Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrP^C) to the disease‐related scrapie isoform (PrP^Sc). Copper(II) coordination to PrP^C has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrP^C. In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG₁₁)₃PrP(60‐114) [Ac=acetyl] as a model for the whole N‐terminus of the PrP^C metal‐binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide‐bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide‐bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal‐binding‐affinity order observed in our previous studies.     

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

Copper is an essential nutrient for the normal development of the brain and nervous system, although the hallmark of several neurological diseases is a change in copper concentrations in the brain and central nervous system. Prion protein (PrP) is a copper‐binding, cell‐surface glycoprotein that exists in two alternatively folded conformations: a normal isoform (PrP^C) and a disease‐associated isoform (PrP^Sc). Prion diseases are a group of lethal neurodegenerative disorders that develop as a result of conformational conversion of PrP^C into PrP^Sc. The pathogenic mechanism that triggers this conformational transformation with the subsequent development of prion diseases remains unclear. It has, however, been shown repeatedly that copper plays a significant functional role in the conformational conversion of prion proteins. In this review, we focus on current research that seeks to clarify the conformational changes associated with prion diseases and the role of copper in this mechanism, with emphasis on the latest applications of NMR and EPR spectroscopy to probe the interactions of copper with prion proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

High‐resolution differentiation of transmissible spongiform encephalopathy strains by quantitative N‐terminal amino acid profiling (N‐TAAP) of PK‐digested abnormal prion protein

New forms of transmissible spongiform encephalopathy (TSE) continue to be identified, and consequently sensitive differential diagnosis is increasingly important both for the management of disease in humans and livestock and in providing confidence in the safety of the food chain. TSE diseases are associated with accumulation of protease‐resistant prion protein (PrP^Sc) and detection of this marker protein is central to diagnosis. Proteolysis by proteinase K (PK) generates protease‐resistant products (PrP^res) with partially variable N‐termini. The conformation(s) of PrP^Sc and thus the points of PK cleavage are thought to be dependent on the strain of prion disease. Western blot (WB) analysis of PrP^res gives characteristic migration patterns that can be used to diagnose TSEs, but the relatively low resolution of this technique limits its ability to differentiate certain disease strains. Mass spectrometry (MS) has the capability to resolve these various PK cleavage sites to the level of individual amino acid residues. In the present study multiple selected reaction monitoring (mSRM) was used to detect and quantify PrP^res N‐terminal tryptic peptides by MS and thus to define the N‐terminal amino acid profiles (N‐TAAPs) of PrP^res characteristic for various TSEs in sheep. The fragmentation behaviour of the N‐terminal tryptic peptides was studied to allow selection of the transitions specific for each peptide. Different PrP^res preparation methods were evaluated and the most effective approach applied to differentiate the N‐TAAPs corresponding to various sheep TSE isolates. Marked differences were identified between the N‐TAAPs of bovine spongiform encephalopathy (BSE) and classical scrapie, and between classical scrapie and the experimental strains SSBP/1 and CH1641, thereby validating this approach as a means of TSE‐strain specific diagnosis. Copyright © 2008 John Wiley & Sons, Ltd.     

Side‐chain glycylglycine‐based polymer for simultaneous sensing and removal of copper(II) from aqueous medium

Since glycylglycine (Gly‐Gly) residue in the N‐terminal region of human prion protein, a copper binding protein, binds with Cu(II), N‐terminus Gly‐Gly side‐chain containing water soluble block copolymer was synthesized and used for simultaneous sensing and removal of Cu(II) ion from aqueous medium. The polymer has amide nitrogen atom and ester carbonyl group as potential binding sites in the side‐chain Gly‐Gly pendants. Job's plot experiment confirms 2:1 binding stoichiometry of polymer with Cu(II). This polymer is able to sense parts per billion level of Cu(II) very selectively in an aqueous medium and remove Cu(II) ions quantitatively by precipitating out the Cu(II) via complex formation in the pH range 7–9. The binding mode of polymer with Cu(II) in polymer‐Cu(II) complex was characterized by ¹H NMR, FTIR, and UV–vis spectroscopy. The attachment of Cu(II) in the polymer‐Cu(II) complex was confirmed by cyclic voltammetry experiment. Cu(II) release from the complex was achieved at pH 5 due to the protonation of amide nitrogen atoms in the Gly‐Gly moiety. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 914–921     

Environmental Effects on a Prion's Helix II Domain: Copper(II) and Membrane Interactions with PrP180–193 and Its Analogues

