Copper-mediated Amyloid-β Toxicity Is Associated with an Intermolecular Histidine Bridge
2006; Elsevier BV; Volume: 281; Issue: 22 Linguagem: Inglês
10.1074/jbc.m600417200
ISSN1083-351X
AutoresDavid P. Smith, Danielle G. Smith, Cyril C. Curtain, John F. Boas, John R. Pilbrow, Giuseppe D. Ciccotosto, Tong‐Lay Lau, Deborah J. Tew, Keyla Perez, John D. Wade, Ashley I. Bush, Simon C. Drew, Frances Separovic, Colin L. Masters, Roberto Cappai, Kevin J. Barnham,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoAmyloid-β peptide (Aβ) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Aβ-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Aβ-Cu2+ complex to cultured primary cortical neurons was attenuated when either the π -or τ-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Aβ and Aβ-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Aβ toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge. Amyloid-β peptide (Aβ) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Aβ-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Aβ-Cu2+ complex to cultured primary cortical neurons was attenuated when either the π -or τ-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Aβ and Aβ-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Aβ toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge. The interaction of metals with the amyloid-β peptide (Aβ) 4The abbreviations used are: Aβ, amyloid peptide; PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine]; His-π-Me, amyloid peptide 1-42 where the His residues have been methylated at π-nitrogen of the imidazole side chain; His-τ-Me, amyloid peptide 1-42 where the His residues have been methylated at τ-nitrogen of the imidazole side chain; AD, Alzheimer disease; CQ, clioquinol; DPH, diphenyl-1,3,5-hexatriene; HFIP, hexaflouro-2-propanol; LUVs, large unilamellar vesicles; ROS, reactive oxygen species; PBS, phosphate-buffered saline; ThT, thioflavin-T; WT, wild type; Fmoc, N-(9-fluorenyl)methoxycarbonyl; MTS, 3-(4,5,-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.4The abbreviations used are: Aβ, amyloid peptide; PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine]; His-π-Me, amyloid peptide 1-42 where the His residues have been methylated at π-nitrogen of the imidazole side chain; His-τ-Me, amyloid peptide 1-42 where the His residues have been methylated at τ-nitrogen of the imidazole side chain; AD, Alzheimer disease; CQ, clioquinol; DPH, diphenyl-1,3,5-hexatriene; HFIP, hexaflouro-2-propanol; LUVs, large unilamellar vesicles; ROS, reactive oxygen species; PBS, phosphate-buffered saline; ThT, thioflavin-T; WT, wild type; Fmoc, N-(9-fluorenyl)methoxycarbonyl; MTS, 3-(4,5,-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. plays a pivotal role in the pathogenesis of Alzheimer disease (AD). Transition metals, such as copper (Cu2+), zinc (Zn2+), and iron (Fe3+), are enriched in Aβ plaques (1Bush A.I. Trends Neurosci. 2003; 26: 207-214Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar), and the Aβ peptide possesses both high and low affinity Cu2+ and Zn2+ binding sites in vitro. These interactions with Cu2+, Zn2+, and to a lesser extent Fe3+ have been reported to control the aggregation state of Aβ. Moreover, the Aβ-Cu2+ complexes are redoxactive, suggesting that the oxidative stress observed in AD patients is related to the production of reactive oxygen species (ROS) by metal-bound forms of Aβ and may be central to the pathological mechanism of AD (reviewed by Bush (1Bush A.I. Trends Neurosci. 