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The cellular prion protein, PrPC, is a copper‐binding protein abundantly expressed in the brain, particularly by neurons, and its conformational conversion into the amyloidogenic isoform, PrPSc, plays a key pathogenic role in prion diseases. However, the role of copper binding to PrPC in prion diseases remains unclear. Here, we fed mice with a low‐copper or regular diet and intracerebrally inoculated them with two different mouse‐adapted RML scrapie and BSE prions. Mice with a low‐copper diet developed disease significantly but only slightly later than those with a regular diet after inoculation with BSE prions, but not with RML prions, suggesting that copper could play a minor role in BSE prion pathogenesis, but not in RML prion pathogenesis. We then generated two lines of transgenic mice expressing mouse PrP with copper‐binding histidine (His) residues in the N‐terminal domain replaced with alanine residues, termed TgPrP(5H > A)‐7342/Prnp0/0 and TgPrP(5H > A)‐7524/Prnp0/0 mice, and similarly inoculated RML and BSE prions into them. Due to 2‐fold higher expression of PrP(5H > A) than PrPC in wild‐type (WT) mice, TgPrP(5H > A)‐7524/Prnp0/0 mice were highly susceptible to these prions, compared to WT mice. However, TgPrP(5H > A)‐7342/Prnp0/0 mice, which express PrP(5H > A) 1.2‐fold as high as PrPC in WT mice, succumbed to disease slightly, but not significantly, later than WT mice after inoculation with RML prions, but significantly so after inoculation with BSE prions. Subsequent secondary inoculation experiments revealed that amino acid sequence differences between PrP(5H > A) and WT PrPSc created no prion transmission barrier to BSE prions. These results suggest that copper‐binding His residues in PrPC are dispensable for RML prion pathogenesis but have a minor effect on BSE prion pathogenesis. Taken together, our current results suggest that copper could have a minor effect on prion pathogenesis in a strain‐dependent manner through binding to His residues in the N‐terminal domain of PrPC. [ABSTRACT FROM AUTHOR]

Histidine are one of the most common residues involved in transition metal ion binding in the active sites of metalloenzymes. In order to mimic enzymatic metal binding sites, it is crucial to understand the basic coordination modes of histidine residues, distributed at different positions in the peptide sequence. We show that: (i) the separation of two histidines has a large effect on complex stability - a sequence with adjusting histidine residues forms more stable complexes with Zn(II) than the one in which the residues are separated, while the contrary is observed for Cu(II) complexes, in which amide nitrogens participate in metal binding. No pronounced effect is observed for Ni(II) complexes, where the amides participate in binding at higher pH; (ii) non-coordinating amino acid residues (basic, acidic and aromatic ones) have a significant impact on complex stability; charged and aromatic residues may enhance Zn(II) binding, while the contrary is observed for the amide-binding Cu(II); (iii) cysteine containing sequences are much more effective Zn(II) and Ni(II) binding motifs at pH above 8, while histidine containing ligands are more suitable for effective Zn(II) and Ni(II) binding at lower pH., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

To understand the role of His101 in protein structure stabilization of goose-type (G-type) lysozyme, we conducted thermal unfolding/refolding experiments using native G-type lysozyme from ostrich egg white (nOEL), the recombinant G-type lysozyme (rOEL), and the mutant lysozyme, in which His101 is mutated to alanine (H101A-OEL). Thermal stability on lytic activity and in-gel refolding experiments provided similar profiles for all three OELs. Circular dichroism (CD) spectroscopy was used to determine the secondary structure of three OELs as a function of temperature. Unfolding/refolding experiments (30-90 °C) monitored by CD spectroscopy revealed an unfolding transition at 65-67 °C and a complete refolding at almost the same temperature. Notably, a slightly lower thermal stability was observed for H101A-OEL, corresponding to the calculated difference in transition free energy of thermal unfolding (∆∆ G ) between rOEL and H101A-OEL of −0.63 kcal/mol. To assess the effects of H101A mutation on the electrostatic behavior, we examined the pH-activity profile of the three OELs. nOEL and rOEL exhibit bimodal relationship between pH and lytic activity showing optima at pH 3.0 and 7.0, while optima for H101A-OEL activity were pH 4.0 and 6.0. Electrostatic environment surrounding His101 was affected by the H101A mutation resulting in the slightly lower thermal stability. [ABSTRACT FROM AUTHOR]

Zinc is an important catalytic element for more than 300 enzymes and plays a structural role in the stabilization of many proteins. Protein domain analysis showed that identified Zn transporter proteins belong to the ZIP protein family (PF02535). Zn transporter sequences were found to have similar molecular weights (33.1-51.4 kDa) and amino acid lengths (306-478 amino acids) with 5.31-8.92 pI values. Subcellular localization of Zn transporters was predicted as the plasma membrane. They had 6-9 putative transmembrane domains with a variable region between TM-3 and TM-4, which could contain a potential histidine-rich metal-binding domain. Moreover, alignment analysis showed that the TM-2, -4, and -5 domains could be potential metal-binding sites because they contain highly conserved His residues. Based on a homology search, the retrieved sequences were identified as corresponding homologs of either Arabidopsis thaliana or Oryza sativa. Phylogenetic analysis also supported that A. thaliana and O. sativa sequences could be used as a reference/benchmark to identify Zn transporter homologs in various plant species. The findings of this study will be valuable theoretical knowledge for feature studies in terms of understanding the gene and protein features of Zn transporters in various plants. [ABSTRACT FROM AUTHOR]

A goose-type lysozyme from ostrich egg white (OEL) was produced byEscherichia coliexpression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)6. Although the binding of tri-N-acetylchitotriose, (GlcNAc)3, to OEL perturbed several backbone resonances in the1H–15N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKavalues of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3binding. Thermal unfolding experiments and modeling study of (GlcNAc)6hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites −3 and −2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction. His101 of a goose-type lysozyme from ostrich egg white (OEL) appears to play multiple roles in the substrate binding and the catalytic reaction. [ABSTRACT FROM PUBLISHER]

Polymerized micelles obtained by photopolymerization of diacetylenic surfactants and which are forming polydiacetylenic systems (PDAs) have recently gained interest as stabilized monodisperse systems showing potential for the delivery of hydrophobic drugs as well as of larger biomolecules such as nucleic acids. Introduction of pH-sensitive histidine groups at the surface of the micellar PDA systems allows for efficient delivery of siRNA resulting in specific gene silencing through RNA interference. Here, we describe the detailed experimental procedure for the reproducible preparation of these photopolymerized PDA micelles. We provide physicochemical characterization of these nanomaterials by dynamic light scattering, transmission electron microscopy, and diffusion ordered spectroscopy. Moreover, we describe standardized biological tests to evaluate the silencing efficiency by the use of a cell line constitutively expressing the luciferase reporter gene.