Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

Bacterial model membranes under the harsh subsurface conditions of Mars.

Biomembranes are a key component of all living systems. Most research on membranes is restricted to ambient physiological conditions. However, the influence of extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments, is less well understood. The deep subsurface of Mars is thought to harbour high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments. Here, we investigated the combined effects of high concentrations of Mars-relevant salts, including sodium and magnesium perchlorate and sulphate, and high hydrostatic pressure on the stability, structure, and function of a bacterial model membrane. To this end, several biophysical techniques have been employed, including calorimetry, fluorescence and CD spectroscopy, confocal microscopy, and small-angle X-ray scattering. We demonstrate that sulphate and perchlorate salts affect the properties of the membrane differently, depending on the counterion present (Na+vs. Mg2+). We found that the perchlorates, which are believed to be abundant salts in the Martian environment, induce a more hydrated and less ordered membrane, strongly favouring the physiologically relevant fluid-like phase of the membrane even under high-pressure stress. Moreover, we show that the activity of the phospholipase A2 is strongly modulated by both high pressure and salt. Compellingly, in the presence of the chaotropic perchlorate, the enzymatic reaction proceeded at a reasonable rate even in the presence of condensing Mg2+ and at high pressure, suggesting that bacterial membranes could still persist when challenged to function in such a highly stressed Martian environment.

Modulation of protein-saccharide interactions by deep-sea osmolytes under high pressure stress.

Deep-sea organisms must cope with high hydrostatic pressures (HHP) up to the kbar regime to control their biomolecular processes. To alleviate the adverse effects of HHP on protein stability most organisms use high amounts of osmolytes. Little is known about the effects of these high concentrations on ligand binding. We studied the effect of the deep-sea osmolytes trimethylamine-N-oxide, glycine, and glycine betaine on the binding between lysozyme and the tri-saccharide NAG3, employing experimental and theoretical tools to reveal the combined effect of osmolytes and HHP on the conformational dynamics, hydration changes, and thermodynamics of the binding process. Due to their different chemical makeup, these cosolutes modulate the protein-sugar interaction in different ways, leading to significant changes in the binding constant and its pressure dependence. These findings suggest that deep-sea organisms may down- and up-regulate reactions in response to HHP stress by altering the concentration and type of the intracellular osmolyte.

The anticancer peptide LL-III binds with nanomolar affinity to human telomeric and cMyc G-quadruplexes.

LL-III is a natural anticancer peptide able to cross the membrane of cancer cells and to localize in the nucleolus, but its intracellular target is unknown. Here, we show that LL-III is able to bind with nM affinity to specific G-quadruplex structures known to be relevant anticancer targets.

The impact of N-glycosylation on the properties of the antimicrobial peptide LL-III.

The misuse of antibiotics has led to the emergence of drug-resistant pathogens. Antimicrobial peptides (AMPs) may represent valuable alternative to antibiotics; nevertheless, the easy degradation due to environmental stress and proteolytic enzyme action, limits their use. So far, different strategies have been developed to overcome this drawback. Among them, glycosylation of AMPs represents a promising approach. In this work, we synthesized and characterized the N-glycosilated form of the antimicrobial peptide LL-III (g-LL-III). The N-acetylglucosamine (NAG) was covalently linked to the Asn residue and the interaction of g-LL-III with bacterial model membranes, together with its resistance to proteases, were investigated. Glycosylation did not affect the peptide mechanism of action and its biological activity against both bacteria and eukaryotic cells. Interestingly, a higher resistance to the activity of proteolytic enzymes was achieved. The reported results pave the way for the successful application of AMPs in medicine and biotechnological fields.

The anticancer peptide LL-III alters the physico-chemical properties of a model tumor membrane promoting lipid bilayer permeabilization.

LL-III is an anticancer peptide and has the ability to translocate across tumor cell membranes, which indicates that its action mechanism could be non-membranolytic. However, the exact mechanism through which the peptide gains access into the cell cytoplasm is still unknown. Here, we use a plethora of physico-chemical techniques to characterize the interaction of LL-III with liposomes mimicking the lipid matrix of the tumor cell membrane and its effect on the microstructure and thermotropic properties of the membrane. Furthermore, the effect of the presence of Ca2+ cations at physiological concentration was also investigated. For comparison, the interaction of LL-III with liposomes mimicking the normal cell membrane was also studied. Our results show that the peptide selectively interacts with the model tumor cell membrane. This interaction does not disrupt the lipid bilayer but deeply alters its properties by promoting lipid lateral reorganization and increasing membrane permeability. Overall, our data provide a molecular level description of the interaction of the peptide with the model tumor membrane and are fully consistent with the non-membranolytic action mechanism.

