Article This article is licensed under CC-BY 4.0 www.acsnano.org Amyloid Fibrils Formed by Short Prion- Inspired Peptides Are Metalloenzymes Susanna Navarro, Marta Díaz-Caballero, Francesca Peccati, Lorena Roldán-Martín, Mariona Sodupe, and Salvador Ventura* Cite This: ACS Nano 2023, 17, 16968−16979 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Enzymes typically fold into defined 3D protein structures exhibiting a high catalytic efficiency and selectivity. It has been proposed that the earliest enzymes may have arisen from the self-assembly of short peptides into supramolecular amyloid-like structures. Several artificial amyloids have been shown to display catalytic activity while offering advantages over natural enzymes in terms of modularity, flexibility, stability, and reusability. Hydrolases, especially esterases, are the most common artificial amyloid-like nanozymes with some reported to act as carbonic anhydrases (CA). Their hydrolytic activity is often dependent on the binding of metallic cofactors through a coordination triad composed of His residues in the β- strands, which mimic the arrangement found in natural metalloenzymes. Tyr residues contribute to the coordination of metal ions in the active center of metalloproteins; however, their use has been mostly neglected in the design of metal-containing amyloid-based nanozymes. We recently reported that four different polar prion-inspired heptapeptides spontaneously self-assembled into amyloid fibrils. Their sequences lack His but contain three alternate Tyr residues exposed to solvent. We combine experiments and simulations to demonstrate that the amyloid fibrils formed by these peptides can efficiently coordinate and retain different divalent metal cations, functioning as both metal scavengers and nanozymes. The metallized fibrils exhibit esterase and CA activities without the need for a histidine triad. These findings highlight the functional versatility of prion-inspired peptide assemblies and provide a new sequential context for the creation of artificial metalloenzymes. Furthermore, our data support amyloid-like structures acting as ancestral catalysts at the origin of life. KEYWORDS: peptides, amyloid fibrils, self-assembly, nanoenzymes, biocatalytic nanomaterials, bioremediation INTRODUCTION nanozymes endow them with advantages,16,17 relative to their Enzymes are the molecular entities responsible for most natural counterparts, including lower synthesis and modification biological reactions. Modern enzymes are typically proteins of costs. at least 100 residues that fold into defined 3D native structures About one-third of modern enzymes are metalloenzymes that showcasing high catalytic efficiency and selectivity.1−3 exploit the coordination of metal ions to catalyze some of the It has been hypothesized that the earliest enzymes on Earth most complex chemical reactions in nature.18 Among them, might have arisen from the spontaneous self-assembly of short many hydrolases depend on the coordination of divalent peptides to form supramolecular structures.4−6 Specifically, it metallic cations, frequently Zn2+,19 to exert their activities, like has been suggested that amyloids may have acted as the scaffold in carbonic anhydrases (CAs). Coordination of Zn2+ lowers the supporting primitive catalytic activities.7,8 As amyloids use pKa of a bound water, facilitating the formation of a hydroxide amino acid sequences as their building blocks, and their assembly is driven and sustained by noncovalent interactions, they share similarities with natural proteins.9 In favor of this Received: May 9, 2023 hypothesis, several artificial amyloids formed by short, and often Accepted: August 23, 2023 repetitive, amino acid sequences have been shown to act as Published: August 30, 2023 enzyme mimetics.10−14 Although, in general terms, they are catalytically less efficient than modern enzymes, 15 the modularity, flexible design, stability, and reusability of these © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsnano.3c04164 16968 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article for nucleophilic attack of the substrate.20 CAs are ubiquitous and hydroxyls to the solvent (Figure S1), suggesting the possibility highly efficient metalloenzymes that participate in distinct that the fibrils could bind to divalent metal cations. biological reactions, including the capture of CO 2 in To elucidate whether NY7, QY7, SY7, and GY7 fibrils could erythrocytes via its hydration into bicarbonate (HCO3−), with bind Cu2+, Ni2+, Co2+, and Zn2+, they were incubated in the the production of protons (H+).21,22 This CO2 sequestration presence of different concentrations of CuCl2, NiSO4, CoCl2, ability, together with their esterase activity, makes CA enzymes and ZnCl2. Their retention capacity was determined by of high interest in biotechnology.23 exploiting the metal colorations. Upon centrifugation, the fibrils Among artificial amyloid-like nanozymes, the most common rendered colored pellets that were visible to the naked eye are those exhibiting hydrolytic activity, especially es- (Figure 1), indicative of the binding of metal cations. Cu2+- terases,8,10,24−29 with some of them reported to act as CAs.24,27,30−32 In a large majority of cases, their function is strictly dependent on the presence of a metal cation, with Zn2+ being used on most occasions. In natural enzymes, the Zn2+ binding site often encompasses two His residues projecting from the same β-strand and a third His ligand from an adjacent strand.33 This elementary interstrand 3-His metal-coordinating site is typically found in CAs.34 This β-sheet architecture makes the amyloid fold ideal for building up artificial esterases and CAs,10,24 although unlike in modern enzymes, the fibrils’ catalytic sites are surface-exposed and not buried in the protein core. All metal-dependent amyloid-based esterases reported so far rely strictly on coordinating metal ions by His residues, although they differ in the placement of these residues in the peptide building block. In some cases, His are intercalated with hydrophobic residues that provide the