An abnormal interaction between copper and the prion protein is believed to play a pivotal role in the pathogenesis of prion diseases. Copper binding has been mainly attributed to the N‐terminal domain of the prion protein, but this hypothesis has recently been challenged in some papers which suggest that the C‐terminal domain might also compete for metal anchoring. In particular, the segment corresponding to the helix II region of the prion protein, namely PrP180–193, has been shown both to bind copper and to exhibit a copper‐enhanced cytotoxicity, as well as to interact with artificial membranes. The present work is aimed at extending these results by choosing the most representative model of this domain and by determining its copper affinity. With this aim, the different role played by the electrostatic properties of the C‐ and N‐termini of PrP180–193 (VNITIKQHTVTTTT) in determining its conformational behaviour, copper coordination and ability to perturb model membranes was investigated. Owing to the low solubility of PrP180–193, its copper affinity was evaluated by using the shorter PrPAc184–188NH₂ (IKQHT) analogue as a model. ESI‐MS, ESR, UV/Vis, and CD measurements were carried out on the copper(II )/PrPAc184–188NH₂ and copper(II )/PrP180–193NH₂ systems, and showed that PrPAc184–188NH₂ is a reliable model for the metal interaction with the helix II domain. The affinity of copper(II ) for the helix II fragment is higher than that for the octarepeat and PrP106–126 peptides. Finally, the different ability of PrP180–193 analogues to perturb the DPPC model membrane was assessed by DSC measurements. The possible biological consequences of these findings are also discussed briefly.     

Ubiquitin Associates with the N‐Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI‐MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF_1–14, which is a short model peptide encompassing the first 14 N‐terminal residues of NGF, binds the copper‐binding regions of Ub (K_D=8.6 10^?5 m ). Moreover, the peptide undergoes a random coil–polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF_1–14/Ub interactions. Further experiments performed with the full‐length NGF confirmed the existence of a copper(II)‐dependent association between Ub and NGF and indicated that the N‐terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full‐length protein.     

Biological inorganic and bioinorganic chemistry of neurodegeneration based on prion and Alzheimer diseases

A change of the prion protein conformation results in a class of neurodegenerative diseases called the transmissible spongiform encephalopathies (like mad cow and Creutzfeld-Jakob diseases). The function of the normal prion protein is unknown, although much of recent research demonstrates the it may be a copper binding protein selective for Cu(II). Amyloid precursor protein (APP) releases the 39-42 amino acid peptide, a major constituent of the deposit in plaques of Alzheimer disease brain. Also APP is a metal binding protein, including copper ions. The link between copper and both proteins may provide insight into the role of metals in neurodegenerative pathologies.     

N‐(tert‐Butoxycarbonyl)‐O‐allyl‐l‐seryl‐α‐aminoisobutyryl‐l‐valine methyl ester: a protected tripeptide with an allylated serine residue

The title compound [systematic name (6S,12S)‐methyl 6‐(allyloxymethyl)‐12‐isopropyl‐2,2,9,9‐tetramethyl‐4,7,10‐trioxo‐3‐oxa‐5,8,11‐triazatridecan‐13‐oate], C₂₁H₃₇N₃O₇, containing the little studied O‐allyl‐l ‐serine residue [Ser(All)], crystallizes in the monoclinic space group C2 with one molecule in the asymmetric unit. The compound is an analogue of the Ser140‐Val142 segment of the water channel aquaporin‐4 (AQP4). It forms a distorted type‐II β‐turn with a P_II–3_10L–P_II backbone conformation (P_II is polyproline II). The overall backbone conformation is markedly different from that of the CO(Pro139)–Val142 stretch of rat AQP4, but is quite similar to the corresponding segment of human AQP4, despite significant differences at the level of the individual residues. The side chain of the Ser(All) residue adopts a gauche conformation relative to the backbone CO—C^α and C^α—N bonds. The H atoms of the two CH₂ groups in the Ser(All) side chain are almost eclipsed. The crystal packing of the title compound is divided into one‐molecule‐thick layers, each layer having a hydrophilic core and distinct hydrophobic interfaces on either side.     