Conformational stability of ageritin, a metal binding ribotoxin-like protein of fungal origin.

Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn2+). Here we investigated the conformational stability of ageritin in the pH range 4.0-7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C. At pH 5.0 we calculated a value of 36 kJ mol-1 for the unfolding Gibbs energy at 25 °C. We also analysed the thermodynamic and catalytic behaviour of S-pyridylethylated form, obtained by alkylating the single Cys18 residue, which is predicted to bind Zn2+. We show that this form possesses the same activity and structure of ageritin, but lower stability. In fact, the corresponding values of 52 °C and 14 kJ mol-1 were found. Conservation of activity is consistent with the location of alkylation site on the opposite site of the catalytic site cleft. Inasmuch as Cys18 is part of a structurally stabilizing zinc-binding site, disrupted by cysteine alkylation, our results point to an important role of metal ions in ageritin stability.

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex.

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.

The C-terminus of the GKY20 antimicrobial peptide, derived from human thrombin, plays a key role in its membrane perturbation capability.

Previously, we characterized in detail the mechanism of action of the antimicrobial peptide GKY20, showing that it selectively perturbs the bacterial-like membrane employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. Here, we used a combination of biophysical techniques to similarly characterize the antimicrobial activity as well as the membrane perturbing capability of GKY10, a much shorter version of the GKY20 peptide. GKY10 is only half of the parent peptide and consists of the last 10 amino acids (starting from the C-terminus) of the full-length peptide. Despite a large difference in length, we found that GKY10, like the parent peptide, retains the ability to adopt a helical structure and to induce lipid segregation upon membrane binding. Overall, our results suggest that the amino acid sequence of GKY10 is responsible for most of the observed behaviors of GKY20. Our results shed further light on the mechanism of action of the full-length peptide and provide useful information for the design and development of new peptides that serve as antimicrobial agents.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

Impact of a Single Point Mutation on the Antimicrobial and Fibrillogenic Properties of Cryptides from Human Apolipoprotein B.

Host defense peptides (HDPs) are gaining increasing interest, since they are endowed with multiple activities, are often effective on multidrug resistant bacteria and do not generally lead to the development of resistance phenotypes. Cryptic HDPs have been recently identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, with anti-biofilm, wound healing and immunomodulatory properties, and with the ability to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, a multidisciplinary approach was used, including time killing curves, differential scanning calorimetry, circular dichroism, ThT binding assays, and transmission electron microscopy analyses. The effects of a single point mutation (Pro → Ala in position 7) on the biological properties of ApoB-derived peptide r(P)ApoBLPro have been evaluated. Although the two versions of the peptide share similar antimicrobial and anti-biofilm properties, only r(P)ApoBLAla peptide was found to exert bactericidal effects. Interestingly, antimicrobial activity of both peptide versions appears to be dependent from their interaction with specific components of bacterial surfaces, such as LPS or LTA, which induce peptides to form ß-sheet-rich amyloid-like structures. Altogether, obtained data indicate a correlation between ApoB-derived peptides self-assembling state and their antibacterial activity.

Toxicity and membrane perturbation properties of the ribotoxin-like protein Ageritin.

Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.

A Super Stable Mutant of the Plant Protein Monellin Endowed with Enhanced Sweetness.

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 °C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI.

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III.

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.

Covalently bonded hopanoid-Lipid A from Bradyrhizobium: The role of unusual molecular structure and calcium ions in regulating the lipid bilayers organization.

