The dramatic increase in antimicrobial resistance has led to active research for new treatments. Antimicrobial peptides (AMPs) are small 7 to 100 amino acids [1, 2]. One discovered mode of action of AMPs is membrane disruptive activity, associated with the interaction with membrane phospholipids [3]. Another way of action of AMPs is intracellular targeting. Some AMPs can cross the cell barriers and reach targets in the cytoplasm [2]. For some AMPs, the occurrence of bivalent metal ions (such as Zn2+ and Cu2+) affects their activity or mode of action either because of binding metal ions, so that microbes cannot get enough nutrients essential for their life and virulence [4] or because AMPs need the metal ion as a booster of their antimicrobial activity, affecting the charge or the structure of the peptide [5]. Here, we discuss the action mechanism of Holothuroidin I (HLGHHALDHLLK) [6], a natural derived peptide from Mediterranean Sea cucumber Holothuria tubulosa, of the interactions with important metal ions located in mammals (such as Zn2+ and Cu2+).
Zinc (II), is the second most abundant transition metal in living organisms, [7] and present in superoxide dismutases, central enzymes in bacteria and fungi, associated with the detoxification of ROS generated by host cells during host-pathogen interactions [8]. Copper is another essential metal ion, which can switch between oxidized (Cu2+) and reduced (Cu+) states. It is essential for enzymes involved in cellular respiration, iron transport, and superoxide dismutation. Besides, the redox activity of copper may also contribute to its toxicity to microbes (and mammals) through classic copper-catalyzed Fenton chemistry and an increase in reactive oxygen species [9].
The action mechanism evaluation was performed using potentiometric titrations, conductivity studies, UV-Vis studies, voltammetric methods, and density function theory studies, giving valuable inside into the mechanism of interactions of Holothuroidin I with copper and zinc.

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[2] F. H. Waghu, R. S. Barai, P. Gurung and S. Idicula-Thomas, Nucleic Acids Res., 2016, 44, D1094–D1097.
[3] J. D. F. Hale and R. E. W. Hancock, Expert Rev. Anti-Infect. Ther., 2007, 5 (6), 951–959.
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[6] Domenico Schillaci, Maria Grazia Cusimano, Vincenzo Cunsolo, Rosaria Saletti, Debora Russo, Mirella Vazzana, Maria Vitale and Vincenzo Arizza, AMB Express 2013, 3:35.
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Protein farnesylation is a post-translational modification where a 15 carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by Farnesyltransferase (FTase). In the canonical understanding of FTase, the isoprenoids are attached to the Cysteine residue of a four amino acid CaaX box sequence. However, recent work has shown that five amino acid sequences can be recognized, such as the pentapeptide CMIIM. This new discovery greatly increases the number of potential FTase substrates, as the FTase is already known to tolerate a wide variety of amino acids in the canonical CaaX sequence. Due to the large number of potential substrates it is difficult to assay for novel CaaX sequences. With the goal of developing a more rapid and methodical method to evaluate potential substrates, we envisioned using MALDI to assay libraries of 10 peptides at a time, varying one amino acid in the CaaX box to all 20 canonical amino acids over two libraries. Through this method we observed 30 hits in the mass spectrum and chose eight for further evaluation. Seven of these sequences are novel substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Additionally, in vivo experiments in yeast demonstrate that proteins bearing these sequences can be efficiently prenylated in a biological context.

Peptide secondary structures have privileged roles in molecular recognition and therapeutic potential. α-Amino lactam residues have been commonly used as conformational constraints to study peptide structure-activity relationship for drug discovery [1]. N-Aminoimidazolone (Nai) residues offer similar means for constraining peptide backbone geometry [1, 2]. In model peptides, (4-methyl)Nai residues were found to adopt the central position of β- and γ-turn secondary structures. The addition of substituents at the 4- and 5-positions of the Nai residues may be used to mimic side chain function and orientation [3, 4]. Our presentation will feature the synthesis and application of Nai residues in the study of peptide structure-activity relationships [5].