The Propensity of α‐Aminoisobutyric Acid (=2‐Methylalanine; Aib) to Induce Helical Secondary Structure in an α‐Heptapeptide: A Computational Study

We present a molecular‐dynamics simulation study of an α‐heptapeptide containing an α‐aminoisobutyric acid (=2‐methylalanine; Aib) residue, Val¹‐Ala²‐Leu³‐Aib⁴‐Ile⁵‐Met⁶‐Phe⁷, and a quantum‐mechanical (QM) study of simplified models to investigate the propensity of the Aib residue to induce 3₁₀/α‐helical conformation. For comparison, we have also performed simulations of three analogues of the peptide with the Aib residue being replaced by L ‐Ala, D ‐Ala, and Gly, respectively, which provide information on the subtitution effect at C(α) (two Me groups for Aib, one for L ‐Ala and D ‐Ala, and zero for Gly). Our simulations suggest that, in MeOH, the heptapeptide hardly folds into canonical helical conformations, but appears to populate multiple conformations, i.e., C₇ and 3₁₀‐helical ones, which is in agreement with results from the QM calculations and NMR experiments. The populations of these conformations depend on the polarity of the solvent. Our study confirms that a short peptide, though with the presence of an Aib residue in the middle of the chain, does not have to fold to an α‐helical secondary structure. To generate a helical conformation for a linear peptide, several Aib residues should be present in the peptide, either sequentially or alternatively, to enhance the propensity of Aib‐containing peptides towards the helical conformation. A correction of a few of the published NMR data is reported.     

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR. On the basis of the analysis of variable temperature FTIR spectra of these peptides in solution, the organization of strands within the beta-sheets has been determined; at equilibrium, the strands form a beta-sheet in which the hydrophobic core (112-122) participates in the sheet structure, resulting in the alignment of residue 117 in all of the strands. The peptides initially form a kinetically trapped intermediate beta-sheet, with a distribution of strand alignments, which can be rearranged into the stable equilibrium conformation by an annealing cycle. These observations are discussed in terms of the biological significance of residue 117 of the prion protein and the mechanism of beta-aggregate nucleation in prion proteins.     

Construction and Characterization of an Anti‐Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Abstract

A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrP^c). After five rounds of panning against recombinant bovine prion protein (rb‐PrP^c) and ELISA test, two positive clones with high affinity to rb‐PrP^c, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrP^c were 3.24×10^?8 M, 8.82×10^?8M, and 8.10×10^?9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrP^c with detection limit of 1 pg/ml.     

Mapping of possible prion protein self-interaction domains using peptide arrays

Background  

The common event in transmissible spongiform encephalopathies (TSEs) or prion diseases is the conversion of host-encoded protease sensitive cellular prion protein (PrP^C) into strain dependent isoforms of scrapie associated protease resistant isoform (PrP^Sc) of prion protein (PrP). These processes are determined by similarities as well as strain dependent variations in the PrP structure. Selective self-interaction between PrP molecules is the most probable basis for initiation of these processes, potentially influenced by chaperone molecules, however the mechanisms behind these processes are far from understood. We previously determined that polymorphisms do not affect initial PrP^C to PrP^Sc binding but rather modulate a subsequent step in the conversion process. Determining possible sites of self-interaction could elucidate which amino acid(s) or amino acid sequences contribute to binding and further conversion into other isoforms. To this end, ovine – and bovine PrP peptide-arrays consisting of 15-mer overlapping peptides were probed with recombinant sheep PrP^C fused to maltose binding protein (MBP-PrP).     

New Insight in Copper‐Ion Binding to Human Islet Amyloid: The Contribution of Metal‐Complex Speciation To Reveal the Polypeptide Toxicity

Type‐2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace‐element serum level is significantly influenced during the development of diabetes. In particular, the dys‐homeostasis of Cu²⁺ ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu²⁺ ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal‐ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17–29 and 14–22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17–29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu²⁺ ions with Ac‐PEG‐hIAPP(17–29)‐NH₂, Ac‐rIAPP(17–29)R18H‐NH₂, and Ac‐PEG‐hIAPP(14–22)‐NH₂ were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu²⁺ ions starting from the His18 imidazole nitrogen atom toward the N‐terminus domain. The stability constants of copper(II) complexes with Ac‐PEG‐hIAPP(14–22)‐NH₂ were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu²⁺ ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu²⁺ ions present in phosphate‐buffered saline (PBS), and 3) a reducing fluorogenic probe on H₂O₂ production attributed to the polypeptide alone.     

