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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.)

The cellular prion protein, PrP C , is a copper-binding protein abundantly expressed in the brain, particularly by neurons, and its conformational conversion into the amyloidogenic isoform, PrP Sc , plays a key pathogenic role in prion diseases. However, the role of copper binding to PrP C 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/Prnp 0/0 and TgPrP(5H > A)-7524/Prnp 0/0 mice, and similarly inoculated RML and BSE prions into them. Due to 2-fold higher expression of PrP(5H > A) than PrP C in wild-type (WT) mice, TgPrP(5H > A)-7524/Prnp 0/0 mice were highly susceptible to these prions, compared to WT mice. However, TgPrP(5H > A)-7342/Prnp 0/0 mice, which express PrP(5H > A) 1.2-fold as high as PrP C 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 PrP Sc created no prion transmission barrier to BSE prions. These results suggest that copper-binding His residues in PrP C 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 PrP C ., (© 2023 International Society for Neurochemistry.)

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.

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 m ) 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.

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression 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 the (1)H-(15)N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKa values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3 binding. Thermal unfolding experiments and modeling study of (GlcNAc)6 hydrolysis 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.

All members of the sulfotransferase (SOT, EC 2.8.2.-) protein family transfer a sulfuryl group from the donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an appropriate hydroxyl group of several classes of substrates. The primary structure of these enzymes is characterized by a histidine residue in the active site, defined PAPS binding sites and a longer SOT domain. Proteins with this SOT domain occur in all organisms from all three domains, usually as a multi-protein family. Arabidopsis thaliana SOTs, the best characterized SOT multi-protein family, contains 21 members. The substrates for several plant enzymes have already been identified, such as glucosinolates, brassinosteroids, jasmonates, flavonoids, and salicylic acid. Much information has been gathered on desulfo-glucosinolate (dsGl) SOTs in A. thaliana. The three cytosolic dsGl SOTs show slightly different expression patterns. The recombinant proteins reveal differences in their affinity to indolic and aliphatic dsGls. Also the respective recombinant dsGl SOTs from different A. thaliana ecotypes differ in their kinetic properties. However, determinants of substrate specificity and the exact reaction mechanism still need to be clarified. Probably, the three-dimensional structures of more plant proteins need to be solved to analyze the mode of action and the responsible amino acids for substrate binding. In addition to A. thaliana, more plant species from several families need to be investigated to fully elucidate the diversity of sulfated molecules and the way of biosynthesis catalyzed by SOT enzymes.

Stimuli-responsive drug delivery systems are highly desirable for improved therapeutic efficacy and minimized adverse effects of drugs. Mesoporous silica nanoparticles (MSNs) functionalized with pentadentate ligands, N-(3-trimethoxysilylpropyl)ethylenediamine triacetate (TSP-DATA), in the presence of metal ions with and without myoglobin (Mb)-containing surface-accessible histidine residues, were constructed for pH-triggered controlled release. The DATA ligands immobilized on the MSN pore outlets could encapsulate cargo within the pores by metal latching across pore openings, and release efficiency increased with the increase of surface density of the DATA ligands. The release efficiencies for the metal-chelating protein nanogates, through multiple-site binding of Mb with the metal-chelating ligands, were higher than those for the metal-latching ligand nanogates but were almost independent of surface density of the ligands investigated. Both the metal-latching ligands and the metal-chelating proteins played a synergetic role in gating MSNs for high-loading drug delivery and stimuli-responsive controlled release. The constructed Mb-Cu(2+)-gated MSN delivery system has promising applications in targeted drug therapy of tumors.