Beyond copper: examining the significance of His-mutations in mycobacterial GroEL1 HRCT for Ni(II) complex stability and formation.

Recently, we have studied the coordination chemistry of the Cu(II)-histidine-rich C-terminal tail (HRCT) complex of the mycobacterial GroEL1 protein. The structure of this domain differs significantly compared to the well-known methionine-glycine-rich GroEL chaperonin - it was predicted that mycobacterial GroEL1 could play a significant role in the metal homeostasis of Mycobacteria, especially copper. However, we found that this particular domain's pattern also repeats in a number of Ni(II)-binding proteins. Here, we present the studies concerning the properties of GroEL1 HRCT as a ligand for Ni(II) ions. For this purpose, we chose eight model peptides: L1 - Ac-DHDHHHGHAH, L2 - Ac-DKPAKAEDHDHHHGHAH, and 6 mutants of the latter in the pH range of 2-11. We examined the stoichiometry, stability, and spectroscopic features of copper complexes. We noticed that similar to the Cu(II)-complex, the presence of a Lys5 residue significantly increases the stability of the system. The impact of His mutations was also examined and carefully studied using NMR spectroscopy. His9 and His13 are the crucial residues for Ni(II) binding, whereas His12 has minimal relevance in complex formation.

Selective Formation of Pd-DNA Hybrids Using Tailored Palladium-Mediated Base Pairs: Towards Heteroleptic Pd-DNA Systems.

The formation of highly organized metal-DNA structures has significant implications in bioinorganic chemistry, molecular biology and material science due to their unique properties and potential applications. In this study, we report on the conversion of single-stranded polydeoxycytidine (dC15 ) into a Pd-DNA supramolecular structure using the [Pd(Aqa)] complex (Aqa=8-amino-4-hydroxyquinoline-2-carboxylic acid) through a self-assembly process. The resulting Pd-DNA assembly closely resembles a natural double helix, with continuous [Pd(Aqa)(C)] (C=cytosine) units serving as palladium-mediated base pairs, forming interbase hydrogen bonds and intrastrand stacking interactions. Notably, the design of the [Pd(Aqa)] complex favours the interaction with cytosine, distinguishing it from our previously reported [Pd(Cheld)] complex (Cheld=chelidamic acid). This finding opens possibilities for creating heteroleptic Pd-DNA hybrids where different complexes specifically bind to nucleobases. We confirmed the Pd-DNA supramolecular structural assembly and selective binding of the complexes using NMR spectroscopy, circular dichroism, mass spectrometry, isothermal titration calorimetry, and DFT calculations.

Novel method for rapid monitoring of OPFRs by LLE and GC-MS as a tool for assessing biodegradation: validation and applicability.

Organophosphate flame retardants (OPFRs) are high-production volume chemicals widely present in environmental compartments. The presence of water-soluble OPFRs (tri-n-butyl phosphate (TnBP), tris(2-butoxyethyl) phosphate (TBEP), tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCPP), and triethyl phosphate (TEP)) in water compartments evidences the struggle of conventional wastewater treatment plants (WWTPs) to effectively eliminate these toxic compounds. This study reports for the first time the use of white-rot fungi as a promising alternative for the removal of these OPFRs. To accomplish this, a simple and cost-efficient quantification method for rapid monitoring of these contaminants' concentrations by GC-MS while accounting for matrix effects was developed. The method proved to be valid and reliable for all the tested parameters. Sample stability was examined under various storage conditions, showing the original samples to be stable after 60 days of freezing, while post-extraction storage techniques were also effective. Finally, a screening of fungal degraders while assessing the influence of the glucose regime on OPFR removal was performed. Longer chain organophosphate flame retardants, TBP and TBEP, could be easily and completely removed by the fungus Ganoderma lucidum after only 4 days. This fungus also stood out as the sole organism capable of partially degrading TCEP (35% removal). The other chlorinated compound, TCPP, was more easily degraded and 70% of its main isomer was removed by T. versicolor. However, chlorinated compounds were only partially degraded under nutrient-limiting conditions. TEP was either not degraded or poorly degraded, and it is likely that it is a transformation product from another OPFR's degradation. These results suggest that degradation of chlorinated compounds is dependent on the concentration of the main carbon source and that more polar OPFRs are less susceptible to degradation, given that they are less accessible to radical removal by fungi. Overall, the findings of the present study pave the way for further planned research and a potential application for the degradation of these contaminants in real wastewaters.