3,5‐Bis(4‐methoxy­benzyl­idene)‐1‐methyl‐4‐piperidone and 3,5‐bis(4‐methoxy­benzyl­idene)‐1‐methyl‐4‐oxopiperidinium chloride: potential biophotonic materials

In the title compound 3,5‐bis(4‐methoxy­benzyl­idene)‐1‐methyl‐4‐piperidone, C₂₂H₂₃NO₃, (I), the central heterocyclic ring adopts a flattened boat conformation, while in the related salt 3,5‐bis(4‐methoxy­benzyl­idene)‐1‐methyl‐4‐oxopiperidin­ium chloride, C₂₂H₂₄NO₃⁺·Cl⁻, (II), the ring exhibits a `sofa' conformation in which the N atom deviates from the planar fragment. The pendant benzene rings are twisted from the heterocyclic ring planes in both mol­ecules in the same direction, the range of dihedral angles between the ring planes being 24.5 (2)–32.7 (2)°. The dominant packing motif in (I) involves centrosymmetric dimers bound by weak intermolecular C—H⋯O hydrogen bonds. In (II), cations and anions are linked by strong N—H⋯Cl hydrogen bonds, while weak C—H⋯O and C—H⋯Cl hydrogen bonds link the cations and anions into a three‐dimensional framework.     

Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full‐length prion protein amyloid fibrils

Understanding the structural basis that distinguishes the amyloid form of the prion protein from its monomeric homologue is of crucial importance to elucidate the mechanism of the lethal diseases related to this protein. Recently, an in vitro conversion system was established which reproduces the transition of recombinant prion protein PrP(23–230) from its native α‐helical rich form into an aggregated amyloid β‐sheet rich form with physicochemical properties reminiscent to those of the disease‐related isoform of the prion protein, PrP^Sc. To study the tertiary and quaternary structural organization within recombinant amyloid fibrils from mouse, mPrP(23–231)^βf; bovine, bPrP(23–230)^βf; and elk, ePrP(23–230)^βf; we utilized hydrogen/deuterium (H/D) exchange analyzed by matrix‐assisted laser desorption/ionization (MALDI) and nano‐electrospray (nano‐ESI) mass spectrometry. No significant differences were found by measuring the deuterium exchange kinetics of the aggregated fibrillar forms for mPrP(23–231)^βf, bPrP(23–230)^βf and ePrP(23–230)^βf, indicating a similar overall structural organization of the fibrils from all three species. Next, we characterized the solvent accessibility for the soluble and fibrillar forms of the mouse prion protein by hydrogen exchange, pepsin proteolysis and nano‐ESI ion trap mass spectrometry analysis. In its amyloid form, two highly protected regions of mPrP(23–231) comprising residues [24–98] and [182–212] were identified. The residues between the two highly protected stretches were found to be more solvent exposed, but less than in the soluble protein, and might therefore rather form part of a fibrillar interface. Copyright © 2009 John Wiley & Sons, Ltd.     

Square Wave‐Anodic Stripping Voltammetric Determination of Copper at a Bismuth Film/Glassy Carbon Electrode Using 3‐[(2‐Mercapto‐Vinyl)‐Hydrazono]‐ 1,3‐Dihydro‐Indol‐2‐One

This study reports a highly sensitive electrochemical sensor based on Bi film modified glassy carbon electrode (BiF/GCE) for total determination and speciation trace concentrations of copper(II) ions in environmental water samples. Square wave‐adsorptive anodic stripping voltammetric (SW‐ASV) experiment was performed for monitoring selective accumulation of copper(II) with reagent 3‐[(2‐mercapto‐vinyl)‐hydrazono]‐1,3‐dihydro‐indol‐2‐one (MHDI) at pH 9–10. The mechanism of the electrode reaction of Cu²⁺‐MHDI complex was safely assigned. The sensor exhibited a wide linear range (3.22×10⁻⁹–2.0×10⁻⁷ mol L⁻¹) with lower limits of detection (LOD) and quantitation (LOQ) of 9.6×1⁻¹⁰ and 3.22×10⁻⁹ mol L⁻¹, respectively (R²=0.9993). The proposed sensor exhibited interference from active metal ions e. g. Cd, Hg. The performance of the proposed method was compared successfully with most of the reported methods and comparable efficiencies were obtained. The analytical utility of the proposed SW‐ASV method has been successfully validated for trace analysis of copper(II) in environmental water samples. The method offers a precise, accurate approach with good reproducibility, robustness, ruggedness, and cost effectiveness.     