2003; 26: 207-214Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar)). The coordination environment of Cu2+ bound to Aβ has been studied by EPR, Raman, and NMR spectroscopies. The consensus is that the coordination of Cu2+ to Aβ is via the three His residues His6, His13, and His14 and an as yet undefined fourth ligand; options put forward include tyrosine (Tyr10) (2Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 3Curtain C.C. Ali F.E. Smith D.G. Bush A.I. Masters C.L. Barnham K.J. J. Biol. Chem. 2003; 278: 2977-2982Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 4Tickler A. Smith D. Ciccotosto G. Tew D. Curtain C.C. Carrington D. Masters C.L. Bush A.I. Cherny R.A. Cappai R. Wade J. Barnham K.J. J. Biol. Chem. 2005; 280: 13355-13363Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5Huang X. Cuajungco M.P. Atwood C.S. Hartshorn M.A. Tyndall J.D. Hanson G.R. Stokes K.C. Leopold M. Multhaup G. Goldstein L.E. Scarpa R.C. Saunders A.J. Lim J. Moir R.D. Glabe C. Bowden E.F. Masters C.L. Fairlie D.P. Tanzi R.E. Bush A.I. J. Biol. Chem. 1999; 274: 37111-37116Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar, 6Antzutkin O.N. Magn. Reson. Chem. 2004; 42: 231-246Crossref PubMed Scopus (66) Google Scholar), the N-terminal nitrogen (7Syme C.D. Nadal R.C. Rigby S.E. Viles J.H. J. Biol. Chem. 2004; 279: 18169-18177Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), or an as yet unidentified carboxylate side chain (8Karr J.W. Kaupp L.J. Szalai V.A. J. Am. Chem. Soc. 2004; 126: 13534-13538Crossref PubMed Scopus (145) Google Scholar, 9Karr J.W. Akintoye H. Kaupp L.J. Szalai V.A. Biochemistry. 2005; 44: 5478-5487Crossref PubMed Scopus (162) Google Scholar) (Fig. 1a). Recently, Glu11 has been identified as providing the carboxylate side chain when Zn2+ is bound to Aβ 1–16 (10Zirah S. Kozin S.A. Mazur A. Blond A. Cheminant M. Segalas-Milazzo I. Debey P. Rebuffat S. J. Biol. Chem. 2006; 281: 2151-2161Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The EPR studies of Curtain et al. (2Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) showed that in the pH range 6.0 to 7.5 in PBS, an increase in the Cu2+/peptide molar ratio above ∼0.3:1 broadened the mononuclear Cu2+ spectrum observed at low ratios. At Cu2+/peptide molar ratios greater than 0.6:1, a distinctive broad resonance developed near g ∼ 2. This broad resonance was attributed to exchange interactions between two Cu2+ ions ∼6 Ä apart, with the second Cu2+ ion being coordinated to Aβ in a cooperative manner via a His bridge (Fig. 1b), similar to that observed by Ohtsu et al. (11Ohtsu H. Shimazaki Y. Odani A. Yamauchi O. Mori W. Itoh S. Fukuzumi S. J. Am. Chem. Soc. 2000; 122: 5733-5741Crossref Scopus (201) Google Scholar) for Cu2+ bridge imidizolate complexes. Here we report the first observation of resonances at g ∼ 4 in the EPR spectra of Aβ-Cu2+ peptides, where the broad g ∼ 2 line is also observed. Such EPR spectra are diagnostic for the existence of binuclear Cu2+ centers where the Cu2+ ions are coupled by dipolar interactions; simulations presented here demonstrate that these atoms are 6.2 ± 0.2 Ä apart (12Smith T.D. Pilbrow J.R. Coord. Chem. Rev. 1974; 13: 173-278Crossref Scopus (444) Google Scholar). This distance is consistent with a His bridge between the Cu2+ atoms. To further the understanding of the role played by the His bridge and Aβ-lipid interactions in the neurotoxicity of Aβ, we examine the toxic mechanism of both metal-free and Cu2+-bound forms of the Aβ peptide. The link between His bridge formation and neurotoxicity was explored through experiments using Aβ 1-42 peptides modified by the methylation of the His residues at either the π- or τ-nitrogen of the imidazole side chain (Fig. 1c). Correspondingly modified Aβ 1-40 has been shown to be nontoxic, despite being redox-active in a manner similar to the wild type (WT) peptide (4Tickler A. Smith D. Ciccotosto G. Tew D. Curtain C.C. Carrington D. Masters C.L. Bush A.I. Cherny R.A. Cappai R. Wade J. Barnham K.J. J. Biol. Chem. 2005; 280: 13355-13363Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This lack of toxicity has been attributed to these peptides being unable to bind to cell surface membranes, congruent with the notion that Aβ-lipid membrane interactions are central to the cause of neurotoxicity in AD (13Eckert G.P. Wood W.G. Muller W.E. Subcell. Biochem. 2005; 38: 319-337Crossref PubMed Google Scholar, 14Kagan B.L. Hirakura Y. Azimov R. Azimova R. Lin M.C. Peptides. 