out again with filter paper. Grids were exhaustively 39 40 scanned using a JEM 1400 transmission electron microscope (JEOL Ltd, Japan) operating at 80 41 42 43 kV and images were acquired with a CCD GATAN ES1000W Erlangshen camera (Gatan Inc, 44 45 United States). 46 47 Determination of HY6, HY7, HY8 and HY9 fibrils width. TEM micrographs were analyzed 48 49 50 to determine width of HY6, HY7, HY8, and HY9 fibrils. Measurements were performed with 51 52 ImageJ software (NIH, USA), using the corresponding scale. Values correspond to the mean of at 53 54 least 20 different measurements from 3 micrographs. Error corresponds to the standard deviation. 55 56 57 58 59 60 ACS Paragon Plus Environment 30 Page 31 of 46 ACS Catalysis 1 2 3 Dynamic Light Scattering (DLS). Peptides reactions were prepared as described above and 4 5 6 diluted 1:2 in the same buffer for its measurement. DLS measurements were performed at 25°C in 7 8 a Malvern Zetasizer Nano S90 (Malvern, Worcestershire, UK). Each sample was measured 10 9 10 times, each time measured in 10 runs; average distributions are presented, and the standard 11 12 13 deviation was calculated. 14 15 Cell viability assay. Human SH-SY5Y cells were plated into 96-well plates at a density of 4,000 16 17 cells/well (100 μL/well) in F-12 medium supplemented with 10 % FBS and maintained at 37°C 18 19 20 and 5 % CO2 atmosphere. HY7, HY8, and HY9 peptides were prepared at 250 µM in 100 mM Tris 21 22 HCl pH 8.0 and in 100 mM Tris-Citrate pH 4.0 sterile buffers and incubated during 4 days at room 23 24 temperature in quiescent conditions. After incubation, pH 8.0 samples were centrifuged at 12,000 25 26 x g for 30 minutes and soluble and insoluble fraction were separated. Pelleted fibrils were 27 28 29 resuspended in the same volume of the previous buffer. Cells were incubated in the presence of 30 31 10, 25 and 50 μM of soluble and insoluble fraction for pH 8.0 samples and total fraction for pH 32 33 4.0 samples for 72 hours in triplicates. For controls, the same volume of 100 mM Tris HCl pH 8.0 34 35 36 buffer and 100 mM Tris-Citrate pH 4.0 buffer was added respectively in control wells. 37 38 Treated cells were incubated with 10 μL PrestoBlue® Cell Viability Reagent (Invitrogen, United 39 40 States) for 30 minutes. Cell viability was determined by recording fluorescence at 615 nm, using 41 42 43 an excitation wavelength of 531 nm in a Victor3 fluorescent plate reader (Perkin Elmer, United 44 45 States). Data correspond to the mean of two independent experiments and error bars correspond to 46 47 standard deviation. 48 49 50 Esterase activity assay. HY7, HY8, and HY9 peptides were prepared at 250 µM in 100 mM 51 52 Tris HCl pH 8.0 buffer and incubated for 4 days at room temperature in quiescent conditions. 53 54 Samples were centrifuged at 12,000 x g for 30 minutes and the supernatant was removed. For 55 56 57 58 59 60 ACS Paragon Plus Environment 31 ACS Catalysis Page 32 of 46 1 2 3 assays at pH 8.0, pelleted fibers were resuspended in 25 mM Tris HCl pH 8.0. Esterase activity 4 5 6 assay was performed 100 µM peptide final concentration for all the assays. For assays at pH 7.4, 7 8 pelleted fibrils were resuspended in 25 mM Tris HCl at pH 7.4. The reaction was performed in 96- 9 10 well plate and measured every 10 minutes until the plateau is reached in a Victor3 Plate reader 11 12 13 (Perkin Elmer, USA) acquiring absorbance at 405 nm. P-nitrophenyl acetate (pNPA) and p- 14 15 nitrophenol (pNP) (Sigma Aldrich, Germany) stock solutions were freshly prepared at 0.1 M in 16 17 acetonitrile and diluted to desired final concentrations. The molar extinction coefficient was 18 19 20 experimentally obtained from the calibration curve generated from a standard 4-nitrophenol (pNP) 21 22 solution (Sigma Aldrich, United States), corresponding to 13014 M-1 cm-1 at 405 nm. Buffer signals 23 24 was subtracted from all samples. Kinetic parameters were calculated using pNPA concentrations 25 26 ranging from 0.2 to 1.5 mM. Reported results correspond to the average of at least three 27 28 29 independent measurements. Kinetic parameters were obtained by fitting the data to the Michaelis- 30 31 Menten equation [ V0 = kcat·[E]0·[S]0 / (KM + [S]0) ] using GraphPad PRISM 5.0 software. 