A de novo evolved domain improves the cadmium detoxification capacity of limpet metallothioneins.

Metallothioneins (MTs) constitute an important family of metal binding proteins. Mollusk MTs, in particular, have been used as model systems to better understand the evolution of their metal binding features and functional adaptation. In the present study two recombinantly produced MTs, LgiMT1 and LgiMT2, and their de novo evolved γ domain, of the marine limpet Lottia gigantea, were analyzed by electronic spectroscopy and mass spectrometry. Both MT proteins, as well as their γ domains, exhibit a strong binding specificity for Cd(II), but not for Zn(II) or Cu(I). The LgiMTs' γ domain renders an MII4(SCys)10 cluster with an increased Cd stoichiometry (binding 4 instead of 3 Cd2+ ions), representing a novel structural element in the world of MTs, probably featuring an adamantane 3D structure. This cluster significantly improves the Cd(II)-binding performance of the full length proteins and thus contributes to the particularly high Cd coping capacity observed in free-living limpets.

Histidine-Rich C-Terminal Tail of Mycobacterial GroEL1 and Its Copper Complex─The Impact of Point Mutations.

The mycobacterial histidine-rich GroEL1 protein differs significantly compared to the well-known methionine/glycine-rich GroEL chaperonin. It was predicted that mycobacterial GroEL1 can play a significant role in the metal homeostasis of Mycobacteria but not, as its analogue, in protein folding. In this paper, we present the properties of the GroEL1 His-rich C-terminus as a ligand for Cu(II) ions. We studied the stoichiometry, stability, and spectroscopic features of copper complexes of the eight model peptides: L1─Ac-DHDHHHGHAH, L2─Ac-DKPAKAEDHDHHHGHAH, and six mutants of L2 in the pH range of 2-11. We revealed the impact of adjacent residues to the His-rich fragment on the complex stability: the presence of Lys and Asp residues significantly increases the stability of the system. The impact of His mutations was also examined: surprisingly, the exchange of each single His to the Gln residue did not disrupt the ability of the ligand to provide three binding sites for Cu(II) ions. Despite the most possible preference of the Cu(II) ion for the His9-His13 residues (Ac-DKPAKAEDHDHHH-) of the model peptide, especially the His11 residue, the study shows that there is not only one possible binding mode for Cu(II). The significance of this phenomenon is very important for the GroEL1 function─if the single mutation occurs naturally, the protein would be still able to interact with the metal ion.

Exploring the Biological Properties of Zn(II) Bisthiosemicarbazone Helicates.

The design of artificial helicoidal molecules derived from metal ions with biological properties is one of the objectives within metallosupramolecular chemistry. Herein, we report three zinc helicates derived from a family of bisthiosemicarbazone ligands with different terminal groups, Zn2(LMe)2∙2H2O 1, Zn2(LPh)2∙2H2O 2 and Zn2(LPhNO2)23, obtained by an electrochemical methodology. These helicates have been fully characterized by different techniques, including X-ray diffraction. Biological studies of the zinc(II) helicates such as toxicity assays with erythrocytes and interaction studies with proteins and oligonucleotides were performed, demonstrating in all cases low toxicity and an absence of covalent interaction with the proteins and oligonucleotides. The in vitro cytotoxicity of the helicates was tested against MCF-7 (human breast carcinoma), A2780 (human ovarian carcinoma cells), NCI-H460 (human lung carcinoma cells) and MRC-5 (normal human lung fibroblasts), comparing the IC50 values with cisplatin. We will try to demonstrate if the terminal substituent of the ligand precursor exerts any effect in toxicity or in the antitumor activity of the zinc helicates.

