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Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M-1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter., Gly-His-Lys (GHK) is a human bioactive tripeptide thought to be activated by Cu(II) binding, but little is known about the molecular aspects of its action. UV−vis, circular dichroism (CD), EPR, and NMR spectroscopies, and cyclic voltammetry were used to examine the reduction of Cu(II)GHK with glutathione (GSH), the most abundant biological thiol. A semistable GSH-Cu(II)GHK reaction intermediate was discovered, with properties suitable for delivering Cu(I) to biological transport proteins.

The protein misfolding cyclic amplification (PMCA) technique employs repeated cycles of incubation and sonication to amplify minute amounts of misfolded protein conformers. Spontaneous (de novo) prion formation and ultrasonic power level represent two potentially interrelated sources of variation that frustrate attempts to replicate results from different laboratories. We previously established that water splitting during PMCA provides a radical-rich environment leading to oxidative damage to substrate molecules as well as the polypropylene PCR tubes used for sample containment. Here it is shown that the cross-linking agent formaldehyde is generated from buffer ions that are attacked by hydroxyl radicals. In addition, free radical damage to protein, nucleic acid, lipid, and detergent molecules produces a substantial concentration of aldehydes (hundreds of micromolar). The measurement of aldehydes using the Hantzsch reaction provides a reliable and inexpensive method for measuring the power delivered to individual PMCA samples, and for calibrating the power output characteristics of an individual sonicator. The proposed method may also be used to better account for inter-assay and inter-laboratory variation in prion replication and de novo prion generation, the latter of which may correlate with aldehyde-induced cross-linking of substrate molecules.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log c K Cu Cu ( HSA ) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 μM), while proteolysis of Aβ yielded the soluble, Aβ4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aβ4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 μM) further suggests a mechanism for modulating NEP activity, Aβ4-9 production, and Cu2+ homeostasis.

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M−1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M−1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function.

Neurogenesis continues in the post-developmental brain throughout life. The ability to stimulate the production of new neurones requires both quiescent and actively proliferating pools of neural stem cells (NSCs). Actively proliferating NSCs ensure that neurogenic demand can be met, whilst the quiescent pool makes certain NSC reserves do not become depleted. The processes preserving the NSC quiescent pool are only just beginning to be defined. Herein, we identify a switch between NSC proliferation and quiescence through changing intracellular redox signalling. We show that N-terminal post-translational cleavage products of the prion protein (PrP) induce a quiescent state, halting NSC cellular growth, migration, and neurite outgrowth. Quiescence is initiated by the PrP cleavage products through reducing intracellular levels of reactive oxygen species. First, inhibition of redox signalling results in increased mitochondrial fission, which rapidly signals quiescence. Thereafter, quiescence is maintained through downstream increases in the expression and activity of superoxide dismutase-2 that reduces mitochondrial superoxide. We further observe that PrP is predominantly cleaved in quiescent NSCs indicating a homeostatic role for this cascade. Our findings provide new insight into the regulation of NSC quiescence, which potentially could influence brain health throughout adult life. Electronic supplementary material The online version of this article (10.1007/s00018-018-2790-3) contains supplementary material, which is available to authorized users.

Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiological role of CuII binding to hCtr1.

Aβ4-42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu(II) with an affinity approximately 3000 times higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu(II) from Aβ1-40 when acting in its native Zn7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7 MT-3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity Cu(II) complex of Aβ4-16 is resistant to Zn7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aβ4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aβ4-42 as a Cu(II) scavenger in the synaptic cleft.

The oligomerisation of many proteins and peptides is known to be influenced by the binding of transition metal ions such as divalent copper. To investigate the oligomeric state of model peptides related to the N-terminus of α-synuclein (αSyn) in the presence of Cu(II), electron paramagnetic resonance (EPR) spectroscopy and isotopic labelling were recently used to conclude that Cu(II) occupies N-terminal bridging positions within closed-chain αSyn dimers and trimers with a Cu/peptide stoichiometry of 1:1. Herein, a statistical correction is identified and the consequences are evaluated. The analysis reveals that αSyn forms Cu-bridged antiparallel dimers and closed-chain trimers that coexist with Cu(II)-bound monomers (including a "macrochelate") and, depending on metal stoichiometry and protein environment, with open-chain Cu-bridged oligomers and heterodimers. The results demonstrate that the Cu(II) ion can be exploited as a probe of protein quaternary structure, with the potential to delineate heterogeneous oligomeric populations.

