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The natural tripeptide glutathione (GSH) is a ubiquitous compound harboring various biological tasks, among them interacting with essential and toxic metal ions. Yet, although weakly binding the poisonous metal lead (Pb), GSH poorly detoxifies it. β-Mercaptoaspartic acid is a new-to-nature novel amino acid that was found to enhance the Pb-detoxification capability of a synthetic cyclic tetrapeptide. Aiming to explore the advantages of noncanonical amino acids (ncAAs) of this nature, we studied the detoxification capabilities of GSH and three analogue peptides, each of which contains at least one ncAA that harbors both free carboxylate and thiolate groups. A thorough investigation that includes in vitro detoxification and mechanistic evaluations, metal-binding affinity, metal selectivity, and computational studies shows that these ncAAs are highly beneficial in additively enhancing Pb binding and reveals the importance of both high affinity and metal selectivity in synergistically reducing Pb toxicity in cells. Hence, such ncAAs join the chemical toolbox against Pb poisoning and pollution, enabling peptides to strongly and selectively bind the toxic metal ion.

Lead (Pb) is a ubiquitous poisonous metal, affecting the health of vast populations worldwide. Medications to treat Pb poisoning suffer from various limitations and are often toxic owing to insufficient metal selectivity. Here, we report a cyclic tetrapeptide that selectively binds Pb and eradicates its toxic effect on the cellular level, with superior potency than state-of-the-art drugs. The Pb-peptide complex is remarkably strong and was characterized experimentally and computationally. Accompanied by the lack of toxicity and enhanced stability of this peptide, these qualities indicate its merit as a potential remedy for Pb poisoning.

Among the broad applicability of peptides in numerous aspects of life and technologies, their interactions with lead (Pb), one of the most harmful substances to the environment and health, are constantly explored. So far, peptides were developed for environmental remediation of Pb-contaminations by various strategies such as hydrogelation and surface display. They were also designed for Pb detection and sensing by electrochemical and fluorescent methods and for modeling natural proteins that involve in mechanisms by which Pb is toxic. This review aims at summarizing selected examples of these applications, manifesting the enormous potential of peptides in the combat against Pb pollution. Nevertheless, the absence of new medicinal treatments against Pb poisoning that are based on peptides is noticeable. An overview of previous achievements utilizing Pb-peptide interactions towards various goals is presented and can be therefore leveraged to construct a useful toolbox for the design of smart peptides as next-generation therapeutics against Pb.

Functionalizing noble metal nanoparticles is often necessary to tailor their properties, in particular for biological applications such as drug delivery, therapy and imaging. However, this process is highly challenging, due to the complications in maintaining similar geometrical parameters of the nanoparticles as of without functionalization. The heptapeptide H-Lys-[Pro-Gly-Lys]2-NH2 was found to be an efficient additive for the formation and stabilization of small and monodisperse platinum nanoparticles (PtNPs). We show that the same additive peptide can be used as a platform for the functionalization of PtNPs, as demonstrated by the introducion of three different moieties: coumarin for monitoring cellular uptake of the PtNPs; biotin for detecting their stability in human blood serum; and an arginine-containing peptide for enhanced cell penetration. The peptide-coated PtNPs can be functionalized both before and after reduction of the metal ions, in aqueus environment, emphasizing the high robustness of the system.

Peptide-stabilized platinum nanoparticles (PtNPs) were developed that have significantly greater toxicity against hepatic cancer cells (HepG2) than against other cancer cells and non-cancerous liver cells. The peptide H-Lys-Pro-Gly-dLys-NH2 was identified by a combinatorial screening and further optimized to enable the formation of water-soluble, monodisperse PtNPs with average diameters of 2.5 nm that are stable for years. In comparison to cisplatin, the peptide-coated PtNPs are not only more toxic against hepatic cancer cells but have a significantly higher tumor cell selectivity. Cell viability and uptake studies revealed that high cellular uptake and an oxidative environment are key for the selective cytotoxicity of the peptide-coated PtNPs.

Bioconjugate Chemistry, 30 (3), ISSN:1043-1802, ISSN:1520-4812

Ordering π-systems into defined supramolecular structures is important for the development of organic functional materials. In recent years, peptides with defined secondary structures and/or self-assembly properties were introduced as powerful tools to order peptide-chromophore conjugates into different morphologies. This work explores whether or not the directionality of peptides can be used to control the self-assembly. The position of the π-system in conjugates between oligoprolines and perylene monoimide (PMI) chromophores was varied by attaching the PMI moiety to the second-to-last residue from the C- and N-termini, respectively. Microscopic and diffraction analysis revealed that the positional isomers form distinctly different supramolecular architectures that extend into the micrometer regime. NMR spectroscopic studies in solution phase allowed correlation of the self-assembly properties with markedly different conformational preferences of the isomeric building blocks. These insights enabled the design of building blocks with predictable self-assembly properties. Thus, the directionality of peptides offers exciting opportunities for controlling the self-assembly and electronic properties of π-systems.