The Modular Architecture of Metallothioneins Facilitates Domain Rearrangements and Contributes to Their Evolvability in Metal-Accumulating Mollusks.

Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs are bidomain proteins that arose by lineage-specific rearrangements between six putative domains: α, ß1, ß2, ß3, γ and δ. Some domains have been characterized in bivalves and gastropods, but nothing is known about the MTs and their domains of other Mollusca classes. To fill this gap, we investigated the metal-binding features of NpoMT1 of Nautilus pompilius (Cephalopoda class) and FcaMT1 of Falcidens caudatus (Caudofoveata class). Interestingly, whereas NpoMT1 consists of α and ß1 domains and has a prototypical Cd2+ preference, FcaMT1 has a singular preference for Zn2+ ions and a distinct domain composition, including a new Caudofoveata-specific δ domain. Overall, our results suggest that the modular architecture of MTs has contributed to MT evolution during mollusk diversification, and exemplify how modularity increases MT evolvability.

Cell-penetrating peptide-conjugated copper complexes for redox-mediated anticancer therapy.

Metal-based chemotherapeutics like cisplatin are widely employed in cancer treatment. In the last years, the design of redox-active (transition) metal complexes, such as of copper (Cu), has attracted high interest as alternatives to overcome platinum-induced side-effects. However, several challenges are still faced, including optimal aqueous solubility and efficient intracellular delivery, and strategies like the use of cell-penetrating peptides have been encouraging. In this context, we previously designed a Cu(II) scaffold that exhibited significant reactive oxygen species (ROS)-mediated cytotoxicity. Herein, we build upon the promising Cu(II) redox-active metallic core and aim to potentiate its anticancer activity by rationally tailoring it with solubility- and uptake-enhancing functionalizations that do not alter the ROS-generating Cu(II) center. To this end, sulfonate, arginine and arginine-rich cell-penetrating peptide (CPP) derivatives have been prepared and characterized, and all the resulting complexes preserved the parent Cu(II) coordination core, thereby maintaining its reported redox capabilities. Comparative in vitro assays in several cancer cell lines reveal that while specific solubility-targeting derivatizations (i.e., sulfonate or arginine) did not translate into an improved cytotoxicity, increased intracellular copper delivery via CPP-conjugation promoted an enhanced anticancer activity, already detectable at short treatment times. Additionally, immunofluorescence assays show that the Cu(II) peptide-conjugate distributed throughout the cytosol without lysosomal colocalization, suggesting potential avoidance of endosomal entrapment. Overall, the systematic exploration of the tailored modifications enables us to provide further understanding on structure-activity relationships of redox-active metal-based (Cu(II)) cytotoxic complexes, which contributes to rationalize and improve the design of more efficient redox-mediated metal-based anticancer therapy.

Metal-dependent glycosylation in recombinant metallothioneins.

We show for the first time glycosylation of recombinant metallothioneins (MTs) produced in E. coli. Interestingly, our results show that the glycosylation level of the recombinant MTs is inversely proportional to the degree of protein structuration, and reflects their different metal preferences.

Synthesis and In Vitro Studies of Photoactivatable Semisquaraine-type Pt(II) Complexes.

The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.

Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Estrategia de superación profesoral sobre atención integral al adulto mayor / Teaching Improvement Strategy about Comprehensive Care of the Elderly