Lipopolysaccharides (LPS) are complex amphiphilic macromolecules forming the external leaflet of the outer membrane of Gram-negative bacteria. The LPS glycolipid portion, named Lipid A, is characterized by a disaccharide backbone carrying multiple acyl chains. Some Lipid A bear very-long-chain-fatty-acids (VLCFA), biosynthesized to span the entire lipid membrane profile. The synbiontic Bradyrhizobium BTAi1 strain carries an unique Lipid A specie, named HoLA, in which VLCFA terminus is covalently-bonded to hopanoid, a triterpenoid displaying structural similarity with eukaryotic sterols. Here, we investigate the role of HoLA in regulating self-assembly, microstructure and thermotropicity of lipid membranes composed by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1'-rac-glycerol. DLS and SANS indicated the formation of multilamellar aggregates whose size increases when the hopanoid is present in the bilayer. EPR and DSC showed that HoLA induced a more rigid and ordered organization among the lipids in the bilayer, evocative of a mechanical strengthening. Notably, the presence of calcium ions promote an increase of the bilayer thickness and vesicles size, leading to low curvature aggregates. These results highlight the key role of the hopanoid covalently-linked to VLCFA in defining the physico-chemical properties of bacterial envelope, thus offering a robust scientific basis for the interpretation of the biological activity of the considered strain.

KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study.

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5'-AGGGCGGTGTGGGAATAGGGAA-3' (KRAS 22RT) and 5'-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3' (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs.

Synthesis of new riboflavin modified ODNs: Effect of riboflavin moiety on the G-quadruplex arrangement and stability.

In the panorama of modified G-quadruplexes (G4s) with interesting proprieties, here, it has been reported the synthesis of new modified d(TGGGAG) sequences forming G-quadruplexes, with the insertion of a riboflavin unit (Rf, vitamin B2). Exploiting the flavin similarity with the hydrogen bond pattern of guanine and aiming at mimic a typical nucleoside scaffold, the synthesis of the riboflavin building block 3 it has been efficiently carried out. The effect of insertion of riboflavin mimic nucleoside on the G-quadruplex properties has been here, for the first time investigated. A biophysical characterization of Rf-modified sequences (A-D) has been carried out by circular dichroism (CD), fluorescence spectroscopy, differential scanning calorimetry (DSC) and native gel electrophoresis. CD and electrophoresis data have suggested that Rf-modified sequences are able to form parallel tetramolecular G4 structures similar to that of the unmodified sequence. Analysis of the DSC thermograms has revealed that all modified G-quadruplexes have a higher thermal stability compared with the natural sequence, particularly the stabilisation is higher when the Rf residue is introduced at the 3'-end. Further, DSC analysis has revealed that the Rf residues introduced at the 3'-end are able to form additional stabilising interactions, energetically almost comparable to the enthalpic contribution of a G-tetrad. Fluorescence measurement are consistent with this result showing that the Rf residues introduced at 3'-end are able to form stacking interactions with the adjacent bases within the G-quadruplex structure. The whole of data suggested that the introduction of Rf unit can stabilize G-quadruplex structures and can be a promising candidate for future theranostic applications.

Similarities and differences for membranotropic action of three unnatural antimicrobial peptides.

Previously, we described the design and synthesis of three nine-residue AMPs, P9Nal(SS), P9Trp(SS), and P9Nal(SR), showing high stability in serum and broad spectrum antimicrobial activity. The peptides P9Trp(SS) and P9Nal(SR) differ from P9Nal(SS) for the replacement of the two 2Nal residues with Trp residues and for the replacement of the two Cys (StBu) with Cys (tBu) residues, respectively. These changes led to peptides with a lower hydrophobicity respect to the P9Nal(SS). Interestingly, the three peptides have very similar activity against Gram-negative bacteria. Instead, they exhibit a significant difference towards Gram-positive bacteria, being P9Nal(SS) the most active. In order to evaluate the impact of amino acids substitution on membranotropic activity and rationalize the observed effects in vivo, here, we report the detailed biophysical characterization of the interaction between P9Nal(SR) and P9Trp(SS) and liposomes by combining differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The comparison with the results for the previously characterized P9Nal(SS) peptide reveals similarities and differences on the interaction process and perturbation activities. It was found that the three peptides can penetrate at different extent inside the bilayer upon changing their conformation and inducing lipid domains formation, revealing that the formation of lipid domains is fundamental for the activity against Gram-negative bacteria. On the contrary, the dissimilar activity against Gram-positive bacteria well correlate with the different affinity of peptides for the lipoteichoic acid, a component selectively present in the cell wall of Gram-positive bacteria.

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury.

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH2 (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).