2002; 23: 1311-1315Crossref PubMed Scopus (203) Google Scholar, 15Butterfield D.A. Boyd-Kimball D. Brain Pathol. 2004; 14: 426-432Crossref PubMed Scopus (217) Google Scholar, 16Kawahara M. Curr. Alzheimer Res. 2004; 1: 87-95Crossref PubMed Scopus (45) Google Scholar, 17Puglielli L. Friedlich A.L. Setchell K.D. Nagano S. Opazo C. Cherny R.A. Barnham K.J. Wade J.D. Melov S. Kovacs D.M. Bush A.I. J. Clin. Invest. 2005; 115: 2556-2563Crossref PubMed Scopus (123) Google Scholar). Here we describe the Cu2+ coordination, lipid interactions, and aggregation properties of both the neurotoxic WT Aβ 1-42 peptide and Aβ 1-42 where the His residues have been methylated at either the π- or τ-nitrogen of the imidazole side chain. The Cu2+-bound WT Aβ 1-42 peptide was shown to be significantly more toxic, as monitored by the viability of primary cortical neurons, under conditions in which the His bridge was formed. Perturbation of this metal coordination mode leads to a loss in toxicity even in the presence of Cu2+. We also show that toxicity correlates with the ability of the peptides to form dityrosine and induce lipid peroxidation. Toxicity displays a negative correlation with the formation of amyloid material and the ability to perturb the acyl-chain region of lipid bilayer. These results show that the ability to form aCu2+-linked His bridge dimer, resulting ultimately in lipid peroxidation, is central to the metal-meditated toxicity of Aβ. All reagents were purchased from Sigma unless otherwise stated. Peptide Synthesis—The uncommon Nim-protected amino acids, Fmoc-l-His(N-1-Me)-OH (His-π-Me) and Fmoc-l-His(N-3-Me)-OH (His-τ-Me), were purchased from Bachem (Bubendorf, Switzerland); all were used without further purification. All modified peptides were synthesized according to methods outlined previously (18Tickler A.K. Barrow C.J. Wade J.D. J. Pept. Sci. 2001; 7: 488-494Crossref PubMed Scopus (82) Google Scholar). Due to the large degree of similarity between the peptides, large batches of resins were prepared and split at appropriate points in the synthetic pathway such that each peptide was prepared on a scale of 0.03 mmol. WT Aβ 1-42 was purchased from the W. M. Keck Laboratory (Yale University, New Haven, CT). Peptide and Aggregate Preparation—PBS is defined as 10 mm sodium phosphate, 150 mm NaCl at pH 7.4. Cu2+ was present as a solution of 1 part CuCl2 to 6 parts glycine. The addition of a glycine counterion was essential in order to prevent the formation of insoluble phosphate-metal complexes. A 25 mm stock solution was made and diluted in deionized water to the desired concentration as required. Peptide working solutions were prepared by taking a known amount of Aβ 1-42 and then dissolving it in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP). The peptide was incubated at 25 °C for 1 h to remove any preformed structure. Known amounts of peptide were then aliquoted out, and HFIP was removed by evaporation. The resulting peptide film was stored at –80 °C until required. The film was dissolved in 20 mm NaOH and then diluted out in deionized water and 10× PBS at a v/v/v ratio of 2:7:1. All solutions were sonicated in a water bath for 10 min and centrifuged for 10 min at 4 °C at high speed before the addition of 10× PBS. The supernatant was kept on ice for immediate use. The absorbance value at 214 nm was measured, and the concentration of the peptides was determined using a molar extinction coefficient value of 75,887 liters/mol/cm, adjusting to the required concentration with buffer A. CuCl2/Gly was added to the required concentration from a 25 mm stock. Samples were used immediately or incubated at 37 °C with agitation at 200 rpm for either 2 or 24 h. EPR Spectroscopy—Continuous wave EPR spectra were obtained with Bruker ESP380E FT/CW and ECS106 X-band spectrometers. Temperatures around 120 K in both were achieved with a Bruker nitrogen gas flow insert in the standard rectangular TE012 cavity. Spectra acquired at 2.5 K using the ESP380E were achieved using an Oxford Instruments CF935 cryostat with an ER4118 cylindrical cavity insert. The microwave frequency was measured with an EIP microwave 548A frequency counter, and the g factors were determined with reference to the F+ line in CaO (19Wertz J.E. Orton J.W. Auzins P. Discuss. Faraday Soc. 1961; 31: 140-150Crossref Google Scholar). Spectrum simulations were performed with the SOPHE software (version 1.1.4) described by Hanson et al. (20Hanson G.R. Gates K.E. Noble C.J. Griffin M. Mitchell A. Benson S. J. Inorg. Biochem. 