Introducción: El informe mundial sobre envejecimiento y salud tiene en cuenta la sociedad e incluye los cuidados integrales para un envejecimiento saludable, por lo que elevar la calidad de la atención al adulto mayor a través del modelo de gestión docente-asistencial contribuirá a elevar la calidad de vida en estas edades. Objetivo: Diseñar una estrategia de superación profesoral sobre atención integral al adulto mayor dirigida a tutores de la residencia de Medicina General Integral. Método: Investigación aplicada, que tuvo una fase descriptiva-evaluativa y una de evaluación de resultados. La población estudiada fue el universo de tutores de la residencia de Medicina General Integral (12). Se estudiaron diferentes variables y se utilizaron encuestas y guías de desempeño, se aplicó frecuencia absoluta, relativa porcentual y la prueba de Mc Nemar. Resultados: Predominaron las edades de 45-54 años (58,3 por ciento). De los tutores, 41,7 por ciento eran asistentes y el 91,7 por ciento master o poseían categoría de investigador, se incrementaron los buenos conocimientos al 100 por ciento después de la intervención y el desempeño muy satisfactorio al 91,7 por ciento. Conclusiones: La estrategia de superación contribuyó al incremento del nivel de conocimientos y del desempeño sobre atención integral al adulto mayor en los tutores, así como una mayor participación en las actividades de ciencia y técnica relacionadas con la temática(AU)

Introduction: The World Report on Aging and Health takes society into account and includes comprehensive care for healthy aging; therefore, raising the quality of care for the elderly through the teaching-care management model will contribute to raising the quality of life in these ages. Objective: To design a teaching improvement strategy about comprehensive care for the elderly aimed at tutors from the Family Medicine residency. Method: Applied research with a descriptive-evaluative phase and a results assessment phase. The population studied was the universe of tutors from the Family Medicine residency (12). Different variables were studied and surveys and performance guides were used. Absolute frequency, relative percentage and the McNemar test were applied. Results: The ages 45-54 years (58.3 percent) predominated. Of the tutors, 41.7 percent were assistants, while 91.7percent were masters or had the researcher category. Good knowledge increased to 100 percent after the intervention, while very satisfactory performance did so to 91.7 percent. Conclusions: The improvement strategy contributed to the increase in the level of knowledge and performance on comprehensive care for the elderly in the tutors, as well as a greater participation in science and technical activities related to the subject(AU)

Modular Evolution and Population Variability of Oikopleura dioica Metallothioneins.

Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby, a suitable system in which to explore the limits of evolutionary processes. For this reason, and in order to gain new insights on the evolution of protein modularity, we have investigated the organization, function and evolution of multi-modular metallothionein (MT) proteins in O. dioica. MTs are a heterogeneous group of modular proteins defined by their cysteine (C)-rich domains, which confer the capacity of coordinating different transition metal ions. O. dioica has two MTs, a bi-modular OdiMT1 consisting of two domains (t-12C and 12C), and a multi-modular OdiMT2 with six t-12C/12C repeats. By means of mass spectrometry and spectroscopy of metal-protein complexes, we have shown that the 12C domain is able to autonomously bind four divalent metal ions, although the t-12C/12C pair -as it is found in OdiMT1- is the optimized unit for divalent metal binding. We have also shown a direct relationship between the number of the t-12C/12C repeats and the metal-binding capacity of the MTs, which means a stepwise mode of functional and structural evolution for OdiMT2. Finally, after analyzing four different O. dioica populations worldwide distributed, we have detected several OdiMT2 variants with changes in their number of t-12C/12C domain repeats. This finding reveals that the number of repeats fluctuates between current O. dioica populations, which provides a new perspective on the evolution of domain repeat proteins.

Tunicates Illuminate the Enigmatic Evolution of Chordate Metallothioneins by Gene Gains and Losses, Independent Modular Expansions, and Functional Convergences.

To investigate novel patterns and processes of protein evolution, we have focused in the metallothioneins (MTs), a singular group of metal-binding, cysteine-rich proteins that, due to their high degree of sequence diversity, still represents a "black hole" in Evolutionary Biology. We have identified and analyzed more than 160 new MTs in nonvertebrate chordates (especially in 37 species of ascidians, 4 thaliaceans, and 3 appendicularians) showing that prototypic tunicate MTs are mono-modular proteins with a pervasive preference for cadmium ions, whereas vertebrate and cephalochordate MTs are bimodular proteins with diverse metal preferences. These structural and functional differences imply a complex evolutionary history of chordate MTs-including de novo emergence of genes and domains, processes of convergent evolution, events of gene gains and losses, and recurrent amplifications of functional domains-that would stand for an unprecedented case in the field of protein evolution.