driving force for the assembly,8,24,25 whereas on other occasions, the catalytic and assembly moieties are segregated at the two extremes of the sequence.27,31,32 In many instances, the presence of the metal in the assembly solution is a requirement not only for the activity but also to attain a stable fibrillar architecture.8,24 Different studies have shown that L-Tyr can bind to transition metals;35 most coordinations occur at the amino nitrogen or carboxylic oxygen, but the phenolic oxygen can also be involved. Indeed, Tyr residues contribute to coordinate metal ions in the active centers of proteins like ferritin,36 cytochrome oxidase,37 Figure 1. Pelleted fibrils containing divalent metal cations Co2+, lytic polysaccharide monooxygenases,38 and galactose oxidase,39 Cu2+, and Ni2+. Pictures corresponding to control pelleted fibrils often through the phenolic hydroxyl group. Nevertheless, NY7, QY7, SY7, and GY7 incubated only with buffer (translucid) despite this evidence, the use of Tyr has been mostly neglected and incubated with Co2+ (red), Cu2+ (blue), and Ni2+ (yellow), in the design of metal-containing amyloid-based nanozymes. respectively. Recently, we have reported a set of low-complexity, polar heptapeptides inspired by prion-like domains’ composition, incubated fibrils were blue, Ni2+ yellowish, and Co2+ purple for which spontaneously self-assemble into highly stable supra- NY7, QY7, and SY7 and orangish for GY7, which might reflect molecular amyloid fibrils endowed with redox activity.40,41 Here, different Co2+ oxidation states. In the case of Zn2+, detection was we characterized the interaction of these His-devoid but Tyr- not possible by a simple visual inspection because the solution rich peptides with divalent metal cations. Once assembled, these was transparent. peptides bind and become decorated with Co2+, Cu2+, Ni2+, and To confirm the ability of the fibrils to sequester Cu2+, Ni2+, Zn2+. The metallized fibrils act as nanozymes exhibiting esterase and Co2+ we determined the metals’ molar extinction and CA activities without requiring a histidine triad. Overall, our coefficients (ε) in solution (εNi2+393 nm = 48 mM−1·cm−1, data support the hypothesis that amyloid-like structures may εCo2+512 nm = 60 mM−1·cm−1, and εCu2+235 nm = 189 mM−1·cm−1) have played a catalytic role in the emergence of life while (Figure S2). For Zn2+, the absorbance maximum was below 200 demonstrating the functional versatility of prion-inspired nm, limiting its spectroscopic detection. Accordingly, Zincon peptide fibrils. dye, which binds Zn2+ with high affinity, presenting an absorbance maximum at 628 nm, was used (εZincon‑Zn2+628 nm = RESULTS AND DISCUSSION 228 mM−1·cm−1). The retention percentages were calculated by 1. Prion-Inspired Fibrils Retain Cu2+, Ni2+ Co2+, and comparing the absorbance of solutions of each divalent cation Zn2+ Divalent Metal Cations. We have shown that seven- without fibrils with those measured in the supernatants of fibril- residue-long polar sequences inspired in the amino acid containing solutions after centrifugation using the Lambert− composition of prion-like domains, NYNYNYN (NY7), Beer equation. The assayed concentration range was adjusted QYQYQYQ (QY7), SYSYSYS (SY7), and GYGYGYG (GY7), for the different divalent metal cations according to their spontaneously self-assemble into amyloid-like fibrillar structures spectrophotometric properties. Figure S3 illustrates how all the endowed with redox activity.32 Based on the fibril models we fibrils can scavenge Cu2+, Ni2+, Co2+, and Zn2+ from the solution, generated previously,40 the assemblies expose multiple phenolic with the percentage of retention depending on the nature of the 16969 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article Table 1. NY7, QY7, SY7, and GY7 Fibrils’ Divalent Cation Retention Capacitya cation NY7 QY7 SY7 GY7 retention capacity (μmol cation/ μmol peptide) Cu2+ 34.1 ± 8.4 69.2 ± 12.5 103.5 ± 9.3 58.1 ± 2.7 Ni2+ 15.5 ± 1.2 35.8 ± 1.3 72.8 ± 17.3 15.3 ± 11.0 Co2+ 38.2 ± 4.7 68.1 ± 3.0 124.6 ± 24.7 46.2 ± 6.1 Zn2+ 35.0 ± 4.6 62.8 ± 9.8 92.5 ± 26.3 36.9 ± 10.0 a Divalent cations Cu2+, Ni2+, Co2+, and Zn2+ retention capacity was calculated in μmol cation/μmol peptide. Values indicate the mean of duplicates from two independent experiments (± standard deviation). Figure 2. Docking of metals on fibrils. Cu2+ and Zn2+ (II) plausible binding sites in SY7 from docking simulations, in square planar and tetrahedral geometry, respectively, and B3LYP-D3-optimized geometries. Distances are in Å. 16970 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article Figure 3. TEM micrographs of fibrils incubated with Cu2+, Ni2+, Co2+, and Zn2+. Representative transmission electron micrographs of the deposited fibrils onto TEM grids. NY7, QY7, SY7, and GY7 self-assembled fibrils were incubated overnight at 25 °C in the presence of 200 mM CuCl2, NiSO4, CoCl2, and ZnCl2 under soft agitation. All samples were deposited onto carbon-coated copper grids except Cu2+ samples, which were deposited onto carbon-coated gold grids. Scale bars correspond to 1 μm. cation and the heptapeptide that conforms to the fibrils. While the solvent-exposed Tyr residues in the fibrils, we selected those these qualitative spectrophotometric measurements confirmed with the highest specific binding capacity (SY7) and the the fibrils’ metal retention ability, additional assays were biologically relevant Cu2+ and Zn2+ cations. We performed required to quantify the binding more accurately. docking calculations on the fibril models previously built by us40 2. Quantification of Metal Coordination by Prion- and run electronic structure DFT(B3LYP-D3) calculations to Inspired Fibrils. To quantify the metal retention capacity of evaluate whether these binding sites are stable or not. According our prion-inspired fibrils, we used inductively coupled plasma to the coordination properties of these metal ions, we optic emission spectroscopy (ICP-OES). First, we calculated the considered a square planar environment for Cu2+ (3d9) and a concentration of heptapeptides in both the soluble and insoluble