32 33 Chemical polymerization of pyrrole with HY-peptides. HY7, HY8, and HY9 peptides 34 35 36 incubated at pH 8.0 were resuspended in 4 mM CuCl2 in a 1.5 mL tube; additionally, 4 mM CuCl2 37 38 was added to HY7, HY8 and HY9 peptides incubated at pH 4.0. 300 µL of freshly distilled pyrrole 39 40 were added into a 20 mL vial and 1.5 mL tubes were carefully introduced in the corresponding 41 42 43 vials, thus avoiding contact between both liquids to expose them to distilled pyrrole vapor. 44 45 Reactions were incubated for 16 hours. As a reference, a solution of plain CuCl2 with 250 or 750 46 47 μM L-Tyr was exposed to distilled pyrrole vapor. The resulting polymer was then resuspended, 48 49 50 and the UV-vis spectra of the solutions were recorded. Pyrrole and L-Tyr were purchased from 51 52 Sigma-Aldrich, Germany. CuCl2 was purchased from Acros Organics, USA. 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 32 Page 33 of 46 ACS Catalysis 1 2 3 Electropolymerization of pyrrole with HY-peptides. Pyrrole and L-Tyr were purchased from 4 5 6 Sigma-Aldrich. Na2SO4 was obtained from Panreac, USA. CuCl2 was purchased from Acros 7 8 Organics, USA. The electropolymerization of pyrrole was done by chronoamperometry in 0.1 M 9 10 Na2SO4 at pH 8.0 and 4.0 using a lab-made three-electrode cell, with a platinum wire as a counter 11 12 13 electrode, silver wire as a reference electrode, and an indium tin oxide (ITO) electrode as a working 14 15 electrode. The potential applied to trigger the electropolymerization was 0.8 V vs Ag/AgCl. The 16 17 concentration of pyrrole in the solution was 50 mM. The total volume of fibers solution was 1 mL. 18 19 20 The sunken area of ITO electrode was 0.7 cm × 0.8 cm. 21 22 HY8 peptide hydrogel formation. 20 mM HY8 peptide stock solution was resuspended in 100 23 24 mM Tris HCl pH 8.0 in the presence of 50 μM of phenol red pH indicator to a final concentration 25 26 of 5 mM. Since HFIP is acidic, the starting pH of the solution was around 4.0, remaining the 27 28 29 peptide in a fluid state. The Eppendorf tube was led open overnight to allow HFIP evaporation and 30 31 pH increased to 8.0, as determined by the change on color of phenol red from yellow to red/pink. 32 33 HY8 peptide solution was then incubated at room temperature and quiescent conditions for 4 days. 34 35 36 Hydrogel assembled at pH 8.0 was immediately disassembled by adding 200 μL of 100 mM Tris 37 38 HCl 50 mM Citrate buffer at pH 4.0 with 50 μM phenol red. 39 40 HY8 peptide hydrogel catalytic activity. HY8 peptide hydrogel was assembled as described 41 42 43 previously in a final volume of 200 μL, in this case, without phenol red pH indicator. To determine 44 45 the catalytic activity, 20 μL of 1 mM pNPA solution were added into each sample and incubated 46 47 at room temperature for 30 minutes. The acquisition of yellow coloration indicated the 48 49 50 transformation of pNPA into pNP and thus esterase catalytic activity. 51 52 Molecular dynamics simulations. NPT Molecular dynamics simulations at 300 K were 53 54 performed with the Amber suite, using the ff14SB force field.48 To generate all possible parallel 55 56 57 58 59 60 ACS Paragon Plus Environment 33 ACS Catalysis Page 34 of 46 1 2 3 fibrillar architectures of HY8d and H8Ye, we used an in-house code that computes the Cartesian 4 5 6 coordinates of steric zippers of any length applying rigid rotations and translations to the 7 8 coordinates of an individual strand. The length of each β-sheet was set to 20 strands, so that each 9 10 fibril model is composed of 40 strands. For HY8d and H8Ye, we ran molecular dynamics 11 12 13 simulations on the six non-equivalent parallel fibrillar architectures (Figure S13) and computed 14 15 their relative energies on 100 frames sampled with a constant stride. See Supplementary 16 17 Information for more details. 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 34 Page 35 of 46 ACS Catalysis 1 2 3 ASSOCIATED CONTENT 4 5 6 7 Supporting Information. 8 9 10 Computational methods. 11 12 Figure S1. Conformational characterization of HY-peptides incubated at pH 4.0 and pH 8.0. 13 14 Figure S2. Size characterization of the HY6, HY7, HY8, and HY9 assemblies at pH 8.0 by DLS. 15 16 17 Figure S3. Characterization of the amyloid-like properties for HY6 peptide incubated at pH 4.0 18 19 and pH 8.0. 