1. St-Cyr D.J., García-Ramos Y., Doan N.D., Lubell W.D. Peptidomimetics I. Springer; Cham, Switzerland: 2017. Aminolactam, N-Aminoimidazolone, and N-Aminoimdazolidinone Peptide Mimics; pp. 125–175.
2. Proulx, C.; Lubell, W. D. "N-Amino-imidazolin-2-one Peptide Mimic Synthesis and Conformational Analysis" Lett., 2012, 14, 4552.
3. Poupart, J., Doan-Ngoc, D., Bérubé, D., Hamdane, Y., Medena, C., Lubell, W. D. "Palladium-Catalyzed Arylation of N-Aminoimidazol-2-ones towards Synthesis of Constrained Phenylalanine Dipeptide Mimics" Heterocycles, 2019, 99, 279-293
4. Poupart, J.; Hamdane, Y.; Lubell, W.D. "Synthesis of enantiomerically enriched 4,5-disubstituted N-aminoimidazol-2-one (Nai) peptide turn mimics" J. Chem. 2020, 98, 278–284.
5. Hamdane, Y.; Chauhan, S. P.; Vutla, S.; Mulumba, D.; Ong, H.; Lubell, W. D. Submitted.

Peptides are promising tools for designing sensitive and stable biosensors. For example, the redox self-assembled monolayer (SAM) based on the sequence Fc-Glu-(Ala)₂-Cys-NH₂ was successful evaluated as transducing interface in electrochemical biosensors. The design of such peptides includes: 1) cysteine to bind covalently the peptide to the gold electrode; 2) glutamic acid in N-terminal position to bind the ferrocene (Fc) in the amine group, and the antibody in the δ-carboxyl group, and 3) alanine to form a hydrophobic layer. Herein, we present the solid-phase synthesis of three different peptides with structure Fc-Glu-(X)₂-Cys-NH₂ (X=Ser, Gly or Phe) and the electrochemical behavior of the obtained SAMs. The Gly was chosen because of its smallest side chain, while Ser and Phe present hydroxyl groups (for H-bonds) and aromatic (for π-π interaction), respectively. The synthesis successful of the HPLC purified peptides were confirmed by mass spectroscopy. From cyclic voltammetry and impedance-derived electrochemical capacitance spectroscopy results, all peptides present reversible redox processes, and electron transfer rates (k_ET ) ranging from 17 to 31 s^-1. Since the peptide with Gly residues presented both the highest surface coverage (Γ = 2.6x10^-10 mol/cm²) and electrochemical capacitance (C_µ = 270 µF/cm²) values, it can be potentially applied for biosensors designing.

The heavy chalcogen tellurium (⁵²Te) is a versatile element with many potential applications in chemical biology and biochemistry, including mass cytometry, fluorescence imaging, and protein structure determination. Using L-tellurienylalanine (TePhe), a mimic of the natural amino acid L-phenylalanine (Phe) in which the phenyl side chain is replaced by a nearly isosteric tellurophene ring, tellurium can be covalently incorporated into the proteome of prokaryotes and eukaryotes by endogenous translation machinery. Our goal is to generate proteins with near stoichiometric levels of Phe to TePhe substitutions, verify preservation of protein structure and activity upon TePhe incorporation, and ultimately exploit the site-specific tellurium centres as handles for crystallographic phasing, protein NMR spectroscopy, and bio-orthogonal reactivity.

Here we report conditions for the expression of TePhe containing proteins in a standard E. coli expression system and validate the ability of TePhe to act as an effective Phe analogue within a folded protein. Our target for TePhe incorporation is the streptococcal immunoglobulin-binding Protein G B1 domain (GB1), a remarkably heat-stable 56-residue domain containing 2 Phe residues which pack against one another within the domain’s hydrophobic core. In Phe-deficient media containing glyphosate as an inhibitor of aromatic amino acid biosynthesis, we obtained a GB1 mixture in which approximately 1 in 2 Phe sites were substituted by TePhe. Fractionation by reverse-phase HPLC allowed us to obtain a sample with 85% TePhe substitution as evidenced by amino acid analysis. Using ¹H-¹⁵N HSQC and circular dichroism spectroscopy, we find that TePhe effectively takes on the role of Phe, and alters the melting temperature of the protein by less than 5 °C.