tetrahedral one for Zn2+ (3d10).44 Docking simulations indicate fractions of the self-assembled reactions. Next, based on the that the maximum number of Tyr residues that can divalent metal cations’ quantification by ICP-OES in centrifuged simultaneously coordinate to either Cu2+ or Zn2+ is three fibrils, after their incubation with 200 mM Cu2+, Ni2+, Co2+, and (Figure 2). The vacant site is thus filled with a water molecule. Zn2+ (Table S1), we calculated the specific retention capacity for For Cu2+ two different sets of results, with a coordination each metal/peptide combination (Table 1). sphere formed by Tyr from either two or three (Figure 2) The results enabled us to rank the four fibrils according to different strands, were obtained. For Zn2+, however, in a their cation retention. Overall, SY7 fibrils displayed the highest tetrahedral environment, only the coordination of Tyr from two retention capacity, followed by QY7, while GY7 and NY7 different strands was encountered. Quantum chemical calcu- showed lower retention. The binding capacity of all fibrils was lations at the B3LYP-D3 level of theory confirmed that these found to be remarkable, with each μmol of peptide retaining at binding modes are stable, metal−oxygen(Tyr) bonding least 15 μmol of the corresponding divalent metallic cation and distances being about 2.1 Å, similar to the values 2.2−2.3 Å up to 125 μmol for some metal/peptide combinations. These identified in metalloproteins.44 Interaction of metal cations with results demonstrate that the prion-inspired amyloid scaffolds the π system of two phenolic moieties was also explored, but function as effective metallic traps, as observed in previous calculations indicate that such coordination (Figure S4) is studies for β-lactoglobulin fibrils, which, in contrast to our significantly less stable (17 kcal mol−1 for Cu2+ and 42 kcal mol−1 assemblies, were formed upon extensive denaturation at pH 2.0 for Zn2+) than with the oxygens of Tyr shown in Figure 2. and 90 °C.42,43 To further assess the availability of Tyr side chains for metal 3. Docking of Divalent Metallic Metals on Prion- ion binding, we computed the average buried area and pKa of Inspired Fibrils. To identify how metal ions can coordinate to Tyr residues along a molecular dynamics simulation of the SY7 16971 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article Figure 4. Kinetic curve fitting to the Michaelis−Menten equation for heptapeptide fibril esterase activity in the presence of Cu2+ and Zn2+. Esterase activity was detected by measuring the increasing absorbance at 405 nm along the time corresponding to the appearance of pNP yellow-colored product in 25 mM HEPES pH 8.0 with 1 mM CuCl2 and 25 mM Tris-HCl pH 8.0 with 1 mM ZnCl2, respectively. Obtained data were fitted to the Michaelis−Menten equation with GraphPad PRISM 5.0 software for Cu2+ (left graph) and Zn2+ (right graph). Data correspond to the mean of three independent experiments (± standard deviation). fibril.40 The average buried area is 25%, and the average pKa is CaRIne Crystallography Software (France) database. All 11.5, confirming that Tyr residues are mostly exposed and diffraction patterns of metallized fibrils matched the reference available for binding and that further stabilization to the metal− pattern for the respective metal. EDX spectra revealed an fibril interaction could come from a fraction of deprotonated Tyr enrichment in the incubated divalent cation element for all side chains. fibrillar samples: Cu2+ (Figure S10); Ni2+ (Figure S11); Co2+ Overall, computational calculations confirm and explain how (Figure S12); and Zn2+ (Figure S13). Tyr-rich fibrils can coordinate to metallic ions. Altogether, the results in this section provided solid evidence 4. Characterization of Prion-Inspired Fibrils Bound to for the presence and identity of metal divalent cations Metallic Cations by Electron Microscopy. Having estab- coordinated to NY7, QY7, SY7, and GY7 fibrils. lished that the fibrils can retain Cu2+, Ni2+, Co2+, and Zn2+, we 5. Esterase Activity of Prion-Inspired Fibrils Decorated investigated the impact of metal cations binding on their with Cu2+ and Zn2+. Hydrolytic enzymes split different groups morphology. First, NY7, QY7, SY7, and GY7 fibrils were of biomolecules such as esters, peptides, and glycosides, in many examined by transmission electron microscopy (TEM) prior to cases thanks to the coordination of a metal ion at the catalytic their incubation with metals, showing well-defined fibrils center. Although His is the preferred metal coordinating residue, (Figure S5A) of some micrometers in length, and a width of 8 Tyr can also participate in the binding in some metal- to 20 nm, consistent with our previous data.41 Additionally, loproteins.36−39 His−metal coordination has been exploited Thioflavin-T (Th-T) binding confirmed the amyloid nature of for the design of artificial nanozymes.5,10,26,31 We assayed if our these assemblies (Figure S5B). peptides may act as a hydrolytic enzyme-like scaffold when Next, NY7, QY7, SY7, and GY7 fibrils incubated with Cu2+ bound to Cu2+ and Zn2+ and thus if we could generate an were deposited on carbon-coated gold grids, while NY7, QY7, artificial Tyr-rich hydrolytic amyloid for the first time. SY7, and GY7 fibrils incubated with Ni2+, Co2+, and Zn2+ were Esterase activity is typically assayed using p-nitrophenyl deposited on carbon-coated copper grids and visualized by acetate (pNPA) as the substrate and monitoring the appearance TEM. The presence of fibrils resembling in width those in the of the yellow-colored product p-nitrophenol (pNP). Therefore, absence of metals was visible in all cases (Figure 3). Notably, the we used pNPA to assess the esterase activity of fibrils when binding of metals to the fibrils allowed their visualization coordinated with Cu2+ and Zn2+. Kinetic analysis revealed that without the need of using uranyl acetate as contrasting agent, as all metal-coordinated heptapeptide fibrils exhibited esterase it was needed in the case of nonincubated fibrils. In images of activity (Figure S14), acting thus as hydrolase-like