The A&beta, 4&minus, 42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer&rsquo, s Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in A&beta, 16 and A&beta, 9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)A&beta, x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-A&beta, 16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)A&beta, 16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of A&beta, 16, Pd(II)-A&beta, 16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-A&beta, complexes.

The tripeptide NH2&ndash, Gly&ndash, His&ndash, Lys&ndash, COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12- x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11- x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

X-ray absorption spectroscopy of CuII amyloid-β peptide (Aβ) under in situ electrochemical control (XAS-EC) has allowed elucidation of the redox properties of CuII bound to truncated peptide forms. The Cu binding environment is significantly different for the Aβ1–16 and the N-truncated Aβ4–9, Aβ4–12, and Aβ4–16 (Aβ4–9/12/16) peptides, where the N-truncated sequence (F4R5H6) provides the high-affinity amino-terminal copper nickel (ATCUN) binding motif. Low temperature (ca. 10 K) XAS measurements show the adoption of identical CuII ATCUN-type binding sites (CuIIATCUN) by the first three amino acids (FRH) and a longer-range interaction modeled as an oxygen donor ligand, most likely water, to give a tetragonal pyramid geometry in the Aβ4–9/12/16 peptides not previously reported. Both XAS-EC and EPR measurements show that CuII:Aβ4–16 can be reduced at mildly reducing potentials, similar to that of CuII:Aβ1–16. Reduction of peptides lacking the H13H14 residues, CuII:Aβ4–9/12, require far more forcing conditions...

The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo.

The deposition of insoluble β-amyloid (Aβ) peptide into extracellular senile plaques is a pathological hallmark of Alzheimer’s disease. Fragmented α-synuclein (αSyn) and a relatively high concentration of Cu2+ ions are found within amyloid deposits. Both Aβ and αSyn bind Cu2+ with an apparent dissociation constant in the sub-nanomolar range at physiological pH, fuelling speculation that deleterious redox chemistry and metal-mediated protein misfolding may contribute to disease progression. Binary Cu(Aβ) and Cu(αSyn) complexes have been extensively studied, although the Cu2+ coordination of heterogeneous mixtures of Aβ and αSyn has yet to be characterised. This study used synthetic N-terminal fragments Aβ1–16 and αSyn1–56 to reveal a new Cu(αSyn)(Aβ) coordination mode anchored upon a 5,6-membered chelate supplied by Met1–Asp2 of αSyn, with the fourth equatorial ligand being supplied by a His side chain of Aβ.

α-Factor-1 (WHWLQLKPGQPMY), a peptidic pheromone of Saccharomyces cerevisiae yeast, contains a XHX type copper(II) binding N-terminal site. Using a soluble analogue, WHWSKNR-amide, we demonstrated that the W(1)H(2)W(3) site alone binds copper(II) with a Kd value of 0.18 pM at pH 7.4 and also binds imidazole (Im) in a ternary complex (Kd of 1 mM at pH 7.4). This interaction boosts the ability of the peptide to sequester copper(II) depending on the Im concentration up to a subfemtomolar range, not available for any oligopeptidic system studied before. Therefore, α-factor-1 and other XHX-type peptides are likely copper(II) carriers in biological systems.

The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by deposition of extracellular amyloid plaques comprised from fibrillar aggregates of the amyloid-β peptide (Aβ). Cu2+ interactions with Aβ appear to be involved in both the production of reactive oxygen species and the misfolding of Aβ into oligomeric intermediates including covalently cross-linked dimers. We have previously investigated the Cu2+ coordination of Aβ monomers in detail, whilst others have shown that Aβ fibrils coordinate Cu2+ in a similar manner to Aβ monomers. However, the coordination of low-molecular-weight Aβ species, which are believed to be responsible for neuronal dysfunction in AD, has not been widely investigated. Here, we report the first study of Cu2+ coordination by synthetic Aβ dimers containing an artificial diaminopimelic acid (DAP) or a dityrosine cross-link at residue 10. Our preliminary findings show that dityrosine cross-linking imparts unique structural constraints, resulting in Cu2+ coordination distinct from Aβ monomers and fibrils, which may be relevant to the greater toxicity of low-molecular-weight Aβ oligomers in AD.

Graphene oxide (GO) possesses unusual electronic and mechanical properties, including the ability to stabilize graphene radicals (GRs). However, controlled generation of GRs remains a challenge for applications requiring large-scale production. In this study, we demonstrate controlled production of GRs by UVB irradiation of GO solutions. Electron paramagnetic resonance spectroscopy of GO solutions revealed a dose-dependent exponential growth in radical production as a function of UVB exposure time. The GRs were air-stable over a long period, both in the solution state and in freeze-dried powders, suggesting they are graphene-based phenalenyl-like radicals. The redox activity of GRs was demonstrated by their ability to oxidize the chromophore 3,5,3′,5′-tetramethylbenzidine, with oxidation capacity of GO increasing with GR content.