Functionalized azobenzene platinum(II) complexes as putative anticancer compounds.

The synthesis and characterization of four platinum(II) complexes using azobenzenes conveniently functionalized as ligands has been carried out. The characteristic photochemical behavior of the complexes due to the presence of azobenzene-type ligands and the role of the ligands in the activation of the complexes has been studied. Their promising cytotoxicity observed in HeLa cells prompted us to study the mechanism of action of these complexes as cytostatic agents. The interaction of the compounds with DNA, studied by circular dichroism, revealed a differential activity of the Pt(II) complexes upon irradiation. The intercalation abilities of the complexes as well as their reactivity with common proteins present in the blood stream allows to confirm some of the compounds obtained as good anticancer candidates.

Human absorption of monoterpenes after a 2-h forest exposure: A field experiment in a Mediterranean holm oak forest.

The current body of literature points monoterpenes as one of the determinant factors of the interaction between forests and human health. The present study aims at analyzing the monoterpene absorption by humans during a 2 -hs forest exposure in a Mediterranean holm oak forest focusing on the four most abundant monoterpene compounds: alpha-pinene, beta-pinene, alpha-phellandrene and limonene. Participants' blood samples were collected before and after exposure to forest or urban environment (control). We conducted air and blood sampling using cartridges and head space method and determined the monoterpene compounds through CGMS. We identified the four compounds in forest air during the experimental study being alpha-pinene the monoterpene with the greatest concentration. Results show no significant changes in monoterpene blood concentrations for the forest and control group. However, a negative significant relationship between absorption and baseline blood concentration of the most abundant forest air monoterpenes, alpha-pinene and beta-pinene, was found in individuals visiting the forest, i.e. higher absorption was found the lower the baseline blood concentration was. Although no significant lineal correlation could be spotted between the vital variables and the monoterpene absorption, we found significant correlations between the absorption of the monoterpene compounds. This attempt, first in a Mediterranean holm oak forest, can serve as a starting point and constitute a valuable contribution for further research in regard to experimental design and laboratory analysis.

Single-Stranded DNA as Supramolecular Template for One-Dimensional Palladium(II) Arrays.

Atomic-level control over the position and growth of a single and continuous metal chain is an ambitious goal that often requires complex and costly processes. Herein, we demonstrate that 1Pd-DNA molecules, comprising a continuous single chain of PdII ions, can be prepared by a simple self-assembly reaction between the complex [Pd(Cheld)(CH3 CN)] (1Pd_CH3 CN) (Cheld=chelidamic acid) and single-stranded DNA homopolymers (ss-DNA) containing adenine (A) or 7-deazaadenine (X) bases. The single PdII -base pairs [1Pd(N1-A)] and [1Pd(N1-X)] were synthesized and characterized in solution and solid-state (X-ray diffraction) revealing an arrangement similar to that of natural Watson-Crick base pairs. Subsequently, 1Pd-DNA hybrids were prepared, characterized, and their structures studied by small-angle X-ray scattering (SAXS) and ab-initio calculations. The results indicate that the 1Pd-DNA structures resemble that of double-stranded DNA, with one strand being replaced by a supramolecular stack of continuous PdII complexes.

Copper(II) N,N,O-Chelating Complexes as Potential Anticancer Agents.

Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying aromatic substituents (-H, -Cl, and -Br) have been synthesized and characterized. The experimental and computational data obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC50 about 25 µM in HeLa cells, which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds.

Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins.

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains-α, ß1, ß2, ß3, γ, and δ-in a lineage-specific manner. We have functionally characterized the Neritimorpha ß3ß1 and the Patellogastropoda γß1 MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.

Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats.

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.