catalysts. fibrils incubated with Cu2+ and Ni2+ electrodense dots over the Remarkably, the activity of the soluble L-Tyr amino acid in the fibrils were evidenced, whereas in images of Co2+- and Zn2+- presence of metal cations is low (Cu2+) or negligible (Zn2+), incubated fibrils, the presence of polygonal depositions was implying that it is the three-dimensional structure of the fibril observed. This suggests that not only do the fibrils bind the that provides an appropriate microenvironment for the metal- metals, but they also facilitate their self-assembly into macro- dependent catalytic activity. molecular structures. Kinetic parameters of the esterase activity in the presence of We selected the NY7 and QY7 peptides to demonstrate that metals were obtained by fitting the reactions in a substrate the metalized fibrils bind Th-T (Figure S6) and display a concentration range from 0.2 to 2.5 (Zn2+) or 0.2 to 5 mM predominant β-sheet structure, as measured by Fourier (Cu2+) to the Michaelis−Menten equation (Figure 4). The transform infrared (FTIR) spectroscopy in the amide I region Michaelis constants (KM) and maximal velocity of the reactions of the spectrum (Figures S7 and S8). (Kcat) were calculated (Table S6). To provide further confirmation of the divalent metal cation The four heptapeptides in the presence of Cu2+ presented a attachment in the deposited fibrils, electronic diffraction (ED) KM ranging between 1.4 and 3.8 mM and a kcat around 0.006 s−1. and energy dispersive X-ray diffraction (EDX) techniques were Subtle differences in the catalytic efficiency between the employed. First, the distances of the different diffraction peptides were observed, with GY7 corresponding to the most patterns were measured (Tables S2 to S5) from the ED efficient fibril with a kcat/KM = 3.8 ± 1.4 M−1·s−1. Similarly, the micrographs (Figure S9) and compared to the reference Zn2+-containing heptapeptide fibrils displayed KM in the mM crystalline diffraction pattern for each element available in the range, between 1.1 and 3.0 mM, and kcat around 0.006 s−1. GY7 16972 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article Figure 5. Carbonic anhydrase activity of the fibrils in the presence of Cu2+. (A) Carbonic anhydrase (CA) activity was detected by measuring absorbance of the phenol red pH indicator in the presence of NY7 (blue), QY7 (red), SY7 (green), and GY7 (purple) and positive control with CA at 50 nM (gray). Initial (continuous line) and final (discontinuous line) spectra were acquired between 350 and 650 nm. The image shows the phenol red coloration corresponding to the initial (red) and final (yellow) samples. (B) Time-dependent decrease in absorbance at 560 nm of phenol red incubated with CO2-treated deionized water. Fibril colors as in (A), negative controls of buffer alone (gray), buffer with Cu2+ (black), QY7 fibrils without metal (black discontinuous), and CA at 50 nM (orange) are indicated. exhibited the highest catalytic efficiency, with a kcat/KM = 6.0 ± M−1·s−1) is less efficient than that reported by Korendovych and 2.3 M−1·s−1. co-workers for the more hydrophobic, His-based IHIHIQI Hydrolysis of pNPA by Zn2+-bound prion-inspired fibrils and, heptapeptide (IH7) (kcat/KM = 62 ± 2 M−1·s−1).24 Comparison in particular, by the modeled SY7 scaffold (kcat/KM = 5.8 ± 2.07 of B3LYP-D3 calculations for model systems representing the 16973 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article Figure 6. Carbonic anhydrase activity of the fibrils in the presence of Zn2+. (A) Carbonic anhydrase (CA) activity was detected by measuring absorbance of the phenol red pH indicator in the presence of NY7 (blue), QY7 (red), SY7 (green), and GY7 (purple) and positive control with CA at 50 nM (gray). Initial (continuous line) and final (discontinuous line) spectra were acquired at between 350 and 650 nm. The image shows the phenol red coloration corresponding to the initial (red) and final (yellow) samples. (B) Time-dependent decrease in absorbance at 560 nm of phenol red incubated with CO2-treated deionized water. Fibril colors as in (A), negative controls of buffer alone (gray), buffer with Zn2+ (black), QY7 fibrils without metal (black discontinuous), and CA at 50 nM (orange) are indicated. metal site in IH7 [Zn2+(His)3OH−]+ and SY7 SY7 (19 kcal·mol−1). This is mainly because the Zn−OH− bond [Zn2+(Tyr)3OH−]+ showed that the energy barrier for the is weaker in [Zn2+(His)3OH−]+ than in [Zn2+(Tyr)3OH−]+. OH− attack is considerably lower in IH7 (9 kcal·mol−1) than in Furthermore, deprotonation of coordinated water is more 16974 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article favorable in IH7 due to the presence of a fourth His able to Spectrophotometric and ICP-OES measurements consis- accept the proton. tently demonstrate that prion-inspired fibrils possess remarkable Despite, as expected, His-based fibrils performing better than metal retention capacity. Docking simulations and electronic Tyr-based ones, our data show that all fibrils, when coordinated structure calculations indicate that up to three Tyr residues can with Cu2+ and Zn2+, behave as esterases, with their kinetic simultaneously coordinate to either Cu2+ or Zn2+ through their parameters depending somehow on the nature of differential phenolic rings, with a water molecule occupying the vacant site. polar side chains in each sequence, since the Tyr content and Specifically, the coordination of Cu2+ involves Tyr in two or disposition are common to all of them. three strands, whereas Zn2+ coordinates with Tyr in two 6. Carbonic Anhydrase Activity of Prion-Inspired different strands. Quantum chemical calculations indicate that Fibrils Decorated with Cu2+ and Zn2+. Carbonic anhydrases oxygen (Tyr) bonding distances are similar to those observed in are among the most ubiquitous enzymes since they are involved metalloproteins, lending support to the stable interactions in multiple physiological functions such as pH homeostasis, between fibrils and metallic ions observed experimentally. calcification, bone resorption, and photosynthesis. Moreover, TEM, electronic diffraction, and energy dispersive X-ray they display a pivotal role in the transport of carbon dioxide, by diffraction spectroscopy techniques provided compelling catalyzing the reversible hydration of CO2 into bicarbonate evidence for the presence and identity of metallic divalent (HCO3−) and a proton (H+), its activity being strictly cations coordinated to NY7, QY7 SY7, and GY7 fibrils. The dependent on the coordination of Zn2+ at the catalytic center. images suggested that the fibrils not only bind to metals but also The search for artificial CA mimics has concentrated may facilitate polymerization on the fibril surface into considerable efforts.8,15 However, to the best of our knowledge, electrodense spherical or polygonal structures, a phenomenon a Tyr-based amyloid catalytic system has not yet been yet that likely contributes to the high metal/peptide ratio observed explored. Accordingly, we aimed to investigate if our fibrils could by ICP-OES. This metal ion sequestration capability suggests catalyze the hydration of carbon dioxide and water in the that the fibrils may be useful for environmental remediation, presence of Zn2+ and Cu2+, producing H+ and an acidification of acting as metallic traps for purifying contaminated water and the solution. soil. Importantly, unlike previous amyloid scavengers, whose The CA activity of the fibrils was followed using phenol red, formation requires high temperatures and acidic conditions, our which presents red coloration at alkaline pH and changes to prion-inspired scaffolds self-assemble into highly stable amyloids yellow at acidic pH. Absorbance spectra of self-assembled heptapeptides in the range of 350−650 nm were acquired before under mild conditions, making them environmentally friendly. and 2 min after the addition of carbonated H2O. As shown in The most remarkable feature of the prion-inspired amyloids is Figure 5A and Figure 6A, the initial spectra exhibited a peak at their capacity to function as metalloenzymes when they are 560 nm and a second lower peak at 430 nm, characteristic of an coordinated to metal ions. Unlike most natural enzymes and alkali medium with a pH around 8.0, whereas after incubation it designed catalytic supramolecular assemblies, containing a His displayed a significant decrease in the 560 nm peak and a as the nucleophile, our amyloids coordinate Zn2+ and Cu2+ via maximum at 430 nm, indicating a drop in the pH due to the the aromatic Tyr residues that face the solvent in the β-strand generation of H+. In this experiment, commercial CA protein ladder. Kinetic analysis of the esterase activity reveals that the was used as a positive control of the reaction. heptapeptide fibrils exhibit substrate-concentration-dependent Next, CA kinetics were followed by measuring the decrease in Michaelis−Menten kinetics, thus functioning as true metal- absorbance at 560 nm along time for 2 min (Figures 5B and 6B), loenzymes, despite the absence of a His triad. Notably, the and the relative activity was calculated (Table S7). Negative esterase activity of a soluble L-Tyr amino acid in the presence of controls in the absence of heptapeptide fibrils, with or without metal cations is low, indicating that it is the three-dimensional the metal ions, and fibrils without added metals exhibited structural framework of the fibril that provides an appropriate residual activity. As one would expect, wild-type CA protein, microenvironment for the metal-dependent catalytic activity, used in the experiment as a positive control, promoted a high consistent with computational calculations. and fast reduction in absorbance. Importantly, all Cu2+ and Zn2+ The esterase catalytic efficiency of our Tyr-based fibrils is incubated fibrils were active, displaying significant CA activity, in lower than that of His-based amyloids, which is consistent with some cases resembling that of the natural enzyme in the same the evolutionary selection of His rather than Tyr at the active site conditions (Figures 5B and 6B and Table S7). of current hydrolases. In any case, our findings support the Overall, our results demonstrate that the metallized prion- notion that amyloid-like structures formed by short and low- inspired fibrils are endowed with hydrolytic catalytic activity, complexity peptides could act as catalysts at the origin of life allowing fixation and hydration of CO2. while expanding the amino acid sequence and composition space compatible with the expression of hydrolytic activities in CONCLUSIONS these ancestral polymers. In this study, we have investigated the coordination of Cu2+, Importantly, in many of the reported His-based catalytic Co2+, Ni2+, and Zn2+ ions by NY7, QY7, SY7, and GY7 amyloid amyloids, the presence of the metal cation during the assembly fibrils. Unlike catalytic amyloids reported to date, the short reaction is required for amyloid formation, since it provides peptides forming these fibrils lack the characteristic His residues contacts necessary for interstrand interactions. In contrast, the but are instead enriched with Tyr residues. Given that the peptides described herein self-assemble spontaneously in the phenolate oxygen in Tyr has been reported to coordinate absence of a metallic cofactor but can bind it in their mature divalent metal cations in certain metalloproteins and provided fibrillar form. This property enables them to function as both that a large number of phenolic hydroxyl groups are exposed to metal scavengers and nanoenzymes and may have implications solvent at the surface of our prion-inspired fibrils, we for their activity in an ancient world context. This allows the hypothesized that these scaffolds could coordinate metal ions uncoupling of the site of fibril synthesis from the site where they and thus become metallized. can express their activity; thus, their action may be 16975 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article compartmentalized in microenvironments with appropriate intrinsic fluorescence was acquired on a Jasco FP-8200 fluorescence metal ion concentrations. spectrophotometer (Jasco Corporation, Japan) in the range 280−400 Finally, we have successfully developed for the first time a nm, exciting at 268 nm wavelength and using an excitation and emission minimalistic artificial amyloid-like carbonic anhydrase that bandwidth of 5 nm at 25 °C. Fluorescence emission at 303 nm, which corresponds to the tyrosine emission maximum, was used for utilizes Tyr−metal coordination for its catalytic activity. These quantification, using the equation from the corresponding calibration fibrillar nanoenzymes exhibit a remarkable capacity to capture curve. CO2, opening an avenue for their utilization in a wide range of Spectrophotometric Quantification of Divalent Metal Cati- fields, including biotechnology, environmental science, and ons. Metallized NY7, QY7, SY7, and GY7 fibrils were obtained as biomedicine, where their biodegradable and innocuous nature previously indicated, and their supernatants were preserved for could prove to be advantageous. spectrophotometric analysis. Calibration curves for Cu2+, Ni2+, and Co2+ were obtained measuring absorbance at 235 nm for Cu2+- incubated samples, at 393 nm for Ni2+-incubated samples, and at 512 METHODS nm for Co2+-incubated samples, using increasing concentrations of each Peptide Preparation. Synthetic heptapeptides NY7 (Ac-NYNY- divalent cation (0 to 200 mM for Cu2+, 0 to 500 mM for Ni2+, 0 to 200 NYN-NH2), QY7 (Ac-QYQYQYQ-NH2), SY7 (Ac-SYSYSYS-NH2), mM for Co2+). Molar extinction coefficients (ε) were obtained and GY7 (Ac-GYGYGYG-NH2) were purchased from CASLO ApS experimentally by measuring the absorbance of stock divalent cation (Scion Denmark Technical University). Lyophilized peptides were solution at different concentrations and calculated from the slope of the dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to obtain a 10 mM stock obtained equations: Cu2+ε235 nm = 189 M−1·cm−1; Ni2+ε393 nm = 4.8 M−1· solution, aliquoted, and frozen at −80 °C. cm−1; Co2+ε512 nm = 6.0 M−1·cm−1. For the Zn2+ spectrophotometric Peptide Aggregation Reactions. NY7, QY7, and SY7 peptides quantification, Zincon dye was used since the Zn2+ absorbance were diluted from a 10 mM stock to a final concentration of 250 μM in maximum is under 200 nm. Zincon dye detects Zn2+ in solution, 100 mM potassium phosphate, pH 7.0. Solutions were incubated in showing a color change from red to blue with an absorbance maximum quiescent conditions for 7 days at 25 °C. We have demonstrated at 620 nm in the presence of Zn. A Zincon monosodium salt (Sigma- computationally and experimentally40,41 that the GY7 peptide displays Aldrich, USA) stock solution was prepared at 2 mM in 20 mM NaOH. a higher critical concentration for fibrillation; therefore, it was diluted to The calibration curve for Zn2+ was obtained by incubating increasing a final concentration of 500 μM in 100 mM potassium phosphate, pH Zn2+ concentrations (0 to 80 μM) with 100 μM Zincon and measuring 7.0. absorbance at 620 nm. The molar extinction coefficient for the Zn− Transmission Electron Microscopy. For the preparation of TEM Zincon complex was experimentally calculated from the slope of the samples, 10 μL of NY7, QY7, SY7, and GY7 fibrils incubated with Ni2+, obtained equation: Zn−Zinconε620 nm = 22 800 M−1·cm−1. All of the Co2+, and Zn2+ were deposited onto carbon-coated copper grids, and measurements were performed in a Specord200 Plus spectropho- NY7, QY7, SY7, and GY7 fibrils incubated with Cu2+ were deposited tometer (Analytik Jena, Germany). Data were analyzed calculating the onto carbon-coated gold grids for 10 min. Liquid excess was removed retention percentage for each peptide and cation, tacking as a reference with filter paper. No negative staining was performed. As controls, NY7, each concentration point used to incubate the fibrils, obtaining the final QY7, SY7, and GY7 fibrils without incubation with divalent metal percentage of the cation retained by fibrils. cations were deposited onto carbon-coated copper grids for 10 min, and Fourier Transform Infrared Spectroscopy. NY7 and QY7 fibrils negative staining was performed with 2% w/v uranyl acetate solution for were centrifuged at 12000g for 30 min, resuspended in Milli-Q water, 1 min. Grids were scanned using a JEM 1400 transmission electron and incubated with divalent metal cations Cu2+, Co2+, and Zn2+ at a final microscope (JEOL Ltd., Japan) operating at 80 kV, and micrographs concentration of 200 mM, overnight at RT. Samples were washed twice were acquired with a CCD GATAN ES1000W Erlangshen camera with water, and fibrils were recovered for FTIR analysis. (Gatan Inc., USA). Infrared spectra were collected using a Bruker Platinum Alpha Eco- Thioflavin-T Binding. Thioflavin-T (Th-T) dye binding to non- ATR FTIR spectrometer (Bruker Optics, USA). Fibers were deposited metallized NY7, QY7, SY7, and GY7 fibrils or metallized NY7 and QY7 on the diamond ATR crystal, and solvent was evaporated in the stream fibrils was analyzed to confirm the amyloid character. Self-assembled of nitrogen. FTIR spectra were recorded between 1700 and 1600 cm−1 peptides were diluted 1:10 in 100 mM potassium phosphate, pH 7.0, in 32 acquisitions at a resolution of 1 cm−1. Spectra were corrected for with 25 μM Th-T. The Th-T fluorescence signal was measured on a background absorption and normalized. Jasco FP-8200 fluorescence spectrophotometer (Jasco Corporation, Electronic Diffraction. Grids prepared for TEM visualization were Japan) in the 460−600 nm range, using an excitation wavelength of 445 also used for electronic diffraction in a JEM-2011 transmission electron nm and with an excitation and emission bandwidth of 5 nm. microscope (JEOL Ltd., Japan) operating at 200 kV. Characteristic Divalent Metal Cation Binding to Self-Assembled Heptapep- distances of diffraction patterns of Cu2+, Ni2+, Co2+, and Zn2+ were tide Fibrils. NY7, QY7, SY7, and GY7 self-assembled fibrils were measured from the obtained micrographs using ImageJ software (NIH, centrifuged at 12000g for 30 min and resuspended in Milli-Q water in USA). half the initial volume. Divalent metal cations Cu2+, Ni2+, Co2+, and Energy Dispersive X-ray Spectroscopy. Grids prepared for Zn2+ stock solutions were prepared at 500 mM using