Electron paramagnetic resonance spectroscopy in conjunction with site-selective isotopic labelling provides a valuable tool for probing the local coordination environment of paramagnetic metal ions. Sometimes the metal ligands are inter-molecular in origin, such as during biological catalysis involving enzyme/metal-ion/substrate complexes or within oligomeric forms of peptides and proteins. Inter- and intra-molecular metal binding within peptide oligomers can be delineated by combining two fractions of peptide monomers containing the same amino acid sequence but having different isotopic content. Here, the principles of isotopic dilution in EPR spectroscopy are described and simulations provided to demonstrate its application to the identification of Cu2+-bridging of peptide oligomers.

The N-truncated β-amyloid (Aβ) isoform Aβ4-x is known to bind Cu(2+) via a redox-silent ATCUN motif with a conditional Kd = 30 fM at pH 7.4. This study characterizes the Cu(2+) interactions and redox activity of Aβx-16 (x = 1, 4) and 2-[(dimethylamino)-methyl-8-hydroxyquinoline, a terdentate 8-hydroxyquinoline (8HQ) with a conditional Kd(CuL) = 35 pM at pH 7.4. Metal transfer between Cu(Aβ1-16), CuL, CuL2, and ternary CuL(NIm(Aβ)) was rapid, while the corresponding equilibrium between L and Aβ4-16 occurred slowly via a metastable CuL(NIm(Aβ)) intermediate. Both CuL and CuL2 were redox-silent in the presence of ascorbate, but a CuL(NIm) complex can generate reactive oxygen species. Because the NIm(Aβ) ligand will be readily exchangeable with NIm ligands of ubiquitous protein His side chains in vivo, this class of 8HQ ligand could transfer Cu(2+) from inert Cu(Aβ4-x) to redox-active CuL(NIm). These findings have implications for the use of terdentate 8HQs as therapeutic chelators to treat neurodegenerative disease.

The electron paramagnetic resonance (EPR) spectra of the trigonal prismatic complexes Tc(abt)3, Tc(bdt)3, Re(abt)3 and Re(bdt)3 (abt, O-aminobenzenethiol; bdt, benzene-1,2-dithiol) in dilute frozen solution are interpreted in terms of an axially symmetric spin Hamiltonian, with g values close to two, principal hyperfine couplings of |A Tc| ~ 5–12 × 10−4 cm−1, nuclear quadrupole couplings of |P Tc| ~ 0.3–0.35 × 10−4 cm−1 and the unusual values 5 × 10−4 cm−1 ~ |A Re

Previous studies suggest membrane binding is a key determinant of amyloid β (Aβ) neurotoxicity. However, it is unclear whether this interaction is receptor driven. To address this issue, a D-handed enantiomer of Aβ42 (D-Aβ42) was synthesized and its biophysical and neurotoxic properties were compared to the wild-type Aβ42 (L-Aβ42). The results showed D- and L-Aβ42 are chemically equivalent with respect to copper binding, generation of reactive oxygen species and aggregation profiles. Cell binding studies show both peptides bound to cultured cortical neurons. However, only L-Aβ42 was neurotoxic and inhibited long term potentiation indicating L-Aβ42 requires a stereospecific target to mediate toxicity. We identified the lipid phosphatidylserine, as a potential target. Annexin V, which has very high affinity for externalized phosphatidylserine, significantly inhibited L-Aβ42 but not D-Aβ42 binding to the cultured cortical neurons and significantly rescued L-Aβ42 neurotoxicity. This suggests that Aβ mediated toxicity in Alzheimer disease is dependent upon Aβ binding to phosphatidylserine on neuronal cells.

α-Synuclein (αS) is a small natively unfolded protein whose interactions with Cu2+ have been proposed to play a role in Parkinson’s disease (PD). We recently studied the Cu2+ coordination of recombinant human αS using electron paramagnetic resonance spectroscopy and identified two coordination modes at physiological pH, one anchored upon the amino terminus (mode 1) and the other anchored upon the side chain of His50 (mode 2). Here we report the Cu2+ coordination of the A30P, E46K and A53T mutants associated with inherited forms of PD. At physiological pH, the same two Cu2+ coordination modes were adopted by each of the familial mutants. The spectrum of Cu2+/αS(A53T) was very similar to the spectrum of the native Cu2+/αS complex; however, mode 2 coordination was marginally higher in the spectrum of Cu2+/αS(E46K) and considerably more favored in the Cu2+/αS(A30P) complex. The alteration in only the relative proportion of modes 1 and 2 suggests the familial mutations introduce structural changes of the protein backbone that indirectly affect the stability, but not the identity, of the native Cu2+ coordination modes.

Beta-cleavage of the neurodegenerative disease-associated prion protein (PrP) protects cells from death induced by oxidative insults. The beta-cleavage event produces two fragments, designated N2 and C2. We investigated the role of the N2 fragment (residues 23-89) in cellular stress response, determining mechanisms involved and regions important for this reaction. The N2 fragment differentially modulated the reactive oxygen species (ROS) response induced by serum deprivation, with amelioration when copper bound. Amino acid residues 23-50 alone mediated a ROS reduction response. PrP23-50 ROS reduction was not due to copper binding or direct antioxidant activity, but was instead mediated through proteoglycan binding partners localised in or interacting with cholesterol-rich membrane domains. Furthermore, mutational analyses of both PrP23-50 and N2 showed that their protective capacity requires the sterically constraining double proline motif within the N-terminal polybasic region. Our findings show that N2 is a biologically active fragment that is able to modulate stress-induced intracellular ROS through interaction of its structurally defined N-terminal polybasic region with cell-surface proteoglycans.

The active sites of mononuclear molybdenum-containing enzymes contain a low-symmetry Mo(V)-dithiolene intermediate whose structure can be probed using electron paramagnetic resonance (EPR). The relationship between experimental EPR spectra and the electronic and geometric structure of the active site can be difficult to establish, not least because of the low molecular symmetry. When density functional theory is used, it is possible to assess this relationship by systematically varying the geometric structure and comparing the theoretical EPR parameters with those obtained experimentally. We employed this approach to examine the relationship between the metal-dithiolate fold angle and the monoclinic spin Hamiltonian parameters (g, A, beta) of a prototypical mononuclear molybdenyl model complex. By comparing the experimental EPR parameters with these results, we show that the metal-dithiolate fold angle of the complex in solution may be obtained from the non-coincidence angle beta that transforms the principal axes of g to those of A. This will provide a useful method for probing the structure of the Mo(V) intermediate of mononuclear molybdenum enzymes, where the electronic structure of the active site is modulated by the fold angle of the dithiolate ligand (the "metal-dithiolate folding effect").

Numerous conflicting models have been proposed regarding the nature of the Cu(2+) coordination environment of the amyloid beta (Abeta) peptide, the causative agent of Alzheimer's disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu(2+) and the ligand nuclei of Abeta, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Abeta16 analogues with site-specific (15)N-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu(2+) coordination modes, {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H13)} (component Ia) and {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H14)} (component Ib), between pH 6-7. A third coordination mode (component II) was identified at pH 8.0, and simulation of the superhyperfine resonances indicated a 3N1O coordination sphere involving nitrogen ligation by His6, His13, and His14. No differences were observed upon (17)O-labeling of the phenolic oxygen of Tyr10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific (15)N-labeling, provided additional support for the common role of His6 in components Ia and Ib, and for the assignment of a {O, N(epsilon)(H6), N(epsilon)(H13), N(epsilon)(H14)} coordination sphere to component II. HYSCORE studies of a peptide analogue with selective (13)C-labeling of Asp1 revealed (13)C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu(2+) ligand interactions, together with the key finding that component I is composed of two distinct coordination modes, provides valuable insight into a range of conflicting ligand assignments and highlights the complexity of Cu(2+)/Abeta interactions.

The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) has been proposed to play a role in Parkinson's disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-COO(-), H2O} binding persisted in conjunction with another two binding modes. Metal-ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH2, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.

Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the Cu(II) site of the copper-resistance protein PcoC from Escherichia coli and certain variant forms. Electron spin echo envelope modulation (ESEEM) experiments confirm the presence of two histidine ligands, His1 and His92, at the Cu(II) site of wild-type PcoC, consistent with the available X-ray crystallographic data for the homolog CopC (67% sequence identity) from Pseudomonas syringae pv. tomato. The variants H1F and H92F each lack one of the histidine residues close to the Cu(II) site. The ESEEM data suggest that the surviving histidine residue remains as a ligand. The nA variant features an extra alanine residue at the N terminus, which demotes the His1 ligand to position 2. At least one of the two histidine residues is bound at the Cu(II) site in this form. Simulation of the (14)N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the Cu(II) sites of the wild-type, H92F and nA forms, while the H1F variant has two nitrogen ligands. The spectra of wild-type form can be fitted adequately with a 3N or a 4N model. The former is consistent with the crystal structure of the CopC homolog, where His1 acts as a bidentate ligand. The latter raises the possibility of an additional unidentified nitrogen ligand. The markedly different spectra of the H1F and nA forms compared with the wild-type and H92F proteins further highlight the integral role of the N-terminal histidine residue in the high-affinity Cu(II) site of PcoC.

Reaction of Tp*MoVSCl2 with a variety of phenols and thiols in the presence of triethylamine produces mononuclear, thiomolybdenyl complexes Tp*MoVSX2 [Tp* = hydrotris(3,5-dimethylpyrazol-1-yl)borate; X = 2-(ethylthio)phenolate (etp), 2-(n-propyl)phenolate (pp), phenolate; X2 = benzene-1,2-dithiolate (bdt), 4-methylbenzene-1,2-dithiolate (tdt), benzene-1,2-diolate (cat)]. The complexes have been characterized by microanalysis, mass spectrometry, IR, EPR, and UV-visible spectroscopic data, and X-ray crystallography (for the etp, pp, bdt, and cat derivatives). The mononuclear, six-coordinate, distorted-octahedral Mo centers are coordinated by terminal sulfido (MoS = 2.123(1)-2.1368(8) A), tridentate facial Tp*, and monodentate or bidentate O/S-donor ligands. Multifrequency (S-, X-, Q-band) EPR spectra of the complexes and selected molybdenyl analogues were acquired at 130 K and 295 K and yielded a spin Hamiltonian of Cs symmetry or lower, with gzzgyygxxge and Az'z'Ax'x' approximately Ay'y', and a noncoincidence angle in the range of beta = 24-39 degrees . Multifrequency EPR, especially at S-band, was found to be particularly valuable in the unambiguous assignment of the spin Hamiltonian parameters in these low-symmetry complexes. The weaker pi-donor terminal sulfido ligand yields a smaller SOMO-LUMO gap and reduced g-values for the thiomolybdenyl complexes compared with molybdenyl analogues, supporting existing crystallographic and EPR data for an apically coordinated oxo group in the active site of xanthine oxidase.

Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.

New copper(II) complexes of general empirical formula, Cu(mpsme)X center dot xCH(3)COCH(3) (mpsme = anionic form of the 6-methyl-2-formylpyridine Schiff base of S-methyldithiocarbazate; X = Cl, N-3, NCS, NO3; x = 0, 0.5) have been synthesized and characterized by IR, electronic, EPR and susceptibility measurements. Room temperature mu(eff) values for the complexes are in the range 1.75-2.1 mu(beta) typical of uncoupled or weakly coupled Cu(II) centres. The EPR spectra of the [Cu(mpsme)X] (X = Cl, N-3, NO3, NCS) complexes reveal a tetragonally distorted coordination sphere around the mononuclear Cu(II) centre. We have exploited second derivative EPR spectra in conjunction with Fourier filtering (sine bell and Hamming functions) to extract all of the nitrogen hyperfine coupling matrices. While the X-ray crystallography of [Cu(mpsme)NCS] reveals a linear polymer in which the thiocyanate anion bridges the two copper(II) ions, the EPR spectra in solution are typical of a magnetically isolated monomeric Cu(II) centres indicating dissociation of the polymeric chain in solution. The structures of the free ligand, Hmpsme and the {[Cu(mpsme)NO3] center dot 0.5CH(3)COCH(3)}(2) and [Cu(mpsme)NCS](n) complexes have been determined by X-ray diffraction. The {[Cu(mpsme)NO3]0.5CH(3)COCH(3)}(2) complex is a centrosymmetric dimer in which each copper atom adopts a five-coordinate distorted square-pyramidal geometry with an N2OS2 coordination environment, the Schiff base coordinating as a uninegatively charged tridentate ligand chelating through the pyridine and azomethine nitrogen atoms and the thiolate, an oxygen atom of a unidentate nitrato ligand and a bridging sulfur atom from the second ligand completing the coordination sphere. The [Cu(mpsme)(NCS)](n) complex has a novel staircase-like one dimensional polymeric structure in which the NCS- ligands bridge two adjacent copper(II) ions asymmetrically in an end-to-end fashion providing its nitrogen atom to one copper and the sulfur atom to the other. (c) 2005 Elsevier B.V. All rights reserved.