2004; 98: 903-916Crossref PubMed Scopus (231) Google Scholar). For the EPR measurements, the chloride of 99.99% pure 65Cu (Cambridge Isotopes) was used. The peptides were dissolved from an HFIP-treated stock to 100 μm in deionized water, and 65CuCl2 to the desired Cu2+/peptide molar ratio was added immediately from a 2.0 mm stock solution via a glass microsyringe. The pH was then adjusted to 7.4 by adding concentrated PBS or ethylmorpholine buffer, and after mixing, samples were rapidly transferred to Wilmad “Suprasil” EPR tubes and frozen immediately. Before and after the EPR spectroscopy, metal concentrations were measured by inductively coupled plasma mass spectrometry, and peptide concentrations were determined by quantitative amino acid analysis. High Performance Immobilized Metal Ion Chromatography—All solutions were delivered to the column at a flow rate of 1.5 ml/min. The high performance immobilized metal ion chromatography column (Amersham Biosciences) was swollen in PBS (pH 7.4), washed in EDTA (50 mm) to remove trace metals, and then washed with deionized H2O. The column was charged with Cu2+ by injecting 2 ml of 0.1 m CuCl2/glycine(1:6). The excess metal was removed by washing the column with deionized H2O. The column was then re-equilibrated in PBS (15 ml). The peptides were prepared at 1 mm in 10% 2′,2′,2′-trifluoroethanol/PBS, and 5 μl was injected onto the column. The peptides were eluted using either a pH gradient (7.4–2.5) generated by a linear increase (0–100%) in PBS at pH 2.5 or an imidazole gradient (0–100% 500 mm imidazole in PBS at pH 7.4). Primary Neuronal Cultures—Cortical neuronal cultures were prepared as described previously (21Ciccotosto G.D. Tew D. Curtain C.C. Smith D. Carrington D. Masters C.L. Bush A.I. Cherny R.A. Cappai R. Barnham K.J. J. Biol. Chem. 2004; 279: 42528-42534Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Briefly, embryonic day 14 BL6Jx129sv mouse cortices were removed, dissected free of meninges, and dissociated in 0.025% (w/v) trypsin in Krebs buffer. The dissociated cells were triturated using a filter-plugged fine pipette tip, pelleted, resuspended in plating medium (minimum Eagle's medium, 10% fetal calf serum, 5% horse serum), and counted. Cortical neuronal cells were plated into poly(l-lysine)-coated 48-well plates at a density of 125,000 cells/well in plating medium. All cultures were maintained in an incubator set at 37 °C with 5% CO2. After 2 h, the plating medium was replaced with fresh neurobasal medium containing B27 supplements, Geneticin, and 0.5 mm glutamine (all tissue culture reagents were purchased from Invitrogen unless otherwise stated). This method resulted in cultures highly enriched for neurons (>95% purity) with minimal astrocyte and microglial content as determined by immunostaining of culture preparations using specific marker antibodies. Cell Viability Assay—The neuronal cells were allowed to mature for 6 days in culture before commencing treatment using freshly prepared serum free neurobasal medium plus B27 supplements minus antioxidants. For the treatment of neuronal cultures, peptide was prepared at 50 μm at Cu2+/peptide molar ratios of 0:1, 0.1:1, 1:1, and 10:1 and used immediately. Peptides were diluted to the final concentration of 5 μm in neurobasal medium. The mixtures were then added to neuronal cells for 96 h. Cell survival was monitored by phase-contrast microscopy, and cell viability was quantitated using the MTS assay as described previously (21Ciccotosto G.D. Tew D. Curtain C.C. Smith D. Carrington D. Masters