Remarkable difference of phospholipid molecular chirality in regulating PrP aggregation and cell responses

Prion diseases are fatal neurodegenerative diseases that can cause severe dementia.The misfolding and accumulation of the prion peptide (Pr P)_106–126is crucial,and this process is closely relevant to biological membranes.However,how Pr P_106–126aggregation is affected by the molecular chirality of phospholipid membrane is unknown.Thus,in this study,a pair of L-and D-aspartic acid (Asp)-modified 1,2-dipalmitoyl-sn–glycero-3-phosphoethanolamine (DPPE) were synthesized to const...     

A Designed Three‐Stranded β‐Sheet in an α/β Hybrid Peptide

The incorporation of β‐amino acid residues into the antiparallel β‐strand segments of a multi‐stranded β‐sheet peptide is demonstrated for a 19‐residue peptide, Boc‐LV^βFV^DPGL^βFVVL^DPGLVL^βFVV‐OMe (BBH19). Two centrally positioned ^DPro–Gly segments facilitate formation of a stable three‐stranded β‐sheet, in which β‐phenylalanine (^βPhe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR‐derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well‐defined three‐stranded β‐sheet structure in solution. Cross‐strand interactions between ^βPhe3/^βPhe17 and ^βPhe3/Val15 residues define orientations of these side‐chains. The observation of close contact distances between the side‐chains on the N‐ and C‐terminal strands of the three‐stranded β‐sheet provides strong support for the designed structure. Evidence is presented for multiple side‐chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three‐stranded β‐sheet structures, which in turn influences the conformational interconversion between type I′ and type II′ β‐turns at the two ^DPro–Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc‐LV^βFV^DPGL^βFVV‐OMe (BBH10), which has been previously characterized as a type I′ β‐turn nucleated hairpin, is shown to favour a type II′ β‐turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.     

Lysine residues in the N‐terminal huntingtin amphipathic α‐helix play a key role in peptide aggregation

Huntington's disease is a genetic neurodegenerative disorder caused by an expansion in a polyglutamine domain near the N‐terminus of the huntingtin (htt) protein that results in the formation of protein aggregates. Here, htt aggregate structure has been examined using hydrogen–deuterium exchange techniques coupled with tandem mass spectrometry. The focus of the study is on the 17‐residue N‐terminal flanking region of the peptide that has been shown to alter htt aggregation kinetics and morphology. A top‐down sequencing strategy employing electron transfer dissociation is utilized to determine the location of accessible and protected hydrogens. In these experiments, peptides aggregate in a deuterium‐rich solvent at neutral pH and are subsequently subjected to deuterium–hydrogen back‐exchange followed by rapid quenching, disaggregation, and tandem mass spectrometry analysis. Electrospray ionization of the peptide solution produces the [M + 5H]⁵⁺ to [M + 10H]¹⁰⁺ charge states and reveals the presence of multiple peptide sequences differing by single glutamine residues. The [M + 7H]⁷⁺ to [M + 9]⁹⁺ charge states corresponding to the full peptide are used in the electron transfer dissociation analyses. Evidence for protected residues is observed in the 17‐residue N‐terminal tract and specifically points to lysine residues as potentially playing a significant role in htt aggregation. Copyright © 2015 John Wiley & Sons, Ltd.     

Copper(II) interaction with unstructured prion domain outside the octarepeat region: speciation, stability, and binding details of copper(II) complexes with PrP106-126 peptides

Copper(II) complexes of the neurotoxic peptide fragments of human and chicken prion proteins were studied by potentiometric, UV-vis, CD, and EPR spectroscopic and ESI-MS methods. The peptides included the terminally blocked native and scrambled sequences of HuPrP106-126 (HuPrPAc106-126NH2 and ScrHuPrPAc106-126NH2) and also the nona- and tetrapeptide fragments of both the human and chicken prion proteins (HuPrPAc106-114NH2, ChPrPAc119-127NH2, HuPrPAc109-112NH2, and ChPrPAc122-125NH2). The histidyl imidazole-N donor atoms were found to be the major copper(II) binding sites of all peptides; 3N and 4N complexes containing additional 2 and 3 deprotonated amide-N donors, respectively, are the major species in the physiological pH range. The complex formation processes for nona- and tetrapeptides are very similar, supporting the fact that successive deprotonation and metal ion coordination of amide functions go toward the N-termini in the form of joined six- and five-membered chelates. As a consequence, the peptide sequences investigated here, related to the neurotoxic region of the human PrP106-126 sequence, show a higher metal-binding affinity than the octarepeat fragments. In the case of the HuPrP peptide sequences, a weak pH-dependent binding of the Met109 residue was also detected in the 3N-coordinated complexes.