Electron spin transient nutation (ESTN) experiments show that the spin multiplicity of the ground state of C-60(3-) in frozen solution is a doublet with S = 1/2. In purified samples, there is no evidence for excited states or other species with higher multiplicity. In the anions Of C120On- (n = 2, 3, 4), where the CW EPR experiments have shown that a mixture of species is present, ESTN experiments confirm that a doublet with S = 1/2 is associated with the 3- anion and triplets with S = 1 are associated with the 2- and 4- anions. A weak nutation peak attributable to m(s) = -1/2 1/2 transitions within a quartet state may arise from association of anions with spins of 1/2 and 1 in solute aggregates.

Superhyperfine interactions in inhomogeneously broadened paramagnetic centers are observed using a single high-turn-angle microwave pulse. The free induction signal that follows the hole-burning pulse exhibits oscillations that are distinct from the oscillatory free induction decay observable in some inhomogeneously broadened systems. It contains frequencies characteristic of the superhyperfine splittings, together with a zero frequency component. Experimental examples of the effect in both orientationally disordered (powdered) and structurally disordered (glassy) systems are presented and compared with the conceptually similar Fourier transform electron paramagnetic resonance detected nuclear magnetic resonance experiment, together with numerical simulations.

The oligomerization of α-synuclein (αSyn) is one of the defining features of Parkinson's disease. Binding of divalent copper to the N terminus of αSyn has been implicated in both its function and dysfunction. Herein, the molecular details of the Cu(II) /αSyn binding interface have been revealed using a library of synthetic 56-residue αSyn peptides containing site-specific isotopic labels. Using electron paramagnetic resonance spectroscopy, αSyn is shown to coordinate Cu(II) with high affinity via two pH-dependent coordination modes between pH 6.5-8.5. Most remarkably, the data demonstrate that the dominant mode is associated with binding to oligomers (antiparallel dimers and/or cyclic trimers) in which Cu(II) ions occupy intermolecular bridging sites. The findings provide a molecular link between Cu(II) -bound αSyn and its associated quaternary oligomeric structure.

The roasting of coffee beans generates stable radicals within melanoidins produced by non-enzymatic browning. Roasting coffee beans has further been suggested to increase the antioxidant (AO) capacity of coffee brews. Herein, we have characterized the radical content and AO capacity of brews prepared from Coffea arabica beans sourced directly from an industrial roasting plant. In-tact beans exhibited electron paramagnetic resonance signals arising from Fe3+, Mn2+ and at least three distinct stable radicals as a function of roasting time, whose intensity changed upon grinding and ageing. In coffee brews, the roasting-induced radicals were harboured within the high molecular weight (> 3 kD) melanoidin-containing fraction at a concentration of 15 nM and was associated with aromatic groups within the melanoidins. The low molecular weight (< 3 kD) fraction exhibited the highest AO capacity using DPPH as an oxidant. The AO activity was not mediated by the stable radicals or by metal complexes within the brew. While other non-AO functions of the roasting-induced radical and metal complexes may be possible in vivo, we confirm that the in vitro antiradical activity of brewed coffee is dominated by low molecular weight phenolic compounds.

Field-swept pulsed electron paramagnetic resonance (EPR) spectra of a ZBLAN fluoride glass doped with a low concentration of Cr3+ are obtained using echo-detected EPR and hole-burning free induction decay detection. We review the utility of the pulsed EPR technique in generating field-swept EPR spectra, as well as some of the distorting effects that are peculiar to the pulsed detection method. The application of this technique to Cr3+-doped ZBLAN reveals that much of the broad resonance extending from geff = 5.1 to geff = 1.97, characteristic of X-band continuous wave EPR of Cr3+ in glasses, is absent. We attribute this largely to the variation in nutation frequencies across the spectrum that result from sites possessing large fine structure interactions. The description of the spin dynamics of such sites is complicated and we discuss some possible approaches to the simulation of the pulsed EPR spectra.

Spin Hamiltonian parameters for the neutral trigonal-prismatic Tc(abt)(3) and Re(abt)(3) chelates (abt = o-aminobenzenethiol) are calculated using relativistic density functional theory at the all-electron level. The small magnitude of the calculated g shifts and metal hyperfine interactions is in excellent agreement with previous experimental predictions based upon a ligand-centered ground-state magnetic orbital. The theoretical (14)N ligand hyperfine and quadrupole couplings also reproduce the nuclear frequencies measured by electron spin-echo envelope modulation spectroscopy. The nuclear quadrupole interaction of (187)Re is predicted to be 2 orders of magnitude larger than that of (99)Tc, in agreement with empirical simulation of the continuous-wave electron paramagnetic resonance spectrum. The spectrum of Tc(abt)(3) at high solute concentrations contains a central resonance not predicted for the isolated complex by theoretical calculations. The absence of this resonance at low solute concentrations provides evidence of intermolecular interactions in these systems.

A family of dinuclear cobalt complexes with bridging bis(dioxolene) ligands derived from 3,3,3',3'-tetramethyl-1,1'-spirobis(indane-5,5',6,6'-tetrol) (spiroH4) and ancillary ligands based on tris(2-pyridylmethyl)amine (tpa) has been synthesized and characterized. The bis(dioxolene) bridging ligand is redox-active and accessible in the (spiro(cat-cat))(4-), (spiro(SQ-cat))(3-), and (spiro(SQ-SQ))(2-) forms, (cat = catecholate, SQ = semiquinonate). Variation of the ancillary ligand (Mentpa; n = 0-3) by successive methylation of the 6-position of the pyridine rings influences the redox state of the complex, governing the distribution of electrons between the cobalt centers and the bridging ligands. Pure samples of salts of the complexes [Co2(spiro)(tpa)2](2+) (1), [Co2(spiro)(Metpa)2](2+) (2), [Co2(spiro)(Me2tpa)2](2+) (3), [Co2(spiro)(Me3tpa)2](2+) (4), [Co2(spiro)(tpa)2](3+) (5), and [Co2(spiro)(tpa)2](4+) (6) have been isolated, and 1, 4, and 6 have been characterized by single crystal X-ray diffraction. Studies in the solid and solution states using multiple techniques reveal temperature invariant redox states for 1, 2, and 4-6 and provide clear evidence for four different charge distributions: 1 and 2 are Co(III)-(spiro(cat-cat))-Co(III), 4 is Co(II)-(spiro(SQ-SQ))-Co(II), 5 is Co(III)-(spiro(SQ-cat))-Co(III), and 6 is Co(III)-(spiro(SQ-SQ))-Co(III). Of the six complexes, only 3 shows evidence of temperature dependence of the charge distribution, displaying a rare thermally induced two-step valence tautomeric transition from the Co(III)-(spiro(cat-cat))-Co(III) form to Co(II)-(spiro(SQ-cat))-Co(III) and then to Co(II)-(spiro(SQ-SQ))-Co(II) in both solid and solution states. This is the first time a two-step valence tautomeric (VT) transition has been observed in solution. Partial photoinduction of the VT transition is also possible in the solid. Magnetic and spectroscopic studies of 5 and 6 reveal that spiroconjugation of the bis(dioxolene) ligand allows electronic interaction across the spiro bridge, suggesting that thermally activated vibronic coupling between the two cobalt-dioxolene moieties plays a key role in the two-step transition evident for 3.

8-Hydroxyquinolines (8HQ) have found widespread application in chemistry and biology due to their ability to complex a range of transition metal ions. The family of 2-substituted 8HQs has been proposed for use in the treatment of Alzheimer's disease (AD). Most notably, the therapeutic PBT2 (Prana Biotechnology Ltd.) has been shown to act as an efficient metal chaperone, disaggregate metal-enriched amyloid plaques comprised of the Aβ peptide, inhibit Cu/Aβ redox chemistry, and reverse the AD phenotype in transgenic animal models. Yet surprisingly little is known about the molecular interactions at play. In this study, we show that the homologous ligand 2-[(dimethylamino)methyl]-8-hydroxyquinoline (HL) forms a CuL complex with a conditional (apparent) dissociation constant of 0.33 nM at pH 6.9 and is capable of forming ternary Cu(2+) complexes with neurotransmitters including histamine (HA), glutamic acid (Glu), and glycine (Gly), with glutathione disulfide (GSSG), and with histidine (His) side chains of proteins and peptides including the Aβ peptide. Our findings suggest a molecular basis for the strong metal chaperone activity of PBT2, its ability to attenuate Cu(2+)/Aβ interactions, and its potential to promote neuroprotective and neuroregenerative effects.