C.L. Bush A.I. Cherny R.A. Cappai R. Barnham K.J. J. Biol. Chem. 2004; 279: 42528-42534Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Briefly, the medium was replaced with fresh neurobasal medium supplemented with B27 lacking antioxidants, and 10% (v/v) MTS (Promega, Madison, WI) was added to each well and incubated for 3 h at 37°C in a 5% CO2 incubator. Plates were gently shaken, and a 150-μl aliquot from each well was transferred to separate wells of a 96-well plate. The color change of each well was determined by measuring the absorbance at 490 nm using a Wallac Victor Multireader, and background readings of MTS incubated in cell-free medium were subtracted from each value before calculations. The data were normalized and calculated as a percentage of untreated vehicle control values. Data are shown as mean ± S.E. Statistical comparisons between groups were done using Student's t test. Quantification of Aggregate and Fibril Formation—For determination of fibril growth end points, a discontinuous assay of fibril growth was used. Peptide samples (20 μl) were removed from incubation after 2 or 24 h and added to 600 μl of a 20 μm thioflavin-T (ThT) solution at pH 7.4 in buffer A. The ThT signal was quantified by averaging the fluorescence emission at 480 nm over 10 s when excited at 444 nm using a PerkinElmer Life Sciences LS55 fluorescence spectrophotometer, slit widths for excitation of 2.5 nm and emission of 12.5 nm at 37 °C. Circular Dichroism Spectroscopy—The peptides were prepared as described above diluting to a final concentration of 20 μm in PBS to achieve a measurable CD signal. Cu2+ was titrated into the peptide preparation at 0.5–2.5 peptide mol eq after diluting the peptide, gently mixed, and allowed to equilibrate for 15 min at room temperature. Far UV-CD spectra were collected between 200 and 250 nm at room temperature on a Jasco 810 spectrometer using a quartz cuvette with a 1-cm path length. Measurements were recorded at 50 nm/min with a bandwidth of 1 nm and a response time of 2 s, averaging 10 accumulation scans per measurement. The spectra were background-subtracted, smoothed using the Jasco software (version 32) FFT filter function, and converted to molar ellipticity. All experiments were performed in triplicate. Dityrosine Formation—Peptide was prepared at 10 μm in the presence of 250 μm H2O2 at Cu2+/peptide molar ratios of 1:1. After 24 h of incubation, the reaction was quenched by the addition of 10 μl of 12.5 mm EDTA (final concentration 250 μm). Dityrosine content was quantified using a PerkinElmer Life Sciences LS55 fluorescence spectrophotometer with an excitation wavelength of 320 nm and scanning the emission wavelengths from 350 to 500 nm. Maximum light signal was observed and recorded at 418 nm (22Atwood C.S. Perry G. Zeng H. Kato Y. Jones W.D. Ling K.Q. Huang X. Moir R.D. Wang D. Sayre L.M. Smith M.A. Chen S.G. Bush A.I. Biochemistry. 2004; 43: 560-568Crossref PubMed Scopus (317) Google Scholar). Preparation of Large Unilamellar Vesicles—Large unilamellar vesicles (LUVs) were prepared by initially dissolving equal quantities of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (PS) (Avanti Polar Lipids Inc.) in chloroform. The chloroform was evaporated off, and the lipids were resuspended in buffer A at ∼10 mm and incubated at 37 °C for 1 h with agitation at 200 rpm in the presence of glass beads. The lipid mixture was subjected to five freeze-thaw cycles using liquid nitrogen and a 37 °C water bath. The sample was then passed through an extruder apparatus using a 100-nm filter, resulting in a uniform solution of LUVs with a mean diameter between 120 and 140 nm. Steady-state Polarization of DPH—Diphenyl-1,3,5-hexatriene (DPH) was purchased from Molecular Probes. Briefly, DPH was dissolved at 10 mm in Me2SO and diluted to 16.6 μm in buffer A containing 416 μm LUVs. A 60-μl aliquot of this sample was mixed with 40 μl of Aβ 1-42 at 10 μm, giving final concentrations of 10 μm probe, 250 μm LUVs, and 4 μm peptide. Samples were then