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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]

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]

The authors' intention was to prepare nanometer-sized zinc-phosphate nanoparticles that would be capable of binding histidine-rich TNF-α analogs onto their surface via a coordinative bond. Zinc-phosphate nanoparticles with a size of around 60 nm were prepared by a wet precipitation method and characterized using SEM, EDX, XRD, and DLS. First, BSA was bound as a testing protein, afterward two TNF-α analogs with decreased activity were bound to the described nanoparticles. The efficiency of binding and the existence of coordinative bond were confirmed with SDS-PAGE analysis. During binding, particle storage, and release experiments, the prepared TNF-α analogs retained their biological activity-hence the epitopes necessary for formation of antibodies stayed intact. The particle size did not change within a period of 2 weeks. No significant agglomeration was observed, the particles could be quickly dispersed in ultrasound. The present nanoparticles and the general approach of coordinative binding are widely applicable for natural and engineered histidine-rich proteins. The nanoparticles bearing appropriate TNF-α analogs could also be potentially used for active immunotherapy to tackle the chronic inflammatory diseases associated with pathogenically elevated levels of TNF-α. [ABSTRACT FROM AUTHOR]

Abstract: To explore Cu(II) ion coordination by His186 in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron–electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 Å. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 Å, 18.1 Å, 10.7 Å, and 8.4 Å, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrPC. [Copyright &y& Elsevier]

Abstract: Adrenal cytochrome b561 (cyt b561 ), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b561 (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31–40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (bH) peak were seen with mutation of His92; the largest changes in the low-potential (bL) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g =3.1 signal (bH) but not the g =3.7 signal (bL). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the bH transition; mutations in His110 produced the largest decreases in ΔA 561 for the bL transition. These results indicate that His92 can be considered part of the bH heme center, and His110 part of the bL heme center, in adrenal cyt b561 . [Copyright &y& Elsevier]

Abstract: We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrPC with varying pHs by using the SDSL–ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT∗. The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177–R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrPSc structure in hereditary prion disease. [Copyright &y& Elsevier]

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone. [ABSTRACT FROM AUTHOR]

Glutamate decarboxylase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II. Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241. [Copyright &y& Elsevier]

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn2+. The effect was reversed by hydroxylamine and involved a residue with a pKa of 6.5±0.1. Half activation with Mn2+ was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in Km for arginine or Ki for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn2+ and contained <0.1 Mn2+/subunit, whereas H101N was half active and contained 1.2±0.1 Mn2+/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state. [Copyright &y& Elsevier]