20 21 Figure S4. Cell viability assay in the presence of HY7, HY8, and HY9 peptides. 22 23 24 Figure S5. TEM visualization of HY7, HY8 and HY9 peptides pH-dependent assembly, 25 26 disassembly and reassembly. 27 28 Figure S6. HY8 assembly and disassembly cycles as a function of the pH. 29 30 31 Figure S7. QY7 assembly and disassembly as a function of the pH. 32 33 Figure S8. Lineweaver-Burk plots of pNPA hydrolysis catalyzed by 100 μM of HY7, HY8 and 34 35 HY9 fibrils at pH 8.0. 36 37 38 Figure S9. Catalytic activity of HY7, HY8 and HY9 fibers at pH 7.4. 39 40 Figure S10. Lineweaver-Burk plots of pNPA hydrolysis catalyzed by 100 μM of HY7, HY8 and 41 42 HY9 fibrils at pH 7.4. 43 44 Figure S11. Bioelectric and chemical polymerization of pyrrole by 750 uM L-Tyr at pH pH 8.0. 45 46 47 Figure S12. Bioelectric and chemical polymerization of pyrrole by L-Tyr, HY7, HY8 and HY9 48 49 peptides at pH 4.0. 50 51 Figure S13. Residue organization at the steric zipper of the 6 analyzed parallel fibril models. 52 53 54 Figure S14. HY8 δ-nitrogen protonated (HY8d) fibril models after 100 ns of molecular dynamics. 55 56 Figure S15. HY8 ε-nitrogen protonated (HY8e) fibril models after 100 ns of molecular dynamics. 57 58 59 60 ACS Paragon Plus Environment 35 ACS Catalysis Page 36 of 46 1 2 3 Figure S16. Evolution of the β-sheet twist angles of the six HY8d steric zipper models. 4 5 Figure S17. Evolution of the β-sheet twist angles of the six HY8e steric zipper models. 6 7 Table S1. FTIR secondary structure assignments for HY6, HY7, HY8, and HY9 fibrils. 8 9 10 Table S2. pH transition values obtained from aggregation titter curves for HY7, HY8, and HY9 11 12 peptides. 13 14 Table S3. Kinetic parameters for HY7, HY8, and HY9 amyloid fibrils esterase activity at pH 8.0 15 16 17 and pH 7.4. 18 19 Table S4. Implicit solvent Amber energies (kcal/mol) and corresponding standard deviation of the 20 21 twelve HY8 fibril models. 22 23 24 Table S5. Inter-sheet NCIIs of the twelve HY8 fibril models. 25 26 27 28 AUTHOR INFORMATION 29 30 31 Corresponding Author 32 33 34 *Salvador Ventura - Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica 35 36 i Biologia Molecular; Universitat Autònoma de Barcelona; 08193 Bellaterra (Barcelona), Spain. 37 38 Tel.: 34-93-5868956, Fax: 34-93-5811264, e-mail: salvador.ventura@uab.es 39 40 41 Author Contributions 42 43 44 Conceptualization, M.S., F.T., and S.V.; methodology, M. D.-C., M.N., F.P., L. R.-S. and S.N.; 45 46 47 formal analysis and validation, M. D.-C., S.N. and S.V.; writing—review and editing, M. D.-C., 48 49 S.N. and S.V.; supervision, S.N and S.V.; funding acquisition, S.V. 50 51 52 Funding Sources 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 36 Page 37 of 46 ACS Catalysis 1 2 3 This work was funded by the Spanish Ministry of Economy and Competitiveness BFU2013- 4 5 6 44763-P and BIO2016-78310-R to S.V and by ICREA, ICREA-Academia 2015 to S.V. M. D.-C. 7 8 was supported by the Spanish Ministry of Science and Innovation via a doctoral grant 9 10 (FPU14/05786). 11 12 13 ACKNOWLEDGMENT 14 15 16 17 We acknowledge the technical support from the Cell Culture Unit of Servei de Cultius Cel·lulars 18 19 i Producció d’Anticossos i Citometria (SCAC) and of the Servei de Microscopia from Universitat 20 21 Autònoma de Barcelona (UAB). FP acknowledges the Mizutani Foundation for Glycoscience 22 23 24 (200077). MS and LRS gratefully acknowledge financial support from MINECO (CTQ2017- 25 26 89132-P) and the Generalitat de Catalunya (2017SGR1323). 27 28 29 30 31 32 NOTES 33 34 35 The authors confirm that there are no known conflicts of interest associated with this publication 36 37 38 and there has been no significant financial support for this work that could have influenced its 39 40 outcome. 41 42 43 REFERENCES 44 45 (1) Sorushanova, A.; Delgado, L. M.; Wu, Z.; Shologu, N.; Kshirsagar, A.; Raghunath, R.; 46 47 48 Mullen, A. 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Theory Comput. 2015, 11 (8), 3696–3713. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 44 Page 45 of 46 ACS Catalysis 1 2 3 Table of Contents 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 45 ACS Catalysis Page 46 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Table of contents 26 85x47mm (600 x 600 DPI) 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment