A bioinformatics approach to the design of minimal biomimetic metal-binding peptides

A bioinformatics approach to the design of minimal biomimetic metal-binding peptides | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A bioinformatics approach to the design of minimal biomimetic metal-binding peptides Mun Hon Cheah, Claudia Spallacci, Marco Chino, Antonio Rosato, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6073214/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Oct, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract Nature-inspired or biomimetic catalyst aims to reach the high catalytic performance and selectivity of natural enzymes while possessing the chemical stability and processability of synthetic catalysts. A promising strategy for designing biomimetic catalysts holds on mimicking the structure of the enzyme active site. This can either entail complicated total synthesis of a synthetic catalyst or design of peptide sequences, able to self-assemble in the presence of metal ions, thus forming metallo-peptide complexes that mimic the active sites of natural enzymes. Using a bioinformatics approach, we designed a minimal peptide made up of eight amino acids (H4pep) to act as a functional mimic of the trinuclear Cu site of the laccase enzyme. Cu(II) binding to H4pep results in the formation of a Cu 2+ (H4pep) 2 complex with a β-sheet secondary structure, able to reduce O 2 . Our study demonstrates the viability and potential of using short peptides to mimic the minimal functional site of natural enzymes. Teaser A minimal peptide, designed via bioinformatics, effectively mimics the trinuclear copper site in laccase for O₂ reduction. MAIN TEXT Physical sciences/Chemistry/Chemical synthesis/Biomimetic synthesis Biological sciences/Computational biology and bioinformatics/Protein design Physical sciences/Chemistry/Biochemistry/Peptides Physical sciences/Chemistry/Catalysis/Biocatalysis Biological sciences/Computational biology and bioinformatics/Software Figures Figure 1 Figure 2 Figure 3 Teaser A minimal peptide, designed via bioinformatics, effectively mimics the trinuclear copper site in laccase for O₂ reduction. Introduction The growing demand for sustainable energy, coupled with the climate crisis, motivates the development of efficient devices for storing and producing renewable fuels and chemicals. Nature has taken advantage of billions of years of evolution to carefully optimize enzyme structures for catalyzing specific chemical reactions. Nature’s repertoire of enzyme functions is coupled with the fundamental ability to accept a variety of different substrates – a promiscuity that is at the base of evolution ( 1 ). Metalloenzymes offer a rich library of models for studying and developing artificial catalysts able to drive reactions crucial to the renewable energy transition ( 2 , 3 ). The Protein Data Bank (PDB) is the leading global repository for proteins, nucleic acids, carbohydrates, and other experimentally determined biological complexes ( 4 ). Recent advancements in metalloenzymes research have shown that the residues in close proximity to the metal-binding site are the main determinants in driving catalytic reaction bias and selectivity ( 5 ). In this context, mini-proteins and biomimetic peptide design have emerged as valuable tools to investigate how the properties and configuration of binding residues influence metal selectivity and reactivity ( 6 – 9 ). The appeal of using short peptides as scaffolds to build efficient catalysts lies in their versatility to achieve high catalytic activity rivaling natural enzymes while possessing higher thermal and pH stability ( 10 , 11 ). Additionally, minimal peptides are more amenable towards interfacial electron transfer on electrodes due to their smaller cross-section, thereby achieving higher surface coverage on electrode surfaces. Moreover, short peptides can be easily synthesized in large amounts and at low cost, thanks to the advanced techniques and well-established methods available in solid-phase peptide synthesis (SPPS) ( 12 , 13 ). With this in mind, we have chosen to use natural metalloenzymes as models to design short peptide ligands as a scaffold for supporting the self-assembly of biomimetic catalysts. Focusing on the first coordination sphere around the catalytic cofactor of target metalloenzymes, our goal is to identify the shortest peptide sequence able to bind metals and mimic the model’s site activity. In this regard, the concept of “minimal functional site” (MFS) developed by Andreini et al. is a good starting point for the design, as it describes the minimal environment determining the metal’s chemical behavior ( 14 ). MFSs represent the local three-dimensional environment including all residues within 5 Å distance from any metal-binding ligand, providing a tool for classifying and comparing enzymatic metal sites. Bioinformatic tools can easily extract MFSs information, enabling their manipulation and driving the design of new biomimetic molecules. The potential of bioinformatic approaches for designing bioactive peptides and molecules is well-established, with examples in drug design, vaccine development, and catalysis ( 15 – 18 ). We have thus created a new tool, MetalSite-Analyzer (MeSA, https://metalsite-analyzer.cerm.unifi.it/ ), that enables users to extract relevant sequence motifs for binding a metal of choice. The tool leverages MFS sequence alignments to obtain information on the most conserved residues in metal sites belonging to the protein family of interest. To demonstrate the value of this approach, we have designed a minimal eight-residues peptide, using as a model the trinuclear copper site of laccase. This well-known enzyme was chosen given our interest in redox catalysis. The design of the minimal peptide was based both on sequence conservation analysis, as dictated by MeSA, and on structural modification, led by rational observation of the enzyme’s three-dimensional structure. Our minimal peptide, which we refer to as H4pep, was synthesized via solid-phase peptide synthesis and purified via preparative HPLC. The ability of H4pep to coordinate copper ions was verified via different spectroscopic methods. Notably, the CD data demonstrate that H4pep-Cu 2+ complexes adopt a beta-sheet conformation as it is indeed observed in the MFS of laccase. We have then determined the electrochemical signatures of the Cu 2+ (H4pep) 2 complex, to assess its potential use in electrocatalysis. In line with the model enzyme, the complex has revealed catalytic electrochemical O 2 reduction. Furthermore, spectrophotometric laccase activity assays showed the ability of the Cu 2+ (H4pep) 2 complex to oxidize the 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) substrate in presence of O 2 . Our data demonstrate that, despite its beta-sheet conformation, Cu 2+ (H4pep) 2 does not undergo aggregation, such that we did not detect the formation of fibrils. To the best of our knowledge, this is the first example of a synthetic β-sheet metallo-peptide complex that is stable in solution and features catalytic activity. These findings demonstrate that the MetalSite-Analyzer tool can provide relevant indications for designing bioinspired catalytically active metallo-peptides. Results Bioinformatic tool and peptide design We have created the MeSA tool with the scope of expanding the MFS concept to include sequence conservation analysis. Starting from an input PDB structure, MeSA allows the selection of user-defined mono-/multinuclear metal sites and, from the extracted MFS, runs a PSI-BLAST search of the metal-binding sequence fragments ( 19 ). This step consists of mapping and aligning the binding fragments to all related sequences contained in Uniprot, the world-leading protein sequence database ( 20 ). The output information allows for the analysis of the conservation of the residues in each specific position of the starting MFS sequence. This provides a basis for further rational design of the desired peptide mimics, by highlighting completely conserved residues, which are presumably strictly necessary for function, moderately variable positions, where one of two/three different amino acids can be selected (e.g. based on considerations of stability or ease of synthesis), and highly variable positions, where almost any amino acid can be introduced. The tool has been implemented as a user-friendly web server, requiring no registration ( https://metalsite-analyzer.cerm.unifi.it/ ). To assess our methodological approach, we have selected laccase as a model to design a minimal-length peptide ligand for binding copper ions. Laccases belong to the family of multicopper oxidases, which catalyze one-electron oxidation of different organic substrates, coupling it with the four-electron reduction of O 2 to H 2 O ( 21 ). In these enzymes, substrate oxidation occurs at a mononuclear copper site (type 1), followed by electron transfer to a trinuclear copper cluster (type 2/type 3), where oxygen reduction occurs. The latter is the site we have selected to test our bioinformatic tool. Firstly, we accessed the MetalPDB database ( 22 , 23 ) to gain information on the binding site(s) of the small laccase from Streptomyces viridosporus (PDB ID: 3tbc). In bacterial small laccases, so called because they have fewer domains than fungus laccase, the trinuclear Cu sites are located at the interface between monomers, in a three-fold symmetry. As in a typical multicopper oxidase, they consist of eight histidine residues coordinating the metals, in a ligand-nonligand-ligand motif (LXL) ( 21 , 22 ). In the apo-form of the enzyme, the monomer exposes the coordinating residues to the surrounding environment, making it potentially accessible to solvent and substrate molecules involved in catalysis. This makes the site particularly attractive to inspire the design of artificial biomimetic peptide catalysts. Feeding the pdb structure as input to MeSA and selecting the trinuclear copper site, the tool was able to extract four fragments contributing to the coordination of the Cu ions with two histidine residues each (Fig. 1 A). The conservation of the residues in each fragment was analyzed via the PSI-BLAST algorithm. As a result, four alignments of metal-binding motifs were generated. Finally, a sequence profile ( 26 ) was obtained for each fragment composing the starting site (Fig. 1 B). In our endeavor to obtain a minimal-length peptide mimicking the laccase site’s activity, further rational design steps are needed to implement the information obtained from MeSA. On a closer look at the structure of the site, we have noticed that the binding His residues belong two by two to antiparallel β-sheets, located on adjacent monomers in a C2 symmetry with respect to the axis running across the Cu atoms (Fig. 1 C). The four metal-binding fragments identified by our bioinformatic search thus represent the four β-strands composing this structural motif. In particular, two of the four fragments extracted by the bioinformatic search are considerably shorter (7–9 residues for fragment 1 of chain A and B versus 11–13 for fragment 2 of chain A and B, see Fig. 1 B), thus more suited for our purpose of designing a minimal-length peptide. Owing to their similarity, we have decided to inspire the design of our short peptide from a single fragment of the site. Hence, for the final sequence, the choice of the amino acid residues in each position was driven by the combination of the consensus sequences of these two fragments, with further modifications led by the rational analysis of the model site (See Material and Methods for details). The final minimal-length peptide ligand is eight residues long and was named H4pep (sequence: HTVHYHGH). Binding of metal ions by H4pep H4pep was synthesized via SPPS and purified via reverse-phase high-pressure liquid chromatography (RP-HPLC). To verify the consistency of our design strategy, we preliminarily carried out UV-visible and NMR experiments to confirm the ability of H4pep to bind copper ions. Such experiments were performed at pH 5.6 to prevent copper oxide formation and N-terminal amine deprotonation ( vide infra ), still favoring imidazole deprotonation and binding (pKa ~ 6) ( 27 ). UV-visible spectra of H4pep display the characteristic peak of tyrosine at 276 nm (Fig. 2 A). Upon gradual copper(II) addition, the characteristic band of Cu 2+ d-d transitions arises in the region between 500 and 800 nm. This feature is in good agreement with reported λ max values for histidine coordination in peptide-copper complexes ( 28 ). Notably, with copper concentration up to 1 equivalent, this band could be fitted with two Gaussian functions, suggesting the contribution of multiple species to the absorbance profile (See Stochiometry of H4pep-Cu 2+ complexes section for details). In excess of copper, the absorption peak of unbound copper(II), with maxima at 786.7 nm, becomes noticeable and increasingly contributes to the observed spectrum. Binding of copper(I) was also tested and the affinity of H4pep to Cu + ions was determined via competitive titration with bicinchoninic acid (BCA, Fig. S1 ). The absorption changes at 562 nm, corresponding to the formation of [Cu(BCA) 2 ] 3− , could be fitted satisfactorily assuming a single class of binding sites with K D = 2.4*10 − 12 M. This affinity is comparable to the values found for a series of nitrite reductase mimics featuring a Cu(His) 3 site bound to a triple-stranded α-helical coiled-coil ( 29 , 30 ). To further investigate the coordination of metal ions to H4pep, the peptide’s structure was analyzed by NMR spectroscopy under different experimental conditions. In aqueous solution, the apo-peptide showed only four very broad amide signals of the nine expected (seven backbone and two C-terminal amide protons) due to fast exchange with the solvent, indicating the absence of stable hydrogen bonds and secondary structure elements (Fig. S2A and S3A). In methanol, while amide resonances became observable, their chemical shift dispersion remained limited, suggesting lack of defined tertiary structure in the apo form (Fig. S3B and S4A). The addition of Zn 2+ , which was used as a diamagnetic probe for the paramagnetic Cu 2+ ion, in a 1:1 ratio induced selective changes in the histidine resonances, with downfield shifts (∼0.1 ppm) of the proton signals belonging to the Cδ and Cε of the imidazole ring (Fig. S2B). The broadening of these resonances suggests a chemical exchange process, widely observed in NMR analysis, which primarily involves the histidine residues ( 31 ). This spectral behavior suggests a dynamic equilibrium between multiple zinc-bound species, where either one or two Zn²⁺ ions are alternately coordinated to the two different available binding sites in the peptide scaffold. More dramatic spectral changes were observed upon addition of Cu + (at H4pep:Cu + ratios of 1:1 and 2:1) under anaerobic conditions both in aqueous buffer (Fig. S2C and S5) and methanol (Fig. S4B and S6). All resonances significantly broadened, suggesting that Cu + binding affects the overall peptide conformation more extensively than Zn 2+ , leading to multiple conformational states in exchange. The spectral broadening persisted in the presence of sodium dithionite, excluding contributions from paramagnetic Cu 2+ species. From the above results we can conclude that H4pep binds both Zn 2+ and Cu + ions in solution; binding of Cu 2+ was demonstrated by the UV-visible spectra (Fig. 2 A). Furthermore, the latter UV-visible data along with the NMR spectrum of H4pep-Zn 2+ indicate that binding occurs via the histidine side chains. Secondary structure and pH dependence of metal binding As anticipated by NMR, CD spectroscopy of H4pep in the far-UV range (190–250 nm) reveals only a deep band at 198 nm indicative of a random coil for the apo-peptide in buffer solution (Fig. 2 B). Based on our peptide design strategy, we can reasonably anticipate that binding of Cu 2+ to H4pep will induce the formation of a β-sheet structural motif. Thus, we expect to observe spectral changes characteristic of β-sheet conformation when H4pep is titrated with Cu 2+ . In line with our design and the previous NMR analysis, we expect metal binding to occur via histidine side chain coordination rather than backbone amide coordination, previously reported for 3-residues-Cu systems ( 32 ). Coordination of copper ions via histidine side chains can be further differentiated from backbone amide coordination as histidine side chain coordination will be strongly affected by pH (histidine side chain has pKa ~ 6). CD spectra recorded at different pHs allowed us to test Cu binding ability under different buffer conditions. H4pep was dissolved in acetate buffer solutions at pH 4.4, 4.8, 5.2, and 5.6 respectively (Fig. S7). Higher pH values are excluded to avoid the formation of copper oxide species, limiting Cu 2+ availability to the peptide ligand, and to mitigate terminal amine binding (pKa ⁓7–8). No significant changes were observed in the CD spectra at pH 4.4 and 4.8 in presence of Cu 2+ , indicating unfavorable binding conditions due to the protonated state of the histidine ligands. In contrast, at pH 5.2 and 5.6, a clear change in conformation is visible. Therefore, pH 5.6 buffer conditions were selected to record the titration spectra in Fig. 2 B. Upon Cu 2+ addition, the CD spectrum displays a negative band at 227 nm and a positive one at 210 nm. This suggests that Cu 2+ binding to H4pep induces a β-sheet conformation, consistent with our initial design. These features saturate upon the addition of 0.5 Cu 2+ equivalents and remain essentially unchanged until a 1:1 H4pep:Cu 2+ ratio. As additional copper equivalents are introduced, the spectrum undergoes further changes, with the positive feature increasing and shifting to 205 nm. This suggests the formation of another species, favored at high copper concentrations. The same trend was observed in the visible range of the CD spectra, in which changes in the coordination environment around the metal ion(s) can be monitored (Fig. S8A). Titration of up to 1 equivalent of Cu 2+ results in the gradual increase of a positive peak centered at 719 nm. At higher Cu 2+ equivalents, the peak position shifts to the red, suggesting the formation of a second H4pep-Cu 2+ species. Titration of Cu + to H4pep results in similar changes in the UV region of the CD spectrum (Fig. S8B). There are no observable features in the visible region of the same CD spectrum, consistent with the lack of d-d transitions in Cu + complexes. This confirms that the observed changes in the UV region of the CD spectrum are associated with the coordination of Cu + ions to H4pep to form H4pep-Cu + complexes. Titration experiments via EPR EPR spectroscopy was employed to further analyze copper(II) binding and to elucidate the presence of different H4pep-Cu 2+ species. Titration of H4pep into a Cu 2+ solution revealed the presence of at least two distinct EPR active species (Fig. 2 C). Under a large excess of peptide (e.g. H4pep:Cu 2+ ratio 1:0.3), a single axial signal is observed, with values of g x , g y , and g z respectively at 2.0419, 2.0737, and 2.2447 (Fig. S9). At higher Cu 2+ equivalents (between H4pep:Cu 2+ ratios 1:0.4 to 1:1) an additional axial signal characterized by lower g values could be detected. Presumably, the coordination of an additional Cu 2+ ion into the peptide can cause a small distortion to the local environment of the first Cu 2+ , resulting in a shifted EPR spectrum, which could be isolated by spectral deconvolution (Fig. S10). In this configuration, the two Cu 2+ binding sites are essentially identical, as only one axial Cu 2+ signal is observed. The presence of two distinct EPR signals at different H4pep:Cu 2+ ratios is consistent with CD spectroscopy results, indicating that two H4pep-Cu 2+ species are formed. Furthermore, above H4pep:Cu 2+ ratio of 1:1, an EPR signal associated with unbound Cu 2+ becomes observable. Stoichiometry of H4pep-Cu complexes The CD and EPR spectra obtained from titrations between Cu 2+ and H4pep were deconvoluted by fitting each spectrum with their respective isolated EPR signals or CD spectra and presented in Fig. 2 D. The observation of unbound Cu 2+ species in the EPR spectra above H4pep:Cu 2+ ratio of 1:1, suggests that each peptide can bind a maximum of 1 Cu 2+ equivalent. The formation of a β-sheet conformation upon Cu 2+ binding, as evident from the CD spectra, indicates that at least two peptide units are required to form a Cu 2 + x (H4pep) y complex (where y ≥ 2). It is unlikely that the minimum number of peptides to form a Cu 2 + x (H4pep) y is three, since we would expect most of the H4pep to exist in a β-sheet conformation at H4pep:Cu 2+ ratio of 1:0.33. On the contrary, our experimental observation led to a fitted molar fraction of peptide in random coil conformation of around 0.5 at 1:0.33 H4pep:Cu 2+ ratio (Fig. 2 D). Therefore, our hypothesis is that a Cu 2 + x (H 4 pep) 2 complex is formed upon addition of Cu 2+ ions to H4pep. In this scenario, there are two possible species corresponding to Cu 2 + 1 (H 4 pep) 2 and Cu 2 + 2 (H 4 pep) 2 , designated as 1Cu2Pep and 2Cu2Pep respectively. Deconvolution of UV-visible d-d band spectra could be obtained accordingly (Fig. S11). Fitting of the spectra up to 1 Cu equivalents was possible considering two Gaussian components, representing 1Cu2Pep and 2Cu2Pep . At higher Cu 2+ concentrations, a third component, arising from free Cu 2+ in solution needed to be included for accurate fitting. Observing the resulting trends, it is clear how the isolation of a single Cu 2 + x (H4pep) 2 species is far from trivial. The 1Cu2Pep species seems to be favored at low Cu 2+ concentrations, to 0.5 equivalents, while both 1Cu2Pep and 2Cu2Pep species are present in similar quantities until 1 Cu 2+ equivalent. The 2Cu2Pep species is favored at higher Cu 2+ concentrations, but an increasing amount of free Cu 2+ ions is detected in these conditions. Notably, even in large excess of Cu 2+ , dynamic light scattering (DLS) experiments revealed the absence of large aggregates, confirming the molecular nature of the complexes (Fig. S12). Electrochemistry and testing of activity To examine if 1Cu2Pep and 2Cu2Pep exhibit catalytic O 2 reduction activity, as in the original laccase, a preliminary electrochemical characterization of these two complexes by cyclic voltammetry was performed (Fig. S13A). Cyclic voltammograms of 2Cu2Pep under N 2 atmosphere showed a reduction peak at -0.11 V against Ag/AgCl reference electrode and a corresponding re-oxidation wave at 0.52 V. The large peak-to-peak separation suggests a slow heterogeneous electron transfer rate between the glassy carbon working electrode and the complex. Notably, during the anodic scan, a sharp reoxidation peak at 0.08 V is observed, characteristic of oxidation of metallic Cu deposited on the working electrode. This suggests that during the reduction of 2Cu2Pep , the copper binding affinity of at least one of the binding sites becomes weaker, resulting in the loss of copper from the complex. Since metallic Cu is also known to participate in electrocatalytic O 2 reduction, we will not examine the O 2 reduction activity of 2Cu2Pep as it is not trivial to verify its catalytic O 2 reduction activity in presence of metallic Cu. On the other hand, cyclic voltammograms of 1Cu2Pep show a reduction peak at -0.16 V and the corresponding reoxidation peak at 0.36 V under N 2 atmosphere. Importantly, features associated with the deposition of metallic Cu and its subsequent reoxidation are not observable. This is further verified by rinse test experiments after CV scans of 1Cu2Pep (Fig. S13B). Under O 2 atmosphere, a current enhancement is observable at around − 0.20 V, suggesting catalytic O 2 reduction activity (Fig. S13C). To verify this behavior, bulk electrolysis experiments were performed in a custom-designed cell that incorporates a Clark electrode for sensing of O 2 concentration. Chronoamperometry experiments at -0.20 V result in a steady-state current of approximately − 15 µA with a concomitant decrease in O 2 concentration (Fig. 3 ). The estimated faradaic efficiency in reference to the moles of O 2 consumed is 62%, assuming that all observed current is due to four-electron of O 2 to water. The low faradaic efficiency maybe the result of side reaction involving two-electron reduction of O 2 to hydrogen peroxide, which cannot be excluded at present. Cyclic voltammetry of the 1Cu2Pep solution after bulk electrolysis does not show significant deviation from that recorded prior to bulk electrolysis, with no indication of Cu 0 plated on the electrode surface (Fig. S13D). Furthermore, additional rinse test experiments show a reductive current compared to the background during chronoamperometry and no observable features in the corresponding cyclic voltammogram (Fig. S13E). To further rule out the participation of unbound copper species during catalytic O 2 reduction, negative control experiments were performed with dilute solutions of unbound Cu 2+ (0.03 mM, 7% of Cu 2+ content in 1Cu2Pep ). Chronoamperometry of 1Cu2Pep shows a higher current compared to 7% Cu 2+ (Fig. S14A), indicating only a minimal, if any, contribution of free copper(II) ions to the catalytic activity. Moreover, rinse test of the unbound Cu 2+ solution shows significant reoxidation of metallic copper species at 0.09 V, not observed for the 1Cu2Pep species (Fig. S14B). Therefore, the O 2 reduction activity in the conditions above is likely to originate from 1Cu2Pep. We have then tested the performances of 1Cu2Pep at a more reducing potential (-0.30 V). In this case, an interesting behavior of the chronoamperometry signal was observed: while the recorded current decreases in the first 120 seconds, it then reaches a plateau and starts increasing after 150 seconds (Fig. S15A). No signals of unbound Cu 2+ could be detected in the cyclic voltammogram and rinse test performed after the measurement (Fig. S15B). This result seems to indicate that a more active species of the H4pep:Cu assembly can be formed in these conditions. Finally, laccase activity of 1Cu2Pep was tested via spectrophotometric assay, monitoring the increase in absorbance at 420 nm characteristic of ABTS substrate oxidation ( 33 ). In O 2 saturated atmosphere, the activity was estimated to be 1.5 ± 0.4 U L − 1 . The reported activity of natural laccase enzymes ranges from 3.5 to 484,000 U L − 1 ( 34 ). The value of laccase activity obtained for 1Cu2Pep , although modest, is in agreement with our electrocatalytic activity results, demonstrating the ability of the complex to perform moderate electrocatalytic O 2 reduction. Discussion Designing peptide mimics of enzymatic metal-binding sites is a challenging task. Our approach demonstrates that bioinformatic tools can support the rational design of such mimics via analysis of sequence conservation. Short, tunable peptide ligands represent promising candidates for applying this strategy. Notable examples are minimal Cu-peptide complexes, which are already widely investigated since histidine-containing binding sites are found in several metalloproteins ( 10 , 35 – 38 ). In these smaller systems, metal coordination often involves backbone amide and terminal amine groups. In contrast, our H4pep-Cu 2+ complexes feature exclusive histidine coordination, as confirmed by the resonance shifts and broadening of the NMR signals of imidazole protons upon metal binding, in line with our model design (Figs. S2C and S4B). Based on the analysis of the NMR and EPR data, we propose a rationale for the formation of the observed 1Cu2Pep and 2Cu2Pep species (Fig. S16). At low copper concentrations, we hypothesize that Cu 2+ preferentially binds to a specific site, where the metal atom is bound to two peptide strands ( 1Cu2Pep species). Assuming that the peptide strands associate in an antiparallel β-sheet structure, two symmetrically equivalent binding sites can be identified. In this scenario, although only four out of eight available histidine residues coordinate Cu 2+ at a given time, all of them (His0, His3, His5, His7) are effectively involved in metal binding via conformational exchange to the symmetrically equivalent species. This is reflected in the observed simultaneous broadening of all histidine’s proton resonances in the NMR experiments. On the other hand, a single axial Cu 2+ signal is observed in EPR experiments, consistent with two symmetrically equivalent binding sites. At higher copper concentrations, coordination of a second Cu 2+ can occur, filling the second identical binding site. However, this leads to a small distortion of both binding sites. The resulting 2Cu2Pep species features two symmetrically identical binding sites, each differing slightly from the original site in the 1Cu2Pep species. Therefore, the EPR signature of 2Cu2Pep displays a single axial copper signal, only slightly shifted compared to that of the 1Cu2Pep species, as the two Cu 2+ ions are not magnetically coupled and are virtually identical (Fig. 2 C). The NMR spectra in a 1:1 H4pep:Cu + ratio also show no significant differences compared to the 2:1 H4pep:Cu + ratio, supporting our interpretation (Figs. S5 and S6). Notably, 1Cu2Pep shows moderate O 2 reduction activity, thus demonstrating the potential of our approach toward designing functional mimics of enzyme active sites. H4pep is an example of how even extremely short peptide ligands can provide versatile scaffolds for metal binding and facilitate the study of structure-function relationships. Different three-dimensional motifs have been employed for this purpose, with α-helical structures being the leading example ( 7 , 10 , 29 , 39 – 43 ). Only recently, metallo-β-sheets peptides have been considered as scaffolds for catalytic site mimics, with just a handful of examples ( 44 – 47 ). CD data of our H4pep-Cu 2+ complexes indicate a metal-induced conformational change to a β-sheet motif, offering a new opportunity to study this structural motif in the context of biomimetic catalysis (Fig. 2 B). Work from different groups has demonstrated how β-sheet motifs tend to self-assemble into supramolecular structures and form insoluble aggregates ( 6 , 46 , 48 ). Gaining a deeper understanding of the factors that promote or prevent aggregation is crucial for utilizing these structural motifs across various research fields. For instance, amyloid-β aggregates are currently intensively studied, as they play a role in the rise of neurodegenerative disorders such as Alzheimer’s disease ( 49 , 50 ). Notably, DLS studies of our H4pep-Cu 2+ complexes have revealed the absence of larger assemblies (Fig. S12), confirming the molecular nature of the complexes and making them particularly suitable for the study of functional minimal active site mimics. Our success in reproducing a molecular metallo-β-sheet motif via bioinformatic and structural design, provides a step forward in understanding the sequence-structure relationship of this conformation. We anticipate that further targeted modifications of the amino acid sequence in our H4pep could lead to the assembly of different species than 1Cu2Pep and 2Cu2Pep . Further efforts could focus on designing peptide sequences that self-assemble into a single, well-defined metallo-peptide complex. In this context, more rigid motifs, such as β-hairpins and WW domains, have recently been proposed as more stable architectures for achieving this goal ( 51 – 53 ). However, recent work from Dang et al. has demonstrated that β-hairpin structures are particularly affected by metal binding, which can cause a rearrangement of the β-sheet domains ( 38 ). Nonetheless, mutating just two amino acids in the sequence resulted in a completely different metal-peptide assembly mechanism, underlining the critical importance of sequence analysis in designing selective and stable binding sites. In conclusion, there is still unexplored potential in β-sheet-based and other scaffold designs. Although 1Cu2Pep exhibits only modest activity, it demonstrates that our minimal peptide design strategy is a viable approach to mimic catalytic sites of redox-active enzymes. While most bioinformatics methods, such as Rosetta and Alphafold ( 54 , 55 ), emphasize the design and understanding of large proteins, our Metal-Site Analyzer platform serves as a developing tool optimized for minimal, easily tunable peptide ligands. Our work further highlights the versatility of short peptide-metal assemblies in exploring the sequence-structure relationships of simple metal-binding motifs and their potential applications in designing stable, biomimetic catalysts. Materials and Methods Chemicals and buffers All chemicals and reagents were of analytical grade and used without further purification. Fmoc-amino acids and Oxyma pure were purchased from Novabiochem (Sigma-Aldrich Sweden). Dimethylformamide (DMF), 20% piperidine, N,N′-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), triisopropylsilane (TIS), diethyl ether, acetonitrile (ACN), copper(II) sulfate pentahydrate, sodium dihydrogen phosphate, sodium hydroxide (NaOH), sodium acetate, acetic acid, sodium sulfate (NaSO 4 ) and 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich Sweden. Buffers were prepared starting from sodium dihydrogen phosphate and adjusting the pH with NaOH, or mixing sodium acetate and acetic acid to the desired pH. Cu 2+ stock solutions were obtained by dissolving copper sulfate salt in distilled water or buffer. Bioinformatic tool Metal-Site Analyzer, available at https://metalsite-analyzer.cerm.unifi.it/ , was designed and implemented with the following characteristics. The input consists of a pdb code, or a protein structure in pdb format (e.g., in case the structure is not yet deposited in the PDB). It is also possible to specify the distance threshold that will be used to identify the coordinating atoms to the metal ion(s) and the residues directly involved in the metal binding. If the structure contains a multinuclear site, the user can choose whether to analyze a single metal ion in the site or investigate the entire multinuclear site. In the latter case, it is required to flag the "Aggregate multinuclear site info" option. Then, by clicking on "Search for metals", the tool extracts the list of metal sites in the structure. If the user prefers to limit the search to a specific metal ion, this can be specified in the “Enter a Cofactor” field on the home page of the tool. The second web page displays the list of the sites found in the input structure. The user must select only one site of interest. The "GO!" button will then initiate the site analysis through the steps listed below: The tool extracts the “minimal functional site” (MFS) from the structure ( 14 ); that is, i) the metal(s), ii) the metal-binding residues, and iii) the residues that fall within 5 Å from at least one atom of the metal-binding residues. Usually, the MFS is composed of more than one fragment ( metal-binding fragments hereafter), because the metal site is not continuous in sequence. The residues identified at points ii) and iii) are mapped onto the sequence of the structure. The tool extracts the segment of sequence(s) that contains the entire MFS (from the most N-terminal residue to the most C-terminal residue) ( metal-binding segment hereafter). The metal-binding segment is searched in UniRef50 by using PSI-BLAST (3 iterations) ( 19 , 20 ). The output sequences are filtered to discard those not containing the metal-binding residues of the input site (as they will most likely not bind metal ion(s)). A multiple sequence alignment is generated from the pairwise sequence alignments of the PSI-BLAST output. The metal-binding fragments of the input site are mapped and extracted from the multiple sequence alignment. At the end of the site analysis, the web server will output the metal-binding fragments alignments and the respective Skylign logos ( 26 ). The latter may be used to identify highly conserved residues, which may have a functional role in the metal site and thus be relevant to design purposes. Peptide design The design of the synthetic peptide was based on the small laccase from Streptomyces viridosporus with PDB code 3tbc. The PDB structure was input to MeSA with no additional constraints. The option to aggregate multinuclear site information was chosen, to be able to include the analysis of the trinuclear copper site of interest. The tool individuated three mononuclear type 1 copper sites, respectively belonging to the three monomers that compose the protein (chain A, B, and C), and three trinuclear sites, located at the interface between monomers. All sites are virtually identical, as the monomer unit repeats with a three-fold symmetry in the functional protein structure. By selecting one of the trinuclear MFS, four fragments were extracted, two belonging to one chain (TFHLHGH and WMYHCHVQSHS) and two to another (SLHVHGLDY and WHYHDHVVGTEHG). By looking at the three-dimensional structure of the MFS, we noticed that fragments on the same chain are part of adjacent beta-strands located at the interface of one monomer, while the other fragments (on the second chain) bind the Cu ions from the opposite monomer interface, in a similar beta-sheet conformation. The binding motif His-Xxx-His is highly conserved in all four fragments, as suggested by the MeSA output. For our purpose of designing a minimal-length peptide to mimic the laccase binding site, we have focused on the sequence conservation analysis of the shortest fragments, respectively TFHLHGH belonging to one chain and SLHVHGLDY belonging to the other. The highly conserved binding histidine residues were selected for positions 3 and 5, as well as the glycine in position 6 (Fig. 1 B). A third histidine was assigned to position 7, as it is fairly conserved and capable of also participating in metal binding. Flexible threonine was chosen versus the more rigid proline and assigned to position 1, as the third and second most conserved residue in the two fragments, respectively. The other positions seemed to be less conserved among the fragments and were assigned based on the analysis of the three-dimensional structure of the model site. Tyrosine was strategically included in the sequence as it can be used as UV-visible and redox probe to validate other experiments. The final sequence HTVHYHGH is an 8-residues long peptide representing the shortest fragment mimicking the model site and containing the conserved binding motif. It is important to note that in the MFS of laccase, each Cu atom is coordinated by histidine residues from different fragments; to explore the feasibility of copper binding with a H4pep:Cu ratio of 1:1, an additional His residue at position 0 was introduced to satisfy the requirement of a four-coordination environment. Peptide synthesis Peptide synthesis was performed via Fmoc-chemistry using an automated microwave peptide synthesizer (Biotage Initiator+, Alstra) on a 0.1 mmol scale using Rink-type resin (TentaGel® R RAM) with a polyethylene glycol/polystyrene backbone. The amino acids (Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Thr(tBu)-OH) were weighed in concentrations of 0.5 M and dissolved in DMF. 20% piperidine in DMF was used as the deprotection solution. All coupling steps were performed at 75°C for 5 min, except for the His residues, which were coupled at room temperature for 60 min to avoid racemization. Oxyma (0.5 M in DMF) and DIC (0.5 M in DMF) were used as the activator and activator base, respectively. Final deprotection was performed after the last coupling cycle to obtain a free amine terminus. Cleavage of the peptides from the resin and side chain deprotection was achieved using a 95% trifluoroacetic acid TFA, 2.5% TIS, and 2.5% distilled water cleavage cocktail for 2 hours at room temperature with stirring. After cleavage, the crude peptides were filtered and excess TFA was evaporated under a gentle stream of N 2 gas. The peptides were precipitated, washed with cold diethyl ether, dissolved in water, and lyophilized. The peptides were dissolved in water for purification over a C18 semi-preparative column using an ÄKTA pure chromatography system (Fig. S17). Solvents A was 0.1% TFA in H 2 O, and solvent B was 0.085% TFA in acetonitrile. The peptide was injected in the equilibrated column with 5% B for 5 min, followed by a linear gradient to 20% B over 30 min at a 4 mL/min flow rate. The peptide was eluted at 10% solvent B. The chromatogram was monitored at 280 nm. The purified peptide was lyophilized and stored at − 20°C. Peptide mass and purity were analyzed by LCMS (Agilent Technology) (Fig. S18). Spectroscopic methods: UV-vis, CD, EPR, NMR UV-vis spectra were recorded using a Cary 50 Bio (Varian) spectrometer, in a 10 mm path-length disposable cuvette containing 10 mM acetate buffer at pH = 5.6. The peptide concentration for the near-UV range was 0.4 mM, with addition of 20 µL aliquots of Cu 2+ from a 1 mM copper sulfate stock solution. For the visible range spectra, a peptide concentration of 2.35 mM was used, and a Cu 2+ stock concentration of 22.8 mM. CD spectra were recorded with a Chirascan V100 spectrometer (Applied Photophysics) equipped with a temperature-controlled cell Peltier holder. Smoothing via adjacent averaging was applied when indicated. The spectra were converted from millidegrees to units of mean residue molar ellipticity [Θ] in deg cm 2 dmol − 1 by setting [Θ] = 100*θ obs / Cln , where θ obs is the recorded ellipticity in millidegrees, C is the concentration of protein in mM, l is the cuvette pathlength in cm, and n is the number of amino acids in the sequence ( n = 8). For UV-range measurements, a 1 mm path-length quartz cuvette was used. The peptide was dissolved in 10 mM acetate buffer at pH 5.6 to obtain a 0.1 mM solution. Cu 2+ aliquots were added from a 1 mM Cu 2+ stock solution. For visible-range measurements, a 10 mm path-length quartz cuvette was used, and the sample was prepared with a peptide concentration of 1 mM and a 10 mM Cu 2+ stock solution. In addition, spectra were recorded in 10 mM NaPi buffer solution at pH 5.8 and displayed no significant changes. All EPR spectra were recorded on a Bruker EMX-micro spectrometer equipped with an EMX-Premium bridge and an ER4119HS resonator in connection with an Oxford Instruments continuous flow cryostat. Low temperatures were reached using liquid helium flow through an ITC 503 temperature controller (Oxford Instruments). The EPR samples were prepared in 10 mM acetate buffer at pH 5.6 with a fixed Cu 2+ concentration of 0.1 mM and different peptide concentrations, to reach the selected stochiometric ratio. The tubes were flash-frozen and stored at 77 K prior to EPR analysis. The EasySpin software version 6.0.5 was used for spectral simulation and fitting ( 56 , 57 ). The spectra were recorded at 10 K, microwave power of 50 µW, microwave frequency of 9.31 GHz, modulation amplitude of 10 G, and modulation frequency of 100 MHz. NMR spectra were acquired at 298K on a Bruker Avance 600 Spectrometer equipped with a triple resonance cryoprobe. Samples for NMR analysis were prepared by dissolving H4pep in either sodium phosphate buffer pH 5.8 (10% D 2 O) or CD 3 OH to a final concentration of 0.5 mM. For metal-binding studies, ZnCl 2 or [Cu(CH 3 CN) 4 ]PF 6 were added in stoichiometric amounts. For Cu + experiments, samples were prepared under inert atmosphere, and sodium dithionite was added to maintain reducing conditions. Water suppression was accomplished using an excitation sculpting sequence ( 58 ). Chemical shifts are referenced to TSP. Competitive Titration Studies Cu + binding affinity was determined through competitive titration experiments with BCA (bicinchoninic acid) as a competing ligand (logβ 2 = 17.2) ( 59 ). Experiments were performed by adding small aliquots of BCA (7.2 mM stock solution) to a solution containing H4pep-Cu + complex (prepared with 0.95 equivalents of [Cu(CH 3 CN) 4 ]PF 6 dissolved in acetonitrile up to 10 mM concentration) in 50 mM sodium phosphate buffer at pH 5.8 with 1 mM sodium ascorbate under inert atmosphere. Formation of [Cu(BCA) 2 ] 3− was monitored spectrophotometrically at 562 nm (ε 562 = 7900 M − 1 cm − 1 ). The dissociation constant was determined by fitting the titration data to Eq. (5 9 ): $$\:{\left[L\right]}_{tot}=2\left[M{L}_{2}\right]+\sqrt{\frac{\left[M{L}_{2}\right]}{{K}_{D}{{\beta\:}}_{2}}\left(\frac{{\left[P\right]}_{tot}}{{\left[M\right]}_{tot}-\left[M{L}_{2}\right]}-1\right)}$$ Where [L] tot , [P] tot , [M] tot are the total BCA, peptide, and Cu + concentrations, respectively, and [ML 2 ] is the [Cu(BCA) 2 ] 3− complex concentration. Electrochemical measurements and activity assay All experiments were performed with a Metrohm Autolab potentiostat or a BioLogic SP-300 potentiostat at room temperature, using a low-volume electrochemical cell. For CV measurements, a 3 mm diameter GCE working electrode was used. For bulk electrolysis measurements, the diameter of the exposed working electrode area was 9 mm. A graphite rod was used as counter electrode. An Ag/AgCl (in 3 M KCl) electrode was used as a reference and regularly checked using K 4 [Fe(CN) 6 ] as standard (E° Ag/AgCl = + 0.210 V vs SHE). All potentials are reported versus Ag/AgCl. For all measurements, the glassy carbon working electrodes were polished with 0.05 µm alumina particles and sonicated for 3 minutes in distilled water immediately before use. Measurements were performed in 10 mM sodium phosphate buffer at pH 5.8, in addition of 40 mM Na 2 SO 4 as supporting electrolyte. 1Cu2Pep cyclic voltammetries were performed in presence of 1 mM H4pep and 0.45 mM of Cu 2+ , while 2Cu2Pep measurements were performed in presence of 1 mM H4pep and 0.9 mM of Cu 2+ . The total solution volume was 3 mL. Activity measurements were recorded by integrating a custom-made Clark-type electrode connected to a Unisense control box. Rinse test measurements were performed immediately after the voltammetry/amperometry by removing the active solution, rinsing the electrode with distilled water for 10 seconds, and reassembling the cell with fresh buffer and supporting electrolyte. Laccase activity was determined by spectrophotometric assay by measuring the oxidation of 0.5 mM ABTS (ε 420 = 36000 M − 1 cm − 1 ) in oxygen-saturated phosphate buffer (20 mM) at pH 6. Sample aliquots of 30 µL were prepared by mixing H4pep and Cu 2+ stock solutions in the same buffer. The formation of the product was measured over 5 minutes by recording the change in absorbance at 420 nm. One activity unit (U) is defined as the amount of enzyme oxidizing 1 µmol of substrate per minute. The activity assay was performed in triplicate. Declarations Acknowledgments Funding: Swedish Energy Agency grant 50529-1 (CS, MHC) Italian MUR, Project SEA-WAVE 2020BKK3W9 [CUP_E69J22001140005] (MC, OM, AL) Author contributions: Conceptualization: CS, CA, MHC Methodology: CS, CA, MC, AL, MHC Investigation: CS, MC, OM, PH, CA, MHC Supervision: AL, CA, MHC Writing—original draft: CS, MHC Writing—review & editing: CS, MC, AR, LDA, AL, CA, MHC with input from all authors Competing interests: Authors declare that they have no competing interests Data and materials availability: All data are available in the main text or the supplementary materials. 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Hassabis, Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). S. Stoll, A. Schweiger, EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. Journal of Magnetic Resonance 178, 42–55 (2006). S. Stoll, “Computational modeling and least-squares fitting of EPR spectra” in Handbook of Multifrequency Electron Paramagnetic Resonance: Data and Techniques (Wiley-VCH, Sushil K. Misra., 2014), pp. 69–138. T. L. Hwang, A. J. Shaka, Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients. Journal of Magnetic Resonance, Series A 112, 275–279 (1995). Z. Xiao, A. G. Wedd, The challenges of determining metal–protein affinities. Nat. Prod. Rep. 27, 768–789 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInformationV12finalgenerictemplate.docx Supplementary Materials Please see attached Supplementary Materials Cite Share Download PDF Status: Published Journal Publication published 06 Oct, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6073214","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":421454283,"identity":"07425b08-6224-4a55-abf2-483a008849cd","order_by":0,"name":"Mun Hon Cheah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDACZgY2IGnBwCABYlckgEiitEhAtZwhRgsDshbGtgTC6nXbmZ89+FEhwcA/u/ng58J5aXK67Qxs0jwMdXK4tJgdZjM37DkjwSBx51iy9MxtOcZmh8FaDhvj1sLDJsHbJsFgIJFjIM27rSJxG0TLgcQGPFok//4Dacn//Jt3TkU9VEtdPT4t0rwNYFtAjJwEqMOYcQYE0C9m0jLHJHgkbqSZWfMcSzPcdpix2XKOwWFDnLacP/xM8k2NjRz/jOTHt3lqkuWBIgdvvKmok8dlCwzwILEZgeYbENIwCkbBKBgFowAfAAD98EcZGEsOfAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5732-1524","institution":"Uppsala University","correspondingAuthor":true,"prefix":"","firstName":"Mun","middleName":"Hon","lastName":"Cheah","suffix":""},{"id":421454284,"identity":"6ccbfb6a-4687-4840-adb8-e2d52cfcb479","order_by":1,"name":"Claudia Spallacci","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Spallacci","suffix":""},{"id":421454285,"identity":"6cee1531-f7ec-47b8-865b-abda9df8facb","order_by":2,"name":"Marco Chino","email":"","orcid":"https://orcid.org/0000-0002-0436-3293","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Chino","suffix":""},{"id":421454286,"identity":"a31f0c62-79e9-40b7-8d65-e7b57ebc3cc2","order_by":3,"name":"Antonio Rosato","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Rosato","suffix":""},{"id":421454287,"identity":"bd2854c6-c2ee-41dc-a15d-b0f6c30a56cd","order_by":4,"name":"Ornella Maglio","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Ornella","middleName":"","lastName":"Maglio","suffix":""},{"id":421454288,"identity":"ceffe209-573c-4b44-b199-de04634cdfe0","order_by":5,"name":"Ping Huang","email":"","orcid":"https://orcid.org/0000-0002-7676-6905","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Huang","suffix":""},{"id":421454289,"identity":"955c5a30-20d6-4112-be5b-f37d2b1daabe","order_by":6,"name":"Luca D’Amario","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"D’Amario","suffix":""},{"id":421454290,"identity":"10aa2122-8d7f-4f3e-bccf-23889666ffd0","order_by":7,"name":"Angela Lombardi","email":"","orcid":"https://orcid.org/0000-0002-2013-3009","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Lombardi","suffix":""},{"id":421454291,"identity":"46507e0f-5b3b-4e1f-aa03-f45f174496c7","order_by":8,"name":"Claudia Andreini","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Andreini","suffix":""}],"badges":[],"createdAt":"2025-02-20 15:25:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6073214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6073214/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-025-01702-z","type":"published","date":"2025-10-06T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78130689,"identity":"934fe7bb-f9f8-4d23-aae4-2c6cdde681d1","added_by":"auto","created_at":"2025-03-10 08:48:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":233892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel laccase, MetalSite-Analyzer output and design strategy.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) The small laccase used as a model for our design (PDB: 3tbc) with focus on its minimal functional site (MFS) and binding fragments extracted by MetalSite-Analyzer (MeSA). (\u003cstrong\u003eB\u003c/strong\u003e) Output of MeSA based on the trinuclear copper site of the model small laccase. (\u003cstrong\u003eC\u003c/strong\u003e) The model MFS features a C2 symmetry axis running across the Cu atoms; the shortest fragments (1 from chain A and 1 from chain B) show a high degree of overlap according to this symmetry. Thus, they are selected for the following sequence variability analysis. Conserved residues, including coordinating His, are highlighted (see \u003cem\u003eMaterials and Methods\u003c/em\u003e for details).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6073214/v1/5c86eff17938e83a2a94fa54.png"},{"id":78132174,"identity":"fb897e2a-8c69-4f87-9408-f5a4592ea16c","added_by":"auto","created_at":"2025-03-10 08:56:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural characterization of H4pep-Cu\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e species.\u003c/strong\u003e (\u003cstrong\u003eA, B\u003c/strong\u003e) UV-visible (A, [H4pep] = 0.4 mM, pH 5.6) and UV-CD spectra (B, [H4pep] = 0.1 mM, pH 5.6) of H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e complexes at increasing Cu\u003csup\u003e2+\u003c/sup\u003e equivalents, ranging from 1:0 to 1:3 peptide:copper ratios. Inset in A ([H4pep] = 2.35 mM), focuses on the Cu\u003csup\u003e2+\u003c/sup\u003e d-d band in the range from 500 to 800 nm. Solid lines in (B) highlight significant samples in determining the stoichiometry of the complexes. (\u003cstrong\u003eC\u003c/strong\u003e) EPR spectra of Cu\u003csup\u003e2+\u003c/sup\u003e and H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e species at different H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratios (acetate buffer, pH 5.6). Spectra were recorded in presence of a fixed Cu\u003csup\u003e2+\u003c/sup\u003e concentration (0.1 mM) and increasing concentrations of H4pep. (\u003cstrong\u003eD\u003c/strong\u003e) Speciation diagrams obtained by spectral fitting of UV-visible data, and spectral deconvolution of CD and EPR data. 1Cu2Pep and 2Cu2Pep species are assigned according to the “Stoichiometry of H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e complexes” section.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6073214/v1/e945b636f3c57fdf54d14410.png"},{"id":78134076,"identity":"f618cd14-d63b-48dd-9d69-ce2ef131a320","added_by":"auto","created_at":"2025-03-10 09:12:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrocatalytic activity of 1Cu2Pep.\u003c/strong\u003e Bulk electrolysis of the 1Cu2Pep species recorded at -0.20 V (versus Ag/AgCl reference electrode) for 600 seconds (top), coupled to O\u003csub\u003e2\u003c/sub\u003e sensing via Clark electrode measurement (bottom). Rinse test chronoamperometry does not show significative reductive current, resulting in a flat trace in the Clark electrode response (not shown). Traces are recorded in 10 mM phosphate buffer with the addition of 40 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e supporting electrolyte, in presence of 1 mM H4pep and 0.45 mM Cu\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6073214/v1/fd9e557ccdfb15c5b34536ad.png"},{"id":92833229,"identity":"7be420d1-1423-4c64-a502-69be91068077","added_by":"auto","created_at":"2025-10-06 07:06:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1428490,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6073214/v1/94b84058-57d5-4388-8669-e38e0528c728.pdf"},{"id":78132170,"identity":"64e34a8a-b0fb-4672-81a2-2d20966461ad","added_by":"auto","created_at":"2025-03-10 08:56:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5594741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlease see attached Supplementary Materials\u003c/p\u003e","description":"","filename":"SupportingInformationV12finalgenerictemplate.docx","url":"https://assets-eu.researchsquare.com/files/rs-6073214/v1/14c7c985c589a210498e36c7.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A bioinformatics approach to the design of minimal biomimetic metal-binding peptides","fulltext":[{"header":"Teaser","content":"\u003cp\u003eA minimal peptide, designed via bioinformatics, effectively mimics the trinuclear copper site in laccase for O₂ reduction.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe growing demand for sustainable energy, coupled with the climate crisis, motivates the development of efficient devices for storing and producing renewable fuels and chemicals. Nature has taken advantage of billions of years of evolution to carefully optimize enzyme structures for catalyzing specific chemical reactions. Nature\u0026rsquo;s repertoire of enzyme functions is coupled with the fundamental ability to accept a variety of different substrates \u0026ndash; a promiscuity that is at the base of evolution (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Metalloenzymes offer a rich library of models for studying and developing artificial catalysts able to drive reactions crucial to the renewable energy transition (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The Protein Data Bank (PDB) is the leading global repository for proteins, nucleic acids, carbohydrates, and other experimentally determined biological complexes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Recent advancements in metalloenzymes research have shown that the residues in close proximity to the metal-binding site are the main determinants in driving catalytic reaction bias and selectivity (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In this context, mini-proteins and biomimetic peptide design have emerged as valuable tools to investigate how the properties and configuration of binding residues influence metal selectivity and reactivity (\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The appeal of using short peptides as scaffolds to build efficient catalysts lies in their versatility to achieve high catalytic activity rivaling natural enzymes while possessing higher thermal and pH stability (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Additionally, minimal peptides are more amenable towards interfacial electron transfer on electrodes due to their smaller cross-section, thereby achieving higher surface coverage on electrode surfaces. Moreover, short peptides can be easily synthesized in large amounts and at low cost, thanks to the advanced techniques and well-established methods available in solid-phase peptide synthesis (SPPS) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith this in mind, we have chosen to use natural metalloenzymes as models to design short peptide ligands as a scaffold for supporting the self-assembly of biomimetic catalysts. Focusing on the first coordination sphere around the catalytic cofactor of target metalloenzymes, our goal is to identify the shortest peptide sequence able to bind metals and mimic the model\u0026rsquo;s site activity. In this regard, the concept of \u0026ldquo;minimal functional site\u0026rdquo; (MFS) developed by Andreini \u003cem\u003eet al.\u003c/em\u003e is a good starting point for the design, as it describes the minimal environment determining the metal\u0026rsquo;s chemical behavior (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). MFSs represent the local three-dimensional environment including all residues within 5 \u0026Aring; distance from any metal-binding ligand, providing a tool for classifying and comparing enzymatic metal sites. Bioinformatic tools can easily extract MFSs information, enabling their manipulation and driving the design of new biomimetic molecules. The potential of bioinformatic approaches for designing bioactive peptides and molecules is well-established, with examples in drug design, vaccine development, and catalysis (\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe have thus created a new tool, MetalSite-Analyzer (MeSA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metalsite-analyzer.cerm.unifi.it/\u003c/span\u003e\u003cspan address=\"https://metalsite-analyzer.cerm.unifi.it/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), that enables users to extract relevant sequence motifs for binding a metal of choice. The tool leverages MFS sequence alignments to obtain information on the most conserved residues in metal sites belonging to the protein family of interest. To demonstrate the value of this approach, we have designed a minimal eight-residues peptide, using as a model the trinuclear copper site of laccase. This well-known enzyme was chosen given our interest in redox catalysis. The design of the minimal peptide was based both on sequence conservation analysis, as dictated by MeSA, and on structural modification, led by rational observation of the enzyme\u0026rsquo;s three-dimensional structure. Our minimal peptide, which we refer to as H4pep, was synthesized via solid-phase peptide synthesis and purified via preparative HPLC. The ability of H4pep to coordinate copper ions was verified via different spectroscopic methods. Notably, the CD data demonstrate that H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e complexes adopt a beta-sheet conformation as it is indeed observed in the MFS of laccase. We have then determined the electrochemical signatures of the Cu\u003csup\u003e2+\u003c/sup\u003e(H4pep)\u003csub\u003e2\u003c/sub\u003e complex, to assess its potential use in electrocatalysis. In line with the model enzyme, the complex has revealed catalytic electrochemical O\u003csub\u003e2\u003c/sub\u003e reduction. Furthermore, spectrophotometric laccase activity assays showed the ability of the Cu\u003csup\u003e2+\u003c/sup\u003e(H4pep)\u003csub\u003e2\u003c/sub\u003e complex to oxidize the 2,2\u0026rsquo;-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) substrate in presence of O\u003csub\u003e2\u003c/sub\u003e. Our data demonstrate that, despite its beta-sheet conformation, Cu\u003csup\u003e2+\u003c/sup\u003e(H4pep)\u003csub\u003e2\u003c/sub\u003e does not undergo aggregation, such that we did not detect the formation of fibrils. To the best of our knowledge, this is the first example of a synthetic β-sheet metallo-peptide complex that is stable in solution and features catalytic activity. These findings demonstrate that the MetalSite-Analyzer tool can provide relevant indications for designing bioinspired catalytically active metallo-peptides.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic tool and peptide design\u003c/h2\u003e \u003cp\u003eWe have created the MeSA tool with the scope of expanding the MFS concept to include sequence conservation analysis. Starting from an input PDB structure, MeSA allows the selection of user-defined mono-/multinuclear metal sites and, from the extracted MFS, runs a PSI-BLAST search of the metal-binding sequence fragments (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This step consists of mapping and aligning the binding fragments to all related sequences contained in Uniprot, the world-leading protein sequence database (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The output information allows for the analysis of the conservation of the residues in each specific position of the starting MFS sequence. This provides a basis for further rational design of the desired peptide mimics, by highlighting completely conserved residues, which are presumably strictly necessary for function, moderately variable positions, where one of two/three different amino acids can be selected (e.g. based on considerations of stability or ease of synthesis), and highly variable positions, where almost any amino acid can be introduced. The tool has been implemented as a user-friendly web server, requiring no registration (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metalsite-analyzer.cerm.unifi.it/\u003c/span\u003e\u003cspan address=\"https://metalsite-analyzer.cerm.unifi.it/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo assess our methodological approach, we have selected laccase as a model to design a minimal-length peptide ligand for binding copper ions. Laccases belong to the family of multicopper oxidases, which catalyze one-electron oxidation of different organic substrates, coupling it with the four-electron reduction of O\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In these enzymes, substrate oxidation occurs at a mononuclear copper site (type 1), followed by electron transfer to a trinuclear copper cluster (type 2/type 3), where oxygen reduction occurs. The latter is the site we have selected to test our bioinformatic tool.\u003c/p\u003e \u003cp\u003eFirstly, we accessed the MetalPDB database (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) to gain information on the binding site(s) of the small laccase from \u003cem\u003eStreptomyces viridosporus\u003c/em\u003e (PDB ID: 3tbc). In bacterial small laccases, so called because they have fewer domains than fungus laccase, the trinuclear Cu sites are located at the interface between monomers, in a three-fold symmetry. As in a typical multicopper oxidase, they consist of eight histidine residues coordinating the metals, in a ligand-nonligand-ligand motif (LXL) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In the apo-form of the enzyme, the monomer exposes the coordinating residues to the surrounding environment, making it potentially accessible to solvent and substrate molecules involved in catalysis. This makes the site particularly attractive to inspire the design of artificial biomimetic peptide catalysts. Feeding the pdb structure as input to MeSA and selecting the trinuclear copper site, the tool was able to extract four fragments contributing to the coordination of the Cu ions with two histidine residues each (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The conservation of the residues in each fragment was analyzed via the PSI-BLAST algorithm. As a result, four alignments of metal-binding motifs were generated. Finally, a sequence profile (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) was obtained for each fragment composing the starting site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn our endeavor to obtain a minimal-length peptide mimicking the laccase site\u0026rsquo;s activity, further rational design steps are needed to implement the information obtained from MeSA. On a closer look at the structure of the site, we have noticed that the binding His residues belong two by two to antiparallel β-sheets, located on adjacent monomers in a C2 symmetry with respect to the axis running across the Cu atoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The four metal-binding fragments identified by our bioinformatic search thus represent the four β-strands composing this structural motif. In particular, two of the four fragments extracted by the bioinformatic search are considerably shorter (7\u0026ndash;9 residues for fragment 1 of chain A and B versus 11\u0026ndash;13 for fragment 2 of chain A and B, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), thus more suited for our purpose of designing a minimal-length peptide. Owing to their similarity, we have decided to inspire the design of our short peptide from a single fragment of the site. Hence, for the final sequence, the choice of the amino acid residues in each position was driven by the combination of the consensus sequences of these two fragments, with further modifications led by the rational analysis of the model site (See \u003cem\u003eMaterial and Methods\u003c/em\u003e for details). The final minimal-length peptide ligand is eight residues long and was named H4pep (sequence: HTVHYHGH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBinding of metal ions by H4pep\u003c/h3\u003e\n\u003cp\u003eH4pep was synthesized via SPPS and purified via reverse-phase high-pressure liquid chromatography (RP-HPLC). To verify the consistency of our design strategy, we preliminarily carried out UV-visible and NMR experiments to confirm the ability of H4pep to bind copper ions. Such experiments were performed at pH 5.6 to prevent copper oxide formation and N-terminal amine deprotonation (\u003cem\u003evide infra\u003c/em\u003e), still favoring imidazole deprotonation and binding (pKa\u0026thinsp;~\u0026thinsp;6) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUV-visible spectra of H4pep display the characteristic peak of tyrosine at 276 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Upon gradual copper(II) addition, the characteristic band of Cu\u003csup\u003e2+\u003c/sup\u003e d-d transitions arises in the region between 500 and 800 nm. This feature is in good agreement with reported λ\u003csub\u003emax\u003c/sub\u003e values for histidine coordination in peptide-copper complexes (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Notably, with copper concentration up to 1 equivalent, this band could be fitted with two Gaussian functions, suggesting the contribution of multiple species to the absorbance profile (See \u003cem\u003eStochiometry of H4pep-Cu\u003c/em\u003e\u003csup\u003e\u003cem\u003e2+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ecomplexes\u003c/em\u003e section for details). In excess of copper, the absorption peak of unbound copper(II), with maxima at 786.7 nm, becomes noticeable and increasingly contributes to the observed spectrum.\u003c/p\u003e \u003cp\u003eBinding of copper(I) was also tested and the affinity of H4pep to Cu\u003csup\u003e+\u003c/sup\u003e ions was determined via competitive titration with bicinchoninic acid (BCA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The absorption changes at 562 nm, corresponding to the formation of [Cu(BCA)\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e3\u0026minus;\u003c/sup\u003e, could be fitted satisfactorily assuming a single class of binding sites with K\u003csub\u003eD\u003c/sub\u003e = 2.4*10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e M. This affinity is comparable to the values found for a series of nitrite reductase mimics featuring a Cu(His)\u003csub\u003e3\u003c/sub\u003e site bound to a triple-stranded α-helical coiled-coil (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo further investigate the coordination of metal ions to H4pep, the peptide\u0026rsquo;s structure was analyzed by NMR spectroscopy under different experimental conditions. In aqueous solution, the apo-peptide showed only four very broad amide signals of the nine expected (seven backbone and two C-terminal amide protons) due to fast exchange with the solvent, indicating the absence of stable hydrogen bonds and secondary structure elements (Fig. S2A and S3A). In methanol, while amide resonances became observable, their chemical shift dispersion remained limited, suggesting lack of defined tertiary structure in the apo form (Fig. S3B and S4A). The addition of Zn\u003csup\u003e2+\u003c/sup\u003e, which was used as a diamagnetic probe for the paramagnetic Cu\u003csup\u003e2+\u003c/sup\u003e ion, in a 1:1 ratio induced selective changes in the histidine resonances, with downfield shifts (\u0026sim;0.1 ppm) of the proton signals belonging to the Cδ and Cε of the imidazole ring (Fig. S2B). The broadening of these resonances suggests a chemical exchange process, widely observed in NMR analysis, which primarily involves the histidine residues (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This spectral behavior suggests a dynamic equilibrium between multiple zinc-bound species, where either one or two Zn\u0026sup2;⁺ ions are alternately coordinated to the two different available binding sites in the peptide scaffold. More dramatic spectral changes were observed upon addition of Cu\u003csup\u003e+\u003c/sup\u003e (at H4pep:Cu\u003csup\u003e+\u003c/sup\u003e ratios of 1:1 and 2:1) under anaerobic conditions both in aqueous buffer (Fig. S2C and S5) and methanol (Fig. S4B and S6). All resonances significantly broadened, suggesting that Cu\u003csup\u003e+\u003c/sup\u003e binding affects the overall peptide conformation more extensively than Zn\u003csup\u003e2+\u003c/sup\u003e, leading to multiple conformational states in exchange. The spectral broadening persisted in the presence of sodium dithionite, excluding contributions from paramagnetic Cu\u003csup\u003e2+\u003c/sup\u003e species. From the above results we can conclude that H4pep binds both Zn\u003csup\u003e2+\u003c/sup\u003e and Cu\u003csup\u003e+\u003c/sup\u003e ions in solution; binding of Cu\u003csup\u003e2+\u003c/sup\u003e was demonstrated by the UV-visible spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, the latter UV-visible data along with the NMR spectrum of H4pep-Zn\u003csup\u003e2+\u003c/sup\u003e indicate that binding occurs via the histidine side chains.\u003c/p\u003e\n\u003ch3\u003eSecondary structure and pH dependence of metal binding\u003c/h3\u003e\n\u003cp\u003eAs anticipated by NMR, CD spectroscopy of H4pep in the far-UV range (190\u0026ndash;250 nm) reveals only a deep band at 198 nm indicative of a random coil for the apo-peptide in buffer solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Based on our peptide design strategy, we can reasonably anticipate that binding of Cu\u003csup\u003e2+\u003c/sup\u003e to H4pep will induce the formation of a β-sheet structural motif. Thus, we expect to observe spectral changes characteristic of β-sheet conformation when H4pep is titrated with Cu\u003csup\u003e2+\u003c/sup\u003e. In line with our design and the previous NMR analysis, we expect metal binding to occur via histidine side chain coordination rather than backbone amide coordination, previously reported for 3-residues-Cu systems (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Coordination of copper ions via histidine side chains can be further differentiated from backbone amide coordination as histidine side chain coordination will be strongly affected by pH (histidine side chain has pKa\u0026thinsp;~\u0026thinsp;6). CD spectra recorded at different pHs allowed us to test Cu binding ability under different buffer conditions. H4pep was dissolved in acetate buffer solutions at pH 4.4, 4.8, 5.2, and 5.6 respectively (Fig. S7). Higher pH values are excluded to avoid the formation of copper oxide species, limiting Cu\u003csup\u003e2+\u003c/sup\u003e availability to the peptide ligand, and to mitigate terminal amine binding (pKa ⁓7\u0026ndash;8). No significant changes were observed in the CD spectra at pH 4.4 and 4.8 in presence of Cu\u003csup\u003e2+\u003c/sup\u003e, indicating unfavorable binding conditions due to the protonated state of the histidine ligands. In contrast, at pH 5.2 and 5.6, a clear change in conformation is visible. Therefore, pH 5.6 buffer conditions were selected to record the titration spectra in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Upon Cu\u003csup\u003e2+\u003c/sup\u003e addition, the CD spectrum displays a negative band at 227 nm and a positive one at 210 nm. This suggests that Cu\u003csup\u003e2+\u003c/sup\u003e binding to H4pep induces a β-sheet conformation, consistent with our initial design. These features saturate upon the addition of 0.5 Cu\u003csup\u003e2+\u003c/sup\u003e equivalents and remain essentially unchanged until a 1:1 H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratio. As additional copper equivalents are introduced, the spectrum undergoes further changes, with the positive feature increasing and shifting to 205 nm. This suggests the formation of another species, favored at high copper concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe same trend was observed in the visible range of the CD spectra, in which changes in the coordination environment around the metal ion(s) can be monitored (Fig. S8A). Titration of up to 1 equivalent of Cu\u003csup\u003e2+\u003c/sup\u003e results in the gradual increase of a positive peak centered at 719 nm. At higher Cu\u003csup\u003e2+\u003c/sup\u003e equivalents, the peak position shifts to the red, suggesting the formation of a second H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e species.\u003c/p\u003e \u003cp\u003eTitration of Cu\u003csup\u003e+\u003c/sup\u003e to H4pep results in similar changes in the UV region of the CD spectrum (Fig. S8B). There are no observable features in the visible region of the same CD spectrum, consistent with the lack of d-d transitions in Cu\u003csup\u003e+\u003c/sup\u003e complexes. This confirms that the observed changes in the UV region of the CD spectrum are associated with the coordination of Cu\u003csup\u003e+\u003c/sup\u003e ions to H4pep to form H4pep-Cu\u003csup\u003e+\u003c/sup\u003e complexes.\u003c/p\u003e\n\u003ch3\u003eTitration experiments via EPR\u003c/h3\u003e\n\u003cp\u003eEPR spectroscopy was employed to further analyze copper(II) binding and to elucidate the presence of different H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e species. Titration of H4pep into a Cu\u003csup\u003e2+\u003c/sup\u003e solution revealed the presence of at least two distinct EPR active species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Under a large excess of peptide (e.g. H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratio 1:0.3), a single axial signal is observed, with values of \u003cem\u003eg\u003c/em\u003e\u003csub\u003e\u003cem\u003ex\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eg\u003c/em\u003e\u003csub\u003e\u003cem\u003ey\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eg\u003c/em\u003e\u003csub\u003e\u003cem\u003ez\u003c/em\u003e\u003c/sub\u003e respectively at 2.0419, 2.0737, and 2.2447 (Fig. S9). At higher Cu\u003csup\u003e2+\u003c/sup\u003e equivalents (between H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratios 1:0.4 to 1:1) an additional axial signal characterized by lower g values could be detected. Presumably, the coordination of an additional Cu\u003csup\u003e2+\u003c/sup\u003e ion into the peptide can cause a small distortion to the local environment of the first Cu\u003csup\u003e2+\u003c/sup\u003e, resulting in a shifted EPR spectrum, which could be isolated by spectral deconvolution (Fig. S10). In this configuration, the two Cu\u003csup\u003e2+\u003c/sup\u003e binding sites are essentially identical, as only one axial Cu\u003csup\u003e2+\u003c/sup\u003e signal is observed. The presence of two distinct EPR signals at different H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratios is consistent with CD spectroscopy results, indicating that two H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e species are formed. Furthermore, above H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratio of 1:1, an EPR signal associated with unbound Cu\u003csup\u003e2+\u003c/sup\u003e becomes observable.\u003c/p\u003e\n\u003ch3\u003eStoichiometry of H4pep-Cu complexes\u003c/h3\u003e\n\u003cp\u003eThe CD and EPR spectra obtained from titrations between Cu\u003csup\u003e2+\u003c/sup\u003e and H4pep were deconvoluted by fitting each spectrum with their respective isolated EPR signals or CD spectra and presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. The observation of unbound Cu\u003csup\u003e2+\u003c/sup\u003e species in the EPR spectra above H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratio of 1:1, suggests that each peptide can bind a maximum of 1 Cu\u003csup\u003e2+\u003c/sup\u003e equivalent. The formation of a β-sheet conformation upon Cu\u003csup\u003e2+\u003c/sup\u003e binding, as evident from the CD spectra, indicates that at least two peptide units are required to form a Cu\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ex\u003c/sub\u003e(H4pep)\u003csub\u003ey\u003c/sub\u003e complex (where y\u0026thinsp;\u0026ge;\u0026thinsp;2). It is unlikely that the minimum number of peptides to form a Cu\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ex\u003c/sub\u003e(H4pep)\u003csub\u003ey\u003c/sub\u003e is three, since we would expect most of the H4pep to exist in a β-sheet conformation at H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratio of 1:0.33. On the contrary, our experimental observation led to a fitted molar fraction of peptide in random coil conformation of around 0.5 at 1:0.33 H4pep:Cu\u003csup\u003e2+\u003c/sup\u003e ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Therefore, our hypothesis is that a Cu\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ex\u003c/sub\u003e(H\u003csub\u003e4\u003c/sub\u003epep)\u003csub\u003e2\u003c/sub\u003e complex is formed upon addition of Cu\u003csup\u003e2+\u003c/sup\u003e ions to H4pep. In this scenario, there are two possible species corresponding to Cu\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003e1\u003c/sub\u003e(H\u003csub\u003e4\u003c/sub\u003epep)\u003csub\u003e2\u003c/sub\u003e and Cu\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003e2\u003c/sub\u003e(H\u003csub\u003e4\u003c/sub\u003epep)\u003csub\u003e2\u003c/sub\u003e, designated as \u003cb\u003e1Cu2Pep\u003c/b\u003e and \u003cb\u003e2Cu2Pep\u003c/b\u003e respectively. Deconvolution of UV-visible d-d band spectra could be obtained accordingly (Fig. S11). Fitting of the spectra up to 1 Cu equivalents was possible considering two Gaussian components, representing \u003cb\u003e1Cu2Pep\u003c/b\u003e and \u003cb\u003e2Cu2Pep\u003c/b\u003e. At higher Cu\u003csup\u003e2+\u003c/sup\u003e concentrations, a third component, arising from free Cu\u003csup\u003e2+\u003c/sup\u003e in solution needed to be included for accurate fitting.\u003c/p\u003e \u003cp\u003eObserving the resulting trends, it is clear how the isolation of a single Cu\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ex\u003c/sub\u003e(H4pep)\u003csub\u003e2\u003c/sub\u003e species is far from trivial. The \u003cb\u003e1Cu2Pep\u003c/b\u003e species seems to be favored at low Cu\u003csup\u003e2+\u003c/sup\u003e concentrations, to 0.5 equivalents, while both \u003cb\u003e1Cu2Pep\u003c/b\u003e and \u003cb\u003e2Cu2Pep\u003c/b\u003e species are present in similar quantities until 1 Cu\u003csup\u003e2+\u003c/sup\u003e equivalent. The \u003cb\u003e2Cu2Pep\u003c/b\u003e species is favored at higher Cu\u003csup\u003e2+\u003c/sup\u003e concentrations, but an increasing amount of free Cu\u003csup\u003e2+\u003c/sup\u003e ions is detected in these conditions. Notably, even in large excess of Cu\u003csup\u003e2+\u003c/sup\u003e, dynamic light scattering (DLS) experiments revealed the absence of large aggregates, confirming the molecular nature of the complexes (Fig. S12).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElectrochemistry and testing of activity\u003c/h2\u003e \u003cp\u003eTo examine if \u003cb\u003e1Cu2Pep\u003c/b\u003e and \u003cb\u003e2Cu2Pep\u003c/b\u003e exhibit catalytic O\u003csub\u003e2\u003c/sub\u003e reduction activity, as in the original laccase, a preliminary electrochemical characterization of these two complexes by cyclic voltammetry was performed (Fig. S13A). Cyclic voltammograms of \u003cb\u003e2Cu2Pep\u003c/b\u003e under N\u003csub\u003e2\u003c/sub\u003e atmosphere showed a reduction peak at -0.11 V against Ag/AgCl reference electrode and a corresponding re-oxidation wave at 0.52 V. The large peak-to-peak separation suggests a slow heterogeneous electron transfer rate between the glassy carbon working electrode and the complex. Notably, during the anodic scan, a sharp reoxidation peak at 0.08 V is observed, characteristic of oxidation of metallic Cu deposited on the working electrode. This suggests that during the reduction of \u003cb\u003e2Cu2Pep\u003c/b\u003e, the copper binding affinity of at least one of the binding sites becomes weaker, resulting in the loss of copper from the complex. Since metallic Cu is also known to participate in electrocatalytic O\u003csub\u003e2\u003c/sub\u003e reduction, we will not examine the O\u003csub\u003e2\u003c/sub\u003e reduction activity of \u003cb\u003e2Cu2Pep\u003c/b\u003e as it is not trivial to verify its catalytic O\u003csub\u003e2\u003c/sub\u003e reduction activity in presence of metallic Cu.\u003c/p\u003e \u003cp\u003eOn the other hand, cyclic voltammograms of \u003cb\u003e1Cu2Pep\u003c/b\u003e show a reduction peak at -0.16 V and the corresponding reoxidation peak at 0.36 V under N\u003csub\u003e2\u003c/sub\u003e atmosphere. Importantly, features associated with the deposition of metallic Cu and its subsequent reoxidation are not observable. This is further verified by rinse test experiments after CV scans of \u003cb\u003e1Cu2Pep\u003c/b\u003e (Fig. S13B). Under O\u003csub\u003e2\u003c/sub\u003e atmosphere, a current enhancement is observable at around \u0026minus;\u0026thinsp;0.20 V, suggesting catalytic O\u003csub\u003e2\u003c/sub\u003e reduction activity (Fig. S13C). To verify this behavior, bulk electrolysis experiments were performed in a custom-designed cell that incorporates a Clark electrode for sensing of O\u003csub\u003e2\u003c/sub\u003e concentration. Chronoamperometry experiments at -0.20 V result in a steady-state current of approximately \u0026minus;\u0026thinsp;15 \u0026micro;A with a concomitant decrease in O\u003csub\u003e2\u003c/sub\u003e concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The estimated faradaic efficiency in reference to the moles of O\u003csub\u003e2\u003c/sub\u003e consumed is 62%, assuming that all observed current is due to four-electron of O\u003csub\u003e2\u003c/sub\u003e to water. The low faradaic efficiency maybe the result of side reaction involving two-electron reduction of O\u003csub\u003e2\u003c/sub\u003e to hydrogen peroxide, which cannot be excluded at present. Cyclic voltammetry of the \u003cb\u003e1Cu2Pep\u003c/b\u003e solution after bulk electrolysis does not show significant deviation from that recorded prior to bulk electrolysis, with no indication of Cu\u003csup\u003e0\u003c/sup\u003e plated on the electrode surface (Fig. S13D). Furthermore, additional rinse test experiments show a reductive current compared to the background during chronoamperometry and no observable features in the corresponding cyclic voltammogram (Fig. S13E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further rule out the participation of unbound copper species during catalytic O\u003csub\u003e2\u003c/sub\u003e reduction, negative control experiments were performed with dilute solutions of unbound Cu\u003csup\u003e2+\u003c/sup\u003e (0.03 mM, 7% of Cu\u003csup\u003e2+\u003c/sup\u003e content in \u003cb\u003e1Cu2Pep\u003c/b\u003e). Chronoamperometry of \u003cb\u003e1Cu2Pep\u003c/b\u003e shows a higher current compared to 7% Cu\u003csup\u003e2+\u003c/sup\u003e (Fig. S14A), indicating only a minimal, if any, contribution of free copper(II) ions to the catalytic activity. Moreover, rinse test of the unbound Cu\u003csup\u003e2+\u003c/sup\u003e solution shows significant reoxidation of metallic copper species at 0.09 V, not observed for the \u003cb\u003e1Cu2Pep\u003c/b\u003e species (Fig. S14B). Therefore, the O\u003csub\u003e2\u003c/sub\u003e reduction activity in the conditions above is likely to originate from \u003cb\u003e1Cu2Pep.\u003c/b\u003e We have then tested the performances of \u003cb\u003e1Cu2Pep\u003c/b\u003e at a more reducing potential (-0.30 V). In this case, an interesting behavior of the chronoamperometry signal was observed: while the recorded current decreases in the first 120 seconds, it then reaches a plateau and starts increasing after 150 seconds (Fig. S15A). No signals of unbound Cu\u003csup\u003e2+\u003c/sup\u003e could be detected in the cyclic voltammogram and rinse test performed after the measurement (Fig. S15B). This result seems to indicate that a more active species of the H4pep:Cu assembly can be formed in these conditions.\u003c/p\u003e \u003cp\u003eFinally, laccase activity of \u003cb\u003e1Cu2Pep\u003c/b\u003e was tested via spectrophotometric assay, monitoring the increase in absorbance at 420 nm characteristic of ABTS substrate oxidation (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In O\u003csub\u003e2\u003c/sub\u003e saturated atmosphere, the activity was estimated to be 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 U L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The reported activity of natural laccase enzymes ranges from 3.5 to 484,000 U L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The value of laccase activity obtained for \u003cb\u003e1Cu2Pep\u003c/b\u003e, although modest, is in agreement with our electrocatalytic activity results, demonstrating the ability of the complex to perform moderate electrocatalytic O\u003csub\u003e2\u003c/sub\u003e reduction.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDesigning peptide mimics of enzymatic metal-binding sites is a challenging task. Our approach demonstrates that bioinformatic tools can support the rational design of such mimics via analysis of sequence conservation. Short, tunable peptide ligands represent promising candidates for applying this strategy. Notable examples are minimal Cu-peptide complexes, which are already widely investigated since histidine-containing binding sites are found in several metalloproteins (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In these smaller systems, metal coordination often involves backbone amide and terminal amine groups. In contrast, our H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e complexes feature exclusive histidine coordination, as confirmed by the resonance shifts and broadening of the NMR signals of imidazole protons upon metal binding, in line with our model design (Figs. S2C and S4B).\u003c/p\u003e \u003cp\u003eBased on the analysis of the NMR and EPR data, we propose a rationale for the formation of the observed \u003cb\u003e1Cu2Pep\u003c/b\u003e and \u003cb\u003e2Cu2Pep\u003c/b\u003e species (Fig. S16). At low copper concentrations, we hypothesize that Cu\u003csup\u003e2+\u003c/sup\u003e preferentially binds to a specific site, where the metal atom is bound to two peptide strands (\u003cb\u003e1Cu2Pep\u003c/b\u003e species). Assuming that the peptide strands associate in an antiparallel β-sheet structure, two symmetrically equivalent binding sites can be identified. In this scenario, although only four out of eight available histidine residues coordinate Cu\u003csup\u003e2+\u003c/sup\u003e at a given time, all of them (His0, His3, His5, His7) are effectively involved in metal binding via conformational exchange to the symmetrically equivalent species. This is reflected in the observed simultaneous broadening of all histidine\u0026rsquo;s proton resonances in the NMR experiments. On the other hand, a single axial Cu\u003csup\u003e2+\u003c/sup\u003e signal is observed in EPR experiments, consistent with two symmetrically equivalent binding sites. At higher copper concentrations, coordination of a second Cu\u003csup\u003e2+\u003c/sup\u003e can occur, filling the second identical binding site. However, this leads to a small distortion of both binding sites. The resulting \u003cb\u003e2Cu2Pep\u003c/b\u003e species features two symmetrically identical binding sites, each differing slightly from the original site in the \u003cb\u003e1Cu2Pep\u003c/b\u003e species. Therefore, the EPR signature of \u003cb\u003e2Cu2Pep\u003c/b\u003e displays a single axial copper signal, only slightly shifted compared to that of the \u003cb\u003e1Cu2Pep\u003c/b\u003e species, as the two Cu\u003csup\u003e2+\u003c/sup\u003e ions are not magnetically coupled and are virtually identical (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The NMR spectra in a 1:1 H4pep:Cu\u003csup\u003e+\u003c/sup\u003e ratio also show no significant differences compared to the 2:1 H4pep:Cu\u003csup\u003e+\u003c/sup\u003e ratio, supporting our interpretation (Figs. S5 and S6). Notably, \u003cb\u003e1Cu2Pep\u003c/b\u003e shows moderate O\u003csub\u003e2\u003c/sub\u003e reduction activity, thus demonstrating the potential of our approach toward designing functional mimics of enzyme active sites.\u003c/p\u003e \u003cp\u003eH4pep is an example of how even extremely short peptide ligands can provide versatile scaffolds for metal binding and facilitate the study of structure-function relationships. Different three-dimensional motifs have been employed for this purpose, with α-helical structures being the leading example (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Only recently, metallo-β-sheets peptides have been considered as scaffolds for catalytic site mimics, with just a handful of examples (\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). CD data of our H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e complexes indicate a metal-induced conformational change to a β-sheet motif, offering a new opportunity to study this structural motif in the context of biomimetic catalysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Work from different groups has demonstrated how β-sheet motifs tend to self-assemble into supramolecular structures and form insoluble aggregates (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Gaining a deeper understanding of the factors that promote or prevent aggregation is crucial for utilizing these structural motifs across various research fields. For instance, amyloid-β aggregates are currently intensively studied, as they play a role in the rise of neurodegenerative disorders such as Alzheimer\u0026rsquo;s disease (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Notably, DLS studies of our H4pep-Cu\u003csup\u003e2+\u003c/sup\u003e complexes have revealed the absence of larger assemblies (Fig. S12), confirming the molecular nature of the complexes and making them particularly suitable for the study of functional minimal active site mimics. Our success in reproducing a molecular metallo-β-sheet motif via bioinformatic and structural design, provides a step forward in understanding the sequence-structure relationship of this conformation.\u003c/p\u003e \u003cp\u003eWe anticipate that further targeted modifications of the amino acid sequence in our H4pep could lead to the assembly of different species than \u003cb\u003e1Cu2Pep\u003c/b\u003e and \u003cb\u003e2Cu2Pep\u003c/b\u003e. Further efforts could focus on designing peptide sequences that self-assemble into a single, well-defined metallo-peptide complex. In this context, more rigid motifs, such as β-hairpins and WW domains, have recently been proposed as more stable architectures for achieving this goal (\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). However, recent work from Dang \u003cem\u003eet al.\u003c/em\u003e has demonstrated that β-hairpin structures are particularly affected by metal binding, which can cause a rearrangement of the β-sheet domains (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Nonetheless, mutating just two amino acids in the sequence resulted in a completely different metal-peptide assembly mechanism, underlining the critical importance of sequence analysis in designing selective and stable binding sites.\u003c/p\u003e \u003cp\u003eIn conclusion, there is still unexplored potential in β-sheet-based and other scaffold designs. Although \u003cb\u003e1Cu2Pep\u003c/b\u003e exhibits only modest activity, it demonstrates that our minimal peptide design strategy is a viable approach to mimic catalytic sites of redox-active enzymes. While most bioinformatics methods, such as Rosetta and Alphafold (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), emphasize the design and understanding of large proteins, our Metal-Site Analyzer platform serves as a developing tool optimized for minimal, easily tunable peptide ligands. Our work further highlights the versatility of short peptide-metal assemblies in exploring the sequence-structure relationships of simple metal-binding motifs and their potential applications in designing stable, biomimetic catalysts.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and buffers\u003c/h2\u003e \u003cp\u003eAll chemicals and reagents were of analytical grade and used without further purification. Fmoc-amino acids and Oxyma pure were purchased from Novabiochem (Sigma-Aldrich Sweden). Dimethylformamide (DMF), 20% piperidine, N,N\u0026prime;-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), triisopropylsilane (TIS), diethyl ether, acetonitrile (ACN), copper(II) sulfate pentahydrate, sodium dihydrogen phosphate, sodium hydroxide (NaOH), sodium acetate, acetic acid, sodium sulfate (NaSO\u003csub\u003e4\u003c/sub\u003e) and 2,2\u0026rsquo;-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich Sweden. Buffers were prepared starting from sodium dihydrogen phosphate and adjusting the pH with NaOH, or mixing sodium acetate and acetic acid to the desired pH. Cu\u003csup\u003e2+\u003c/sup\u003e stock solutions were obtained by dissolving copper sulfate salt in distilled water or buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic tool\u003c/h2\u003e \u003cp\u003eMetal-Site Analyzer, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metalsite-analyzer.cerm.unifi.it/\u003c/span\u003e\u003cspan address=\"https://metalsite-analyzer.cerm.unifi.it/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, was designed and implemented with the following characteristics. The input consists of a pdb code, or a protein structure in pdb format (e.g., in case the structure is not yet deposited in the PDB). It is also possible to specify the distance threshold that will be used to identify the coordinating atoms to the metal ion(s) and the residues directly involved in the metal binding. If the structure contains a multinuclear site, the user can choose whether to analyze a single metal ion in the site or investigate the entire multinuclear site. In the latter case, it is required to flag the \"Aggregate multinuclear site info\" option. Then, by clicking on \"Search for metals\", the tool extracts the list of metal sites in the structure. If the user prefers to limit the search to a specific metal ion, this can be specified in the \u0026ldquo;Enter a Cofactor\u0026rdquo; field on the home page of the tool. The second web page displays the list of the sites found in the input structure. The user must select only one site of interest. The \"GO!\" button will then initiate the site analysis through the steps listed below:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe tool extracts the \u0026ldquo;minimal functional site\u0026rdquo; (MFS) from the structure (\u003cem\u003e14\u003c/em\u003e); that is, i) the metal(s), ii) the metal-binding residues, and iii) the residues that fall within 5 \u0026Aring; from at least one atom of the metal-binding residues. Usually, the MFS is composed of more than one fragment (\u003cem\u003emetal-binding fragments\u003c/em\u003e hereafter), because the metal site is not continuous in sequence.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe residues identified at points ii) and iii) are mapped onto the sequence of the structure.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe tool extracts the segment of sequence(s) that contains the entire MFS (from the most N-terminal residue to the most C-terminal residue) (\u003cem\u003emetal-binding segment\u003c/em\u003e hereafter).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe metal-binding segment is searched in UniRef50 by using PSI-BLAST (3 iterations) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe output sequences are filtered to discard those not containing the metal-binding residues of the input site (as they will most likely not bind metal ion(s)).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eA multiple sequence alignment is generated from the pairwise sequence alignments of the PSI-BLAST output.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe metal-binding fragments of the input site are mapped and extracted from the multiple sequence alignment.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eAt the end of the site analysis, the web server will output the metal-binding fragments alignments and the respective Skylign logos (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The latter may be used to identify highly conserved residues, which may have a functional role in the metal site and thus be relevant to design purposes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePeptide design\u003c/h2\u003e \u003cp\u003eThe design of the synthetic peptide was based on the small laccase from \u003cem\u003eStreptomyces viridosporus\u003c/em\u003e with PDB code 3tbc. The PDB structure was input to MeSA with no additional constraints. The option to aggregate multinuclear site information was chosen, to be able to include the analysis of the trinuclear copper site of interest. The tool individuated three mononuclear type 1 copper sites, respectively belonging to the three monomers that compose the protein (chain A, B, and C), and three trinuclear sites, located at the interface between monomers. All sites are virtually identical, as the monomer unit repeats with a three-fold symmetry in the functional protein structure. By selecting one of the trinuclear MFS, four fragments were extracted, two belonging to one chain (TFHLHGH and WMYHCHVQSHS) and two to another (SLHVHGLDY and WHYHDHVVGTEHG). By looking at the three-dimensional structure of the MFS, we noticed that fragments on the same chain are part of adjacent beta-strands located at the interface of one monomer, while the other fragments (on the second chain) bind the Cu ions from the opposite monomer interface, in a similar beta-sheet conformation. The binding motif His-Xxx-His is highly conserved in all four fragments, as suggested by the MeSA output. For our purpose of designing a minimal-length peptide to mimic the laccase binding site, we have focused on the sequence conservation analysis of the shortest fragments, respectively TFHLHGH belonging to one chain and SLHVHGLDY belonging to the other. The highly conserved binding histidine residues were selected for positions 3 and 5, as well as the glycine in position 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A third histidine was assigned to position 7, as it is fairly conserved and capable of also participating in metal binding. Flexible threonine was chosen versus the more rigid proline and assigned to position 1, as the third and second most conserved residue in the two fragments, respectively. The other positions seemed to be less conserved among the fragments and were assigned based on the analysis of the three-dimensional structure of the model site. Tyrosine was strategically included in the sequence as it can be used as UV-visible and redox probe to validate other experiments. The final sequence HTVHYHGH is an 8-residues long peptide representing the shortest fragment mimicking the model site and containing the conserved binding motif. It is important to note that in the MFS of laccase, each Cu atom is coordinated by histidine residues from different fragments; to explore the feasibility of copper binding with a H4pep:Cu ratio of 1:1, an additional His residue at position 0 was introduced to satisfy the requirement of a four-coordination environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePeptide synthesis\u003c/h2\u003e \u003cp\u003ePeptide synthesis was performed via Fmoc-chemistry using an automated microwave peptide synthesizer (Biotage Initiator+, Alstra) on a 0.1 mmol scale using Rink-type resin (TentaGel\u0026reg; R RAM) with a polyethylene glycol/polystyrene backbone. The amino acids (Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Thr(tBu)-OH) were weighed in concentrations of 0.5 M and dissolved in DMF. 20% piperidine in DMF was used as the deprotection solution. All coupling steps were performed at 75\u0026deg;C for 5 min, except for the His residues, which were coupled at room temperature for 60 min to avoid racemization. Oxyma (0.5 M in DMF) and DIC (0.5 M in DMF) were used as the activator and activator base, respectively. Final deprotection was performed after the last coupling cycle to obtain a free amine terminus. Cleavage of the peptides from the resin and side chain deprotection was achieved using a 95% trifluoroacetic acid TFA, 2.5% TIS, and 2.5% distilled water cleavage cocktail for 2 hours at room temperature with stirring. After cleavage, the crude peptides were filtered and excess TFA was evaporated under a gentle stream of N\u003csub\u003e2\u003c/sub\u003e gas. The peptides were precipitated, washed with cold diethyl ether, dissolved in water, and lyophilized. The peptides were dissolved in water for purification over a C18 semi-preparative column using an \u0026Auml;KTA pure chromatography system (Fig. S17). Solvents A was 0.1% TFA in H\u003csub\u003e2\u003c/sub\u003eO, and solvent B was 0.085% TFA in acetonitrile. The peptide was injected in the equilibrated column with 5% B for 5 min, followed by a linear gradient to 20% B over 30 min at a 4 mL/min flow rate. The peptide was eluted at 10% solvent B. The chromatogram was monitored at 280 nm. The purified peptide was lyophilized and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Peptide mass and purity were analyzed by LCMS (Agilent Technology) (Fig. S18).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSpectroscopic methods: UV-vis, CD, EPR, NMR\u003c/h2\u003e \u003cp\u003eUV-vis spectra were recorded using a Cary 50 Bio (Varian) spectrometer, in a 10 mm path-length disposable cuvette containing 10 mM acetate buffer at pH\u0026thinsp;=\u0026thinsp;5.6. The peptide concentration for the near-UV range was 0.4 mM, with addition of 20 \u0026micro;L aliquots of Cu\u003csup\u003e2+\u003c/sup\u003e from a 1 mM copper sulfate stock solution. For the visible range spectra, a peptide concentration of 2.35 mM was used, and a Cu\u003csup\u003e2+\u003c/sup\u003e stock concentration of 22.8 mM.\u003c/p\u003e \u003cp\u003eCD spectra were recorded with a Chirascan V100 spectrometer (Applied Photophysics) equipped with a temperature-controlled cell Peltier holder. Smoothing via adjacent averaging was applied when indicated. The spectra were converted from millidegrees to units of mean residue molar ellipticity [Θ] in deg cm\u003csup\u003e2\u003c/sup\u003e dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by setting [Θ]\u0026thinsp;=\u0026thinsp;100*θ\u003csub\u003eobs\u003c/sub\u003e/\u003cem\u003eCln\u003c/em\u003e, where θ\u003csub\u003eobs\u003c/sub\u003e is the recorded ellipticity in millidegrees, \u003cem\u003eC\u003c/em\u003e is the concentration of protein in mM, \u003cem\u003el\u003c/em\u003e is the cuvette pathlength in cm, and \u003cem\u003en\u003c/em\u003e is the number of amino acids in the sequence (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). For UV-range measurements, a 1 mm path-length quartz cuvette was used. The peptide was dissolved in 10 mM acetate buffer at pH 5.6 to obtain a 0.1 mM solution. Cu\u003csup\u003e2+\u003c/sup\u003e aliquots were added from a 1 mM Cu\u003csup\u003e2+\u003c/sup\u003e stock solution. For visible-range measurements, a 10 mm path-length quartz cuvette was used, and the sample was prepared with a peptide concentration of 1 mM and a 10 mM Cu\u003csup\u003e2+\u003c/sup\u003e stock solution. In addition, spectra were recorded in 10 mM NaPi buffer solution at pH 5.8 and displayed no significant changes.\u003c/p\u003e \u003cp\u003eAll EPR spectra were recorded on a Bruker EMX-micro spectrometer equipped with an EMX-Premium bridge and an ER4119HS resonator in connection with an Oxford Instruments continuous flow cryostat. Low temperatures were reached using liquid helium flow through an ITC 503 temperature controller (Oxford Instruments). The EPR samples were prepared in 10 mM acetate buffer at pH 5.6 with a fixed Cu\u003csup\u003e2+\u003c/sup\u003e concentration of 0.1 mM and different peptide concentrations, to reach the selected stochiometric ratio. The tubes were flash-frozen and stored at 77 K prior to EPR analysis. The EasySpin software version 6.0.5 was used for spectral simulation and fitting (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The spectra were recorded at 10 K, microwave power of 50 \u0026micro;W, microwave frequency of 9.31 GHz, modulation amplitude of 10 G, and modulation frequency of 100 MHz.\u003c/p\u003e \u003cp\u003eNMR spectra were acquired at 298K on a Bruker Avance 600 Spectrometer equipped with a triple resonance cryoprobe. Samples for NMR analysis were prepared by dissolving H4pep in either sodium phosphate buffer pH 5.8 (10% D\u003csub\u003e2\u003c/sub\u003eO) or CD\u003csub\u003e3\u003c/sub\u003eOH to a final concentration of 0.5 mM. For metal-binding studies, ZnCl\u003csub\u003e2\u003c/sub\u003e or [Cu(CH\u003csub\u003e3\u003c/sub\u003eCN)\u003csub\u003e4\u003c/sub\u003e]PF\u003csub\u003e6\u003c/sub\u003e were added in stoichiometric amounts. For Cu\u003csup\u003e+\u003c/sup\u003e experiments, samples were prepared under inert atmosphere, and sodium dithionite was added to maintain reducing conditions. Water suppression was accomplished using an excitation sculpting sequence (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Chemical shifts are referenced to TSP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCompetitive Titration Studies\u003c/h2\u003e \u003cp\u003eCu\u003csup\u003e+\u003c/sup\u003e binding affinity was determined through competitive titration experiments with BCA (bicinchoninic acid) as a competing ligand (logβ\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.2) (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Experiments were performed by adding small aliquots of BCA (7.2 mM stock solution) to a solution containing H4pep-Cu\u003csup\u003e+\u003c/sup\u003e complex (prepared with 0.95 equivalents of [Cu(CH\u003csub\u003e3\u003c/sub\u003eCN)\u003csub\u003e4\u003c/sub\u003e]PF\u003csub\u003e6\u003c/sub\u003e dissolved in acetonitrile up to 10 mM concentration) in 50 mM sodium phosphate buffer at pH 5.8 with 1 mM sodium ascorbate under inert atmosphere. Formation of [Cu(BCA)\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e3\u0026minus;\u003c/sup\u003e was monitored spectrophotometrically at 562 nm (ε\u003csub\u003e562\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7900 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The dissociation constant was determined by fitting the titration data to Eq.\u0026nbsp;(5\u003cem\u003e9\u003c/em\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\left[L\\right]}_{tot}=2\\left[M{L}_{2}\\right]+\\sqrt{\\frac{\\left[M{L}_{2}\\right]}{{K}_{D}{{\\beta\\:}}_{2}}\\left(\\frac{{\\left[P\\right]}_{tot}}{{\\left[M\\right]}_{tot}-\\left[M{L}_{2}\\right]}-1\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere [L]\u003csub\u003etot\u003c/sub\u003e, [P]\u003csub\u003etot\u003c/sub\u003e, [M]\u003csub\u003etot\u003c/sub\u003e are the total BCA, peptide, and Cu\u003csup\u003e+\u003c/sup\u003e concentrations, respectively, and [ML\u003csub\u003e2\u003c/sub\u003e] is the [Cu(BCA)\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e3\u0026minus;\u003c/sup\u003e complex concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eElectrochemical measurements and activity assay\u003c/h2\u003e \u003cp\u003eAll experiments were performed with a Metrohm Autolab potentiostat or a BioLogic SP-300 potentiostat at room temperature, using a low-volume electrochemical cell. For CV measurements, a 3 mm diameter GCE working electrode was used. For bulk electrolysis measurements, the diameter of the exposed working electrode area was 9 mm. A graphite rod was used as counter electrode. An Ag/AgCl (in 3 M KCl) electrode was used as a reference and regularly checked using K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] as standard (E\u0026deg;\u003csub\u003eAg/AgCl\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.210 V vs SHE). All potentials are reported versus Ag/AgCl. For all measurements, the glassy carbon working electrodes were polished with 0.05 \u0026micro;m alumina particles and sonicated for 3 minutes in distilled water immediately before use. Measurements were performed in 10 mM sodium phosphate buffer at pH 5.8, in addition of 40 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as supporting electrolyte. \u003cb\u003e1Cu2Pep\u003c/b\u003e cyclic voltammetries were performed in presence of 1 mM H4pep and 0.45 mM of Cu\u003csup\u003e2+\u003c/sup\u003e, while \u003cb\u003e2Cu2Pep\u003c/b\u003e measurements were performed in presence of 1 mM H4pep and 0.9 mM of Cu\u003csup\u003e2+\u003c/sup\u003e. The total solution volume was 3 mL. Activity measurements were recorded by integrating a custom-made Clark-type electrode connected to a Unisense control box. Rinse test measurements were performed immediately after the voltammetry/amperometry by removing the active solution, rinsing the electrode with distilled water for 10 seconds, and reassembling the cell with fresh buffer and supporting electrolyte.\u003c/p\u003e \u003cp\u003eLaccase activity was determined by spectrophotometric assay by measuring the oxidation of 0.5 mM ABTS (ε\u003csub\u003e420\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;36000 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in oxygen-saturated phosphate buffer (20 mM) at pH 6. Sample aliquots of 30 \u0026micro;L were prepared by mixing H4pep and Cu\u003csup\u003e2+\u003c/sup\u003e stock solutions in the same buffer. The formation of the product was measured over 5 minutes by recording the change in absorbance at 420 nm. One activity unit (U) is defined as the amount of enzyme oxidizing 1 \u0026micro;mol of substrate per minute. The activity assay was performed in triplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSwedish Energy Agency grant 50529-1 (CS, MHC)\u003c/p\u003e\n\u003cp\u003eItalian MUR, Project SEA-WAVE 2020BKK3W9 [CUP_E69J22001140005] (MC, OM, AL)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: CS, CA, MHC\u003c/p\u003e\n\u003cp\u003eMethodology: CS, CA, MC, AL, MHC\u003c/p\u003e\n\u003cp\u003eInvestigation: CS, MC, OM, PH, CA, MHC\u003c/p\u003e\n\u003cp\u003eSupervision: AL, CA, MHC\u003c/p\u003e\n\u003cp\u003eWriting—original draft: CS, MHC\u003c/p\u003e\n\u003cp\u003eWriting—review \u0026amp; editing: CS, MC, AR, LDA, AL, CA, MHC with input from all authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e All data are available in the main text or the supplementary materials. The Metal-Site Analyzer is available as web interface at https://metalsite-analyzer.cerm.unifi.it/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eO. K. and D. S. Tawfik, Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective. Annual Review of Biochemistry 79, 471\u0026ndash;505 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Chen, F. H. Arnold, Engineering new catalytic activities in enzymes. Nat Catal 3, 203\u0026ndash;213 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. A. Cracknell, K. A. Vincent, F. A. Armstrong, Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis. Chem. Rev. 108, 2439\u0026ndash;2461 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ewwPDB consortium, Protein Data Bank: the single global archive for 3D macromolecular structure data. 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Rep. 27, 768\u0026ndash;789 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6073214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6073214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNature-inspired or biomimetic catalyst aims to reach the high catalytic performance and selectivity of natural enzymes while possessing the chemical stability and processability of synthetic catalysts. A promising strategy for designing biomimetic catalysts holds on mimicking the structure of the enzyme active site. This can either entail complicated total synthesis of a synthetic catalyst or design of peptide sequences, able to self-assemble in the presence of metal ions, thus forming metallo-peptide complexes that mimic the active sites of natural enzymes. Using a bioinformatics approach, we designed a minimal peptide made up of eight amino acids (H4pep) to act as a functional mimic of the trinuclear Cu site of the laccase enzyme. Cu(II) binding to H4pep results in the formation of a Cu\u003csup\u003e2+\u003c/sup\u003e(H4pep)\u003csub\u003e2\u003c/sub\u003e complex with a β-sheet secondary structure, able to reduce O\u003csub\u003e2\u003c/sub\u003e. Our study demonstrates the viability and potential of using short peptides to mimic the minimal functional site of natural enzymes.\u003c/p\u003e \u003cp\u003eTeaser\u003c/p\u003e \u003cp\u003eA minimal peptide, designed via bioinformatics, effectively mimics the trinuclear copper site in laccase for O₂ reduction.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eMAIN TEXT\u003c/span\u003e \u003c/p\u003e","manuscriptTitle":"A bioinformatics approach to the design of minimal biomimetic metal-binding peptides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-10 08:47:58","doi":"10.21203/rs.3.rs-6073214/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"25b8538c-4a7f-445c-aa55-a4ee32f6ac7e","owner":[],"postedDate":"March 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44925953,"name":"Physical sciences/Chemistry/Chemical synthesis/Biomimetic synthesis"},{"id":44925954,"name":"Biological sciences/Computational biology and bioinformatics/Protein design"},{"id":44925955,"name":"Physical sciences/Chemistry/Biochemistry/Peptides"},{"id":44925956,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":44925957,"name":"Biological sciences/Computational biology and bioinformatics/Software"}],"tags":[],"updatedAt":"2025-10-06T07:06:28+00:00","versionOfRecord":{"articleIdentity":"rs-6073214","link":"https://doi.org/10.1038/s42004-025-01702-z","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2025-10-06 04:00:00","publishedOnDateReadable":"October 6th, 2025"},"versionCreatedAt":"2025-03-10 08:47:58","video":"","vorDoi":"10.1038/s42004-025-01702-z","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01702-z","workflowStages":[]},"version":"v1","identity":"rs-6073214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6073214","identity":"rs-6073214","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}