incubated at 37 °C with aggregation at 200 rpm for 20 min prior to reading. The dye was excited at 359 nm, and emission was recorded at 430 nm. Anisotropy was measured on a Varian Eclipses spectrophotometer using internal polarizers and calculated by the equation, r = (Ivv – G·Ivh)/(Ivv + 2G·Ivh), where Ivv and Ivh are the fluorescence intensities when the excitation and emission polarizers are set at v (vertical) and h (horizontal), respectively. The grating factor G = Ihv/Ihh. Solid-state NMR—Solid-state NMR experiments were conducted on a Varian Inova 300 spectrometer operating at a resonance frequency of 121.4 MHz for 31P. 31P spectra were acquired at 28 °C, using a Doty 5-mm double resonance NMR probe with a standard Hahn echo and proton decoupling. Pulse times used were π/2 = 5 μs pulse with a delay of 2 s under magic angle spinning of ∼5 kHz. Typically, 10,000 scans were acquired, and an exponential line broadening of 10 Hz was applied. Chemical shift was referenced using 80% phosphoric acid (H3PO4) as 0 ppm for 31P NMR experiments. Lipid Peroxidation—20 μm peptide was prepared as above at a Cu2+/peptide molar ratio of 0:1 or 1:1. Samples were incubated for 5 min before the addition to 50% PC, 50% PS LUVs and ascorbate. Samples contained a final concentration of 10 μm peptide, 1 mm LUV, and 10 μm ascorbate and were incubated for 24 h at 37 °C with agitation at 200 rpm. CQ was prepared in 100% Me2SO and added to final concentrations of 2, 20, and 50 μm. The final Me2SO concentration was 1% in all cases. The level of lipid peroxidation was quantified via a lipid peroxidation colorimetric assay (Oxford Biomedical Research) as per the manufacturer's instructions. The His Bridge Dimer as Observed by EPR—Most of the experiments described below were performed at pH 7.4 in PBS solutions, because NaCl at a concentration of ∼0.15 m is part of the physiological milieu. Under these conditions of pH, peptide concentration, and buffer, the predominant species observed by EPR depended on the Cu2+/peptide molar ratio. The addition of 65CuCl2 to Aβ 1–16, Aβ 1–28, and WT Aβ 1-42 to give Cu2+/peptide molar ratios of 0–0.5:1 gave EPR signals analogous to that shown in Fig. 2a. These spectra are similar to those reported previously (2Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 3Curtain C.C. Ali F.E. Smith D.G. Bush A.I. Masters C.L. Barnham K.J. J. Biol. Chem. 2003; 278: 2977-2982Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) for low Cu2+/peptide ratios and show a spectrum mainly due to mononuclear Cu2+ in an approximately axially symmetric square planar environment. A simulated spectrum using the spin-Hamiltonian parameters is given in supplemental Table 1. These parameters are typical for Cu2+ coordinated by 2N2O or 3N1O (23Peisach J. Blumberg W.E. Arch. Biochem. Biophys. 1974; 165: 691-708Crossref PubMed Scopus (1168) Google Scholar). At Cu2+/peptide molar ratios of above 0.5:1, a significantly different resonance developed in the g ∼ 2 region, as reported previously (2Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 4Tickler A. Smith D. Ciccotosto G. Tew D. Curtain C.C. Carrington D. Masters C.L. Bush A.I. Cherny R.A. Cappai R. Wade J. Barnham K.J. J. Biol. Chem. 2005; 280: 13355-13363Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and as shown in Fig. 2b. Concurrently, we demonstrate here for the first time that a resonance also develops at g ∼ 4 when very high spectrometer gains are used (Fig. 3). The parallel appearance of the g ∼ 2 and g ∼ 4 resonances is diagnostic for the existence of a binuclear Cu2+ center (i.e. a pair of Cu2+ ions coupled by exchange and/or dipolar interactions) (12Smith T.D. Pilbrow J.R. Coord. Chem. Rev. 1974; 13: 173-278Crossref Scopus (444) Google Scholar). Although the Cu2+ hyperfine structure is not well resolved, its presence is confirmation that the resonance at g ∼ 4 is due to a pair of Cu2+ ions and not to a nonspecific aggregate. At Cu2+/peptide molar ratios of ∼1:1 and greater, another broad resonance developed