Over the past two decades, peptides have expanded the toolbox of additives for the preparation of noble metal nanoparticles. Their functional and structural modularity and accompanying molecular recognition and self-assembly properties offer unique opportunities for the controlled formation of NPs. Within this review, we highlight recent examples for the use of peptides to control a) the size and shape of NPs and b) the spatial arrangement of NPs. The article focuses on examples where the peptides are directly involved in the bottom-up synthesis of the noble metal NPs.

Seleno-substituted model peptides of copper metallochaperone proteins were analyzed for the metal affinity and in vitro anti-oxidative reactivity. An acyclic MTCXXC (X is any amino acid) reference peptide previously analyzed as a potent inhibitor of ROS production underwent substitution of the cysteine residues with selenocysteine to give two singly substituted derivatives C3U and C6U and the doubly substituted analogue C3U/C6U. Presumably due to the softer nature of Se vs. S, all selenocysteine containing peptides demonstrated high affinity to Cu(I), higher than that of the reference peptide, and in the same order of magnitude as that measured for the native protein, Atox1. A stronger impact of residue 3 confirmed previous findings on its more dominant role in metal coordination. In vitro studies on the HT-29 human colon cancer cell line, MEF mice embryonic fibroblasts, and MEF with the knocked-out Atox1 gene (Atox1 −/−) consistently identified C3U/C6U as the most potent inhibitor of ROS cellular production based on the 2′,7′-dichlorodihydrofluorescin diacetate (H2DCF-DA) assay, also in comparison with known drugs employed in the clinic for Wilson's disease. The selenocysteine containing peptides are thus promising drug candidates for chelation therapy of Wilson's disease and related conditions relevant to excessive copper levels.

Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H2 O2 , in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT-29 cancer cells at concentrations 10-100 times higher than the IC50 of the commercial drugs, corroborating its high medicinal potential.

The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural analysis and affinity and oxidation kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidation of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidation rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport.

The first NMR structure of a Cu(I)-bound metallochaperone model with the conserved sequence MT/HCXXC revealed that at pH similar to 3.0 and similar to 6.8 Cu(I) binds through one Cys and the Met rather than the two Cys residues, differently than at pH similar to 8.5. This suggests a possible role of Met in metal transport.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy. NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy. NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

The first NMR structures of Cu(I) and Zn(II) peptide complexes as models of metallochaperones were derived with no predetermined binding mode. The cyclic peptide MDCSGCSRPG was reacted with Cu(I) and Zn(II) at low and moderate pH. This peptide features the conserved sequence of copper chaperones but with Asp at position 2 as appears in the zinc binding domain of ZntA. The structures were compared with those of the Cu(I) complexes of the wild-type sequence peptide MTCSGCSRPG. All analyses were conducted first with no metal-binding constraints to ensure accurate binding ligand assignment. Several structures included metal-Met binding, raising a possible role of Met in the metal transport mechanism. Both Cu(I) and Zn(II) gave different complexes when reacted with the peptide of the native-like sequence under different pH conditions, raising the possibility of pH-dependent transport mechanisms. Cu(I) bound the MTCSGCSRPG peptide through one Cys and the Met under acidic conditions and differently under basic conditions; Zn(II) bound the MDCSGCSRPG peptide through two Cys and the Met residues under acidic conditions and through one Cys and the Met under basic conditions, while Cu(I) bound the non-native Asp mutant peptide through the Asp and one Cys under both conditions, suggesting that Asp may inhibit pH-dependent binding for Cu(I). NOESY and ESI-HRMS supported the presence of an aqua ligand for Zn(II), which likely deprotonated under basic conditions to give a hydroxo group. Coordination similarities were detected among the model system and native proteins, which overall suggest that coordination flexibility is required for the function of metallochaperones.

Transition metal ions can be both beneficial and harmful to biological systems if not carefully regulated. A family of proteins that include a conserved sequence in their binding site of MXCXXC is responsible for delivery and homeostasis of different metals. Model studies present an effective tool for studying the parameters governing metal affinity, selectivity and other mechanistic aspects. Small-molecule, peptide-based, and advanced models will be presented, as well as functional models of potential industrial applications.

Transition metal ions can be both beneficial and harmful to biological systems if not carefully regulated. A family of proteins that include a conserved sequence in their binding site of MXCXXC is responsible for delivery and homeostasis of different metals. Model studies present an effective tool for studying the parameters governing metal affinity, selectivity and other mechanistic aspects. Small-molecule, peptide-based, and advanced models will be presented, as well as functional models of potential industrial applications.