Quadridentate ligand H(2)L binds Cu(I) to form salt [Cu(I)(H(2)L)]BF(4) which undergoes aerial oxidation in solution with isolation of the diamagnetic salt [Cu(II)L˙(-)]BF(4) where π-radical anion L˙(-) is the (1e(-), 2H(+)) oxidation product of H(2)L.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. Within the brain, senile plaques, which comprise extracellular deposits of the amyloid-β peptide (Aβ), are the most common pathological feature of AD. A high concentration of Cu(2+) is found within these plaques, which are also areas under oxidative stress. Laboratory work has shown that in vitro Aβ will react with Cu(2+) to induce peptide aggregation and the production of reactive oxygen species. As such, this interaction offers a possible explanation for two of the defining pathological features observed in the AD brain: the presence of amyloid plaques, which consist largely of insoluble Aβ aggregates, and the abundant oxidative stress therein. Researchers have accordingly put forth the "metals hypothesis" of AD, which postulates that compounds designed to inhibit Cu(2+)/Aβ interactions and redistribute Cu(2+) may offer therapeutic potential for treating AD. Characterization of the pH-dependent Cu(2+) coordination of Aβ is fundamental to understanding the neurological relevance of Cu(2+)/Aβ interactions and aiding the design of new therapeutic agents. In an effort to shed light on the problem, many experimental and theoretical techniques, using a variety of model systems, have been undertaken. The preceding decade has seen numerous conflicting spectroscopic reports concerning the nature of the Cu(2+)/Aβ coordination. As the number of studies has grown, the nature of the pH-dependent ligand environment surrounding the Cu(2+) cation has remained a point of contention. In large part, the difficulties can be attributed to inappropriate choices of the model system or to methods that are incapable of quantitatively delineating the presence and identity of multiple Cu(2+) coordination modes. Electron paramagnetic resonance (EPR) is the method of choice for studying paramagnetic metal-protein interactions. With the introduction of site-specific (15)N, (17)O, and (13)C isotopic labels and the application of advanced techniques, EPR is capable of eliminating much of the ambiguity. Recent EPR studies have produced the most definitive picture of the pH-dependent Cu(2+) coordination modes of Aβ and enabled researchers to address the inconsistencies present in the literature. In this Account, we begin by briefly introducing the evidence for a role of Cu(2+) in AD as well as the potential physiological and therapeutic implications of that role. We then outline the EPR methodology used to resolve the molecular details of the Cu(2+)/Aβ interactions. No drugs are currently available for altering the course of AD, and existing therapies only offer short-term symptomatic relief. This focused picture of the role of Cu(2+) in AD-related plaques offers welcome potential for the development of new methods to combat this devastating disease.

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

Background The amyloid-β (Aβ) peptide is the primary component of the extracellular senile plaques characteristic of Alzheimer's disease (AD). The metals hypothesis implicates redox-active copper ions in the pathogenesis of AD and the Cu2+ coordination of various Aβ peptides has been widely studied. A number of disease-associated modifications involving the first 3 residues are known, including isomerisation, mutation, truncation and cyclisation, but are yet to be characterised in detail. In particular, Aβ in plaques contain a significant amount of truncated pyroglutamate species, which appear to correlate with disease progression. Methodology/Principal Findings We previously characterised three Cu2+/Aβ1–16 coordination modes in the physiological pH range that involve the first two residues. Based upon our finding that the carbonyl of Ala2 is a Cu2+ ligand, here we speculate on a hypothetical Cu2+-mediated intramolecular cleavage mechanism as a source of truncations beginning at residue 3. Using EPR spectroscopy and site-specific isotopic labelling, we have also examined four Aβ peptides with biologically relevant N-terminal modifications, Aβ1[isoAsp]–16, Aβ1–16(A2V), Aβ3–16 and Aβ3[pE]–16. The recessive A2V mutation preserved the first coordination sphere of Cu2+/Aβ, but altered the outer coordination sphere. Isomerisation of Asp1 produced a single dominant species involving a stable 5-membered Cu2+ chelate at the amino terminus. The Aβ3–16 and Aβ3[pE]–16 peptides both exhibited an equilibrium between two Cu2+ coordination modes between pH 6–9 with nominally the same first coordination sphere, but with a dramatically different pH dependence arising from differences in H-bonding interactions at the N-terminus. Conclusions/Significance N-terminal modifications significantly influence the Cu2+ coordination of Aβ, which may be critical for alterations in aggregation propensity, redox-activity, resistance to degradation and the generation of the Aβ3–× (× = 40/42) precursor of disease-associated Aβ3[pE]–x species.

Inclusions of aggregated alpha-synuclein (alpha-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson's disease (PD). In vitro, alpha-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of alpha-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on alpha-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive beta-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of alpha-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the beta-sheet structure found in other pre-fibrillar forms of alpha-syn.