centered at g ∼ 2.14 (Fig. 2c). No resonance at g ∼ 4 appears to be associated with this new signal, which may be attributed to a number of Cu2+ ions within ∼10 Ä of each other but not structurally related. The broken line spectrum in Fig. 2c represents the simulation of an isotropic resonance at g ∼ 2.14, such as might arise from a number of Cu2+ ions within ∼10 Ä of each other without a specific geometrical relationship and where the anisotropic features are averaged by a combination of dipolar and weak exchange interactions. Spectra acquired at 2.5 K of Aβ 1–28 (Cu2+/peptide molar ratio 1:1) exhibited the same resonances as observed at 120 K at g ∼ 2 and g ∼ 4 but with 50 times the intensity, indicating that antiferromagnetic exchange interactions between the Cu2+ ions of the dimer must be ≪10 cm–1. The dimer spectra did not exhibit microwave power saturation up to 25 milliwatts at 2.5 K, indicating the existence of spin-spin relaxation processes as expected for a coupled system. In contrast, the mononuclear spectra exhibited power saturation effects above ∼1 milliwatt at 2.5 K. To verify that the g ∼ 2 and g ∼ 4 spectra were not due to oxo- or phosphate-bridged binuclear Cu2+ centers or to adventitious Fe3+, control spectra were acquired at 120 K of 1.5 mm 65CuCl2 in PBS, of PBS alone, and of the peptide in PBS prior to the addition of 65CuCl2. At pH 7.4, no resonances were observed near either g ∼ 2 or g ∼ 4. As a comparison with WT Aβ 1-42, the spectra of Aβ 1-42 containing His-τ-Me or His-π-Me at positions 6, 13, and 14 were recorded over the range of Cu2+/peptide molar ratios 0.3–1:1. Only mononuclear spectra were observed, similar to those previously published by Tickler et al. (4Tickler A. Smith D. Ciccotosto G. Tew D. Curtain C.C. Carrington D. Masters C.L. Bush A.I. Cherny R.A. Cappai R. Wade J. Barnham K.J. J. Biol. Chem. 2005; 280: 13355-13363Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). There were no indications of the broad resonance at g ∼ 2 characteristic of the binuclear Cu2+ center or for resonances near g ∼ 4, even at spectrometer settings where the g ∼ 4 resonances were clearly observed in the unmodified peptides. Fig. 2d shows the mononuclear spectra obtained with WT Aβ 1-42 at a Cu2+/peptide molar ratio of 0.1:1 and the similarly mononuclear spectra of His-τ-Me obtained at a Cu2+/peptide molar ratio of 1:1 (Fig. 2e). High pressure immobilized affinity chromatography was employed to ascertain if the methylation of the imidazole side chains had a significant effect on the relative Cu2+ binding affinity compared with WT Aβ 1-42. The strength of peptide-metal ion interactions is usually assessed as a function of the pH required to elute the various peptides from a Cu2+-loaded immobilized metal ion affinity chromatography column. However, we were unable to elute any of the peptides from the Cu2+-charged immobilized metal ion affinity chromatography column at pH <2 or by using an imidazole gradient. The peptides could only be eluted by the addition of EDTA to the eluting buffer, indicating that all peptides had a very high affinity for Cu2+. The effect of buffer conditions on Aβ-Cu2+ coordination was investigated by acquiring spectra of Aβ 1–28 in pH 7.4 ethylmorpholine buffer over a range of Cu2+/peptide molar ratios, recreating the conditions of Syme et al. (7Syme C.D. Nadal R.C. Rigby S.E. Viles J.H. J. Biol. Chem. 2004; 279: 18169-18177Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) (supplemental Fig. 1). At Cu2+/peptide molar ratios of 0.1:1 and 1:1, only mononuclear spectra where observed. Spectra in the g ∼ 2 region were identical to those reported at Cu2+/peptide ratios of ≤1:1 by Syme et al. (7Syme C.D. Nadal R.C. Rigby S.E. Viles J.H. J. Biol. Chem. 2004; 279: 18169-18177Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). The broad line near g ∼ 2 was not observed, even at Cu2+ concentrations and spectrometer settings that yielded these resonances in PBS. Therefore,
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