CuCl2, NiSO4, TEM visualization were used for EDX spectroscopy analysis. The EDX CoCl2, and ZnCl2 salts, respectively (Sigma-Aldrich, USA). Centri- spectrum for each sample was acquired in a JEM-2011 transmission fuged NY7, QY7, SY7, and GY7 fibrils were incubated in the presence of electron microscope (JEOL Ltd., Japan) operating at 200 kV, equipped increasing amounts of each divalent cation: Cu2+ was added at 5, 10, 50, with an EDS X-max detector (Oxford Instruments, UK). Spectra were 100, and 200 mM; Ni2+ was added at 50, 100, and 200 mM; Co2+ was analyzed using INCA Software (ETAS group, Germany) and added at 20, 50, 100, and 200 mM; Zn2+ was added at 5, 10, 20, and 50 represented with GraphPad Prism 5.0 (GraphPad Software, USA). mM. All the reactions were incubated at RT with soft agitation. Samples Inductively Coupled Plasma Atomic Optic Emission Spec- were centrifuged at 12000g for 30 min, and pellet and supernatant were trometry. NY7, QY7, SY7, and GY7 assembled fibrils were incubated separated and preserved for further analysis. in the presence of 200 mM of each divalent cation in a 500 μL final Quantification of NY7, QY7, SY7, and GY7 Peptides. volume as previously described, corresponding to a saturation cation Heptapeptide fibrils from day 7 were quantified from the soluble concentration. A 500 μL amount of nonincubated samples with divalent fraction after centrifugation of the reaction. Then the supernatant was metal cations was used as controls. Incubated samples were centrifuged diluted 1:10 and incubated with 2.5 M guanidinium thiocyanate at 12000g for 30 min, and pellets were stored for their further analysis. (GITC) in 100 mM potassium phosphate buffer for a minimum of 12 h Pelleted fibrils were resuspended in an HNO3 65% solution (Merck under soft agitation to reach the equilibrium. Calibration curves using a Suprapur, Germany) and heated in a DINKO D-65 heating block to known concentration of QY7, NY7, SY7, and GY7 peptides ranging promote peptide digestion. Digestion products were injected in an from 0 to 50 μM were incubated under the same conditions. Tyrosine inductively coupled plasma optic emission Optima 4300DV mass 16976 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article spectrometer (PerkinElmer, USA) to quantify divalent metal cation functional method47,48 and applying the Grimme’s correction for content. dispersion (D3).49 The scalar relativistic Stuttgart−Dresden SDD Esterase Activity. NY7, QY7, SY7, and GY7 assembled fibrils were pseudopotential and its associated basis set were used for copper and prepared as described and isolated by centrifugation at 12000g for 30 zinc, and the standard 6-31G(d) basis set was used for the rest of the min. Esterase activity was tested in the presence of Cu2+ and Zn2+, atoms. Optimizations were done fixing the position of the terminal Ca of requiring different buffering conditions: 25 mM HEPES pH 8.0 for each strand to simulate the restraints imposed by the fiber. Solvent Cu2+ and 25 mM Tris HCl pH 8.0 for Zn2+. Samples were incubated in effects were accounted for with the SMD implicit solvation model.50 the presence of 1 mM CuCl2 and 1 mM ZnCl2 at 25 °C in their For NPA hydrolysis simulations we used the larger 6-31+G(d,p) basis respective buffers. Peptide concentration used in all assays corresponds set and the CPCM implicit solvation model. All calculations were to 100 μM. performed using the GAUSSIAN16 software package.51 The reactions were performed in 96-well plates and measured every 10 min until a plateau was reached in a Victor3 plate reader ASSOCIATED CONTENT (PerkinElmer, USA), acquiring absorbance at 405 nm. p-Nitrophenyl acetate (p-NPA) and p-nitrophenol (p-NP) (Sigma-Aldrich, Germany) * sı Supporting Information stock solutions were freshly prepared at 0.1 M in acetonitrile and The Supporting Information is available free of charge at diluted to the desired final concentrations. Extinction molar coefficients https://pubs.acs.org/doi/10.1021/acsnano.3c04164. for p-NP in each buffering condition were experimentally obtained from Table containing divalent cation quantification by ICP- the slope of calibration curves, which were obtained from three independent replicas and measured in duplicate. Obtained values were OES; tables containing electronic diffraction distances as follows: for Cu2+ experiments ε405 nm = 11 670 M−1·cm−1 and for from Cu2+-incubated fibrils, Ni2+-incubated fibrils, Co2+- Zn2+ experiments ε405 nm = 13 409 M−1·cm−1. incubated fibrils, and Zn2+-incubated fibrils; table Preliminary kinetic measurements were performed in the presence of containing kinetic parameters of the fibrils’ esterase 450 μM p-NPA and 100 μM fibrils previously incubated with the activity in the presence of Cu2+; table containing kinetic corresponding divalent metallic cation (Cu2+ or Zn2+). Corresponding parameters of the carbonic anhydrase activity of the self- buffer signal was subtracted from all the samples. A 50 μM assembled fibrils in the presence of Cu2+ and Zn2+; models concentration of L-Tyr was measured as a control. Data were analyzed of GY7 fibrils; experimental determination of molar using GraphPad Prism Software and fitted to a hyperbolic equation. extinction coefficient for divalent cations Ni2+, Cu2+, Co2+, Kinetic parameter calculations were further obtained for both conditions at a final peptide concentration of 100 μM. For Cu2+- and Zn2+ (with Zincon complex); graphical representa- incubated peptides, the substrate (p-NPA) concentration used ranged tion of the fibrils’ retention capacity for divalent cations from 0.2 to 5 mM, and for the Zn2+ substrate (p-NPA) the Ni2+, Cu2+, Co2+, and Zn2+; optimized structures for Cu2+ concentration ranged from 0.2 to 2.5 mM. Reported results correspond and Zn2+ interacting with the p system of tyrosines; to the average of at least three independent measurements. Kinetic biophysical characterization of NY7, QY7, SY7, and GY7 parameters were obtained by fitting the data to the Michaelis−Menten fibrils; fluorescence emission spectra of Th-T in the equation [V0 = kcat·[E]0·[S]0/(KM + [S]0)] using GraphPad Prism 5.0 presence of NY7 and QY7 fibrils incubated with Cu2+, software. Zn2+, and Co2+; FT-IR absorbance spectrum in the amide Carbonic Anhydrase Activity Assay. NY7, QY7, SY7, and GY7 I region of NY7 fibrils alone or incubated with Cu2+, Zn2+, assembled fibrils were prepared as described and isolated by and Co2+; FT-IR absorbance spectrum in the amide I centrifugation at 12000g for 30 min. Carbonic anhydrase was tested in the presence of Cu2+ and Zn2+, requiring different buffering region of QY7 fibrils alone or incubated with Cu2+, Zn2+, conditions: 25 mM HEPES pH 8.0 for Cu2+ and 25 mM Tris HCl pH and Co2+; electronic diffraction pattern micrographs from 8.0 for Zn2+. Samples were incubated in the presence of 1 mM CuCl2 heptapeptide fibrils incubated with Ni2+, Cu2+, Co2+, and and 1 mM ZnCl2 overnight at 25 °C in their respective buffers under Zn2+; electronic dispersive X-ray spectroscopy spectra soft agitation. Peptide concentration used in all assays corresponds to from heptapeptides incubated with Cu2+, Ni2+, Co2+, and 100 μM. Phenol red pH indicator was used to follow the pH Zn2+; fibrils’ esterase activity kinetics in the presence of modification of the reaction at a final concentration of 100 μM in the Cu2+ and Zn2+ (PDF) corresponding buffers. Carbon dioxide (CO2)-saturated water was freshly prepared by bubbling CO2 gas flow into 100 mL of Milli-Q water in a round-bottom AUTHOR INFORMATION crystal flask with a flow rate at 5 psi under ice-cold conditions for 1 h. A Corresponding Author 150 μL amount of 100 μM phenol red in the corresponding buffers was Salvador Ventura − Institut de Biotecnologia i de Biomedicina used to perform the experiments in the presence and in the absence of self-assembled peptides. A 50 μL amount of CO2-saturated water was (IBB) and Departament de Bioquímica i Biologia Molecular, added to the samples. Reactions were performed in a Specord200 Plus Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; spectrophotometer (Analytik Jena, Germany) measuring the change in orcid.org/0000-0002-9652-6351; the absorbance at 560 nm. Additionally, initial and final spectra were Email: salvador.ventura@uab.cat acquired in the 350−650 nm range. Carbonic anhydrase at 50 nM was used as a positive control. Authors Computational Details. Docking calculations were carried out on Susanna Navarro − Institut de Biotecnologia i de Biomedicina our previously built 40-strand fibril model,40 with the GOLD software (IBB) and Departament de Bioquímica i Biologia Molecular, package,45 applying the improvement designed for metal ion docking46 Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; and using the GoldScore (GS) scoring function, a robust scoring for orcid.org/0000-0001-8160-9536 posing predictor. Genetic algorithm (GA) parameters were set to 50 Marta Díaz-Caballero − Institut de Biotecnologia i de GA runs and a minimum of 100 000 operations. The rest of the Biomedicina (IBB) and Departament de Bioquímica i Biologia parameters were set to default. Protein residues were considered rigid, except the tyrosine ones included in the evaluation sphere of 7 Å Molecular, Universitat Autònoma de Barcelona, 08193 centered on the binding site. Barcelona, Spain; orcid.org/0000-0001-9032-9819 Once the possible binding sites were identified, we cut out from the Francesca Peccati − Basque Research and Technology Alliance fiber those strands directly involved in the metal coordination and ran (BRTA), Center for Cooperative Research in Biosciences (CIC quantum chemical calculations with the hybrid B3LYP density bioGUNE), 48160 Derio, Spain 16977 https://doi.org/10.1021/acsnano.3c04164 ACS Nano 2023, 17, 16968−16979 ACS Nano www.acsnano.org Article Lorena Roldán-Martín − Departament de Química, Universitat (14) Arad, E.; Jelinek, R. Catalytic amyloids. Trends in Chemistry 2022, Autònoma de Barcelona, 08193 Barcelona, Spain 4 (10), 907−917. Mariona Sodupe − Departament de Química, Universitat (15) Makam, P.; Yamijala, S.; Tao, K.; Shimon, L. J. W.; Eisenberg, D. Autònoma de Barcelona, 08193 Barcelona, Spain; S.; Sawaya, M. R.; Wong, B. M.; Gazit, E. Non-proteinaceous hydrolase comprised of a phenylalanine metallo-supramolecular amyloid-like orcid.org/0000-0003-0276-0524 structure. Nat. Catal 2019, 2, 977−985. Complete contact information is available at: (16) Diaz-Caballero, M.; Fernandez, M. R.; Navarro, S.; Ventura, S. https://pubs.acs.org/10.1021/acsnano.3c04164 Prion-based nanomaterials and their emerging applications. Prion 2018, 12, 266−272. Notes (17) Diaz-Espinoza, R. Catalytically Active Amyloids as Future Bionanomaterials. Nanomaterials (Basel) 2022, 12, 3802. The authors declare no competing financial interest. (18) Lu, Y.; Yeung, N.; Sieracki, N.; Marshall, N. M. Design of A previous draft of this work was partially published as a functional metalloproteins. Nature 2009, 460, 855−62. preprint: Diaz-Caballero, M. Prion inspired assemblies to build (19) Maret, W. Zinc biochemistry: from a single zinc enzyme to a key up functional bionanomaterials. 2021. Universitat Autònoma de element of life. Adv. Nutr 2013, 4, 82−91. Barcelona. Departament de Bioquimicá i Biologia Molecular. (20) Verpoorte, J. A.; Mehta, S.; Edsall, J. T. Esterase activities of Dipòsit Digital de Dades de la UAB (https://ddd.uab.cat/ human carbonic anhydrases B and C. J. Biol. Chem. 1967, 242, 4221−9. record/238200, accessed Aug 21, 2023). (21) Lindskog, S.; Silverman, D. N. The catalytic mechanism of mammalian carbonic anhydrases. EXS 2000, 175−95. (22) Supuran, C. T. Carbonic anhydrases: novel therapeutic ACKNOWLEDGMENTS applications for inhibitors and activators. Nat. Rev. Drug Discov 2008, This work was funded by the Spanish Ministry of Economy and 7, 168−81. Competitiveness (MINECO) BIO2017-91475-EXP to S.V., by (23) Boone, C. D.; Habibzadegan, A.; Gill, S.; McKenna, R. Carbonic the Ministry of Science and Innovation (MICINN) PID2019- anhydrases and their biotechnological applications. Biomolecules 2013, 105017RB-I00 to S.V. and PID2020-112715GB-I00 to M.S., 3, 553−62. and by ICREA, ICREA-Academia 2015 and 2020 to S.V. 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