NMR Studies of the Zn 2+ Interactions with Rat and Human β-Amyloid...

NMR Studies of the Zn2+ Interactions with Rat and Human â-Amyloid (1-28) Peptides inWater-Micelle Environment

Elena Gaggelli,† Anna Janicka-Klos,‡ Elzbieta Jankowska,§ Henryk Kozlowski,* ,‡

Caterina Migliorini, † Elena Molteni,† Daniela Valensin,† Gianni Valensin,*,† andEwa Wieczerzak§

Department of Chemistry, UniVersity of Siena,Via Aldo Moro, 53-100 Siena, Italy, Faculty of Chemistry,UniVersity of Wrocław F. Joliot-Curie 14, 50-383 Wrocław, Poland, and Faculty of Chemistry, UniVersity ofGdansk, 82-952 Gdan´sk, Poland

ReceiVed: July 3, 2007; In Final Form: October 2, 2007

Alzheimer’s disease is a fatal neurodegenerative disorder involving the abnormal accumulation and depositionof peptides (amyloid-â, Aâ) derived from the amyloid precursor protein. Here, we present the structure andthe Zn2+ binding sites of human and rat Aâ(1-28) fragments in water/sodium dodecyl sulfate (SDS) micellesby using1H NMR spectroscopy. The chemical shift variations measured after Zn2+ addition atT > 310 Kallowed us to assign the binding donor atoms in both rat and human zinc complexes. The Asp-1 amine, His-6Nδ, Glu-11 COO-, and His-13 Nε of rat Aâ28 all enter the metal coordination sphere, while His-6 Nδ, His-13, His-14 Nε, Asp-1 amine, and/or Glu-11 COO- are all bound to Zn2+ in the case of human Aâ28. Finally,a comparison between the rat and human binding abilities was discussed.

The accumulation of protein precipitates at the end ofdegenerating brain neurons is the main feature of Alzheimer’sdisease (AD). Their main constituent is a group of small peptidefragments called amyloid-â (Aâ), with lengths ranging from39 to 43 amino acids, generated by proteolysis of the amyloidprecursor protein (APP), a transmembrane glycoprotein.1 Thefragments with 40 (Aâ40) and 42 (Aâ42) residues are mostcommonly encountered.

Amyloid peptides in solution populate conformational statesstrongly dependent upon solvent and pH.2-8 Membrane mim-icking environments at high (>7) and low (<4) pH favor amonomeric, solubleR-helical or unstructured conformation,whereas water at midrange pH values (between 4 and 7) yieldsa less solubleâ-sheet conformation prone to form oligomersand to eventually precipitate as amyloid. Aâ40 can be consideredas being composed of two parts: (i) the region encompassingresidues 29 to 40, corresponding to the transmembrane regionof APP, is highly hydrophobic, scarcely soluble, and tends toaggregate into a non-amyloid-likeâ-sheet structure regardlessof differences in solvent, pH, or temperature;2 (ii) the (1-28)fragment, occupying the extra cellular region in the APP protein,can produce bothR-helical structures2,6 and oligomericâ-sheetstructures similar to those characteristic of amyloid plaques9,10

and is therefore responsible for the conformational variabilityof the Aâ peptide.

Metal ions as diverse as zinc, copper, and iron have beenimplicated as possible pathogenic agents in AD because of theaccumulation of these metals in amyloid deposits11-13 and inthe cortical tissue of AD patients.14 It has been shown that thesemetals induce Aâ aggregation15-20 and fibril formation21,22 at

high metal concentrations, while low concentrations of Zn2+

and Cu2+ have been shown to selectively destabilize the Aâoligomeric species that are known to be highly toxic.23,24

Recently, a possible mechanism of the Cu2+- or Zn2+-inducedAâ peptide aggregation has been proposed25 where the formationof monomeric metal-Aâ complex induces strong conforma-tional changes that promote interactions of different peptidemolecules.

Numerous studies have suggested a basic role of the threehistidines, present at the N terminus of human Aâ, for thecoordination of copper and zinc;22,24-38 on the contrary, differentresults have been obtained for the fourth metal donor atoms,alternatively assigned to the N-terminal amino for binding ofboth Cu2+ and Zn2+,22,26,27,29,31,35to the Tyr-10 phenolate forboth metals,34,36,37or to the Glu-11 carboxylate only for Zn2+

complexes.33 Evidence for the Glu-11 carboxylate coordinationhas been reached in the NMR structure of the Zn2+-Aâ16

complex,33 but in that case, N acetylation was preventing bindingof zinc to the N-terminal amino group.

Interestingly, amyloid peptides from the rat, bearing only threemutations, (Arg-5f Gly, Tyr-10 f Phe, and His-13f Arg),bind Zn2+ and Cu2+ much less avidly and are less susceptibleto aggregation, such that rats do not develop cerebral Aâdeposition.15,39-42 The NMR solution structure of Zn2+-rat Aâ28

has been obtained in dimethyl sulfoxide (DMSO), showing His-6, Arg-13, and His-14 as the primary binding sites and revealingthe presence of a helical region from Glu-15 to Val-24.43

In the present work, we have investigated the structures andthe Zn2+ metal binding modes of rat and human Aâ28 in water/sodium dodecyl sulfate (SDS) micelles, with particular emphasison the comparison between the binding sites in the two peptidesand on the temperature dependence. The detection of NOEeffects allowed obtaining a NMR three-dimensional (3D)structure of the rat Aâ28-Zn2+ complex substantially differentfrom the one previously determined in DMSO.43 On the otherhand, combination of NMR and CD results allowed to build up

* Corresponding authors. Fax:+390577234233. E-mail: [email protected] (G.V.). Fax: (+48)71-375-7251. E-mail: [email protected](H.K.).

† University of Siena.‡ University of Wrocław.§ University of Gdan´sk.

100 J. Phys. Chem. B2008,112,100-109

10.1021/jp075168m CCC: $40.75 © 2008 American Chemical SocietyPublished on Web 12/12/2007

a Zn2+ binding model for the human Aâ28. The differentaffinities of the human and rat Aâ28 for Zn2+ have beendiscussed in relation to the diverse metal induced aggregationshown by these two peptides.

Materials and Methods

Peptide Synthesis and Purification.Synthesis of human andrat amyloid fragments and their acetylated derivatives (Scheme1) was performed on a solid phase using Fmoc (9-fluorenyl-methoxycarbonyl) strategy with continuous-flow methodology(9050 Plus Millipore peptide synthesizer) on a polystyrene/polyethylene glycol copolymer resin (TentaGel R RAM resin,substitution 0.18 mmol/g).44 Attachment of the first amino acidto the resin and next coupling steps were realized usingdiisopropylcarbodiimide (DIPCI) as a coupling reagent with1-hydroxybenzotriazole (HOBt) as an additive. Removal of theFmoc protecting group during peptide synthesis was achievedby means of 20% piperidine solution in DMFNMP (1:1, v/v)with addition of 1% Triton X-100.45 Acetylation of the N-terminal amino group was performed on the resin using 0.3 Macetylimidazole in DMF.45,46 All peptides were cleaved fromthe resin and deprotected by treatment with the mixturecontaining trifluoroacetic acid, phenol, triisopropylsilane, andwater (88:5:2:5, v/v) for 2 h atroom temperature.45 The resultingcrude peptides were purified by reversed-phase high-perfor-mance liquid chromatography (RP-HPLC) using a C8 semi-preparative Kromasil column (25× 250 mm, 7µm). The purityof the peptides was confirmed by matrix-assisted laser desorp-tion/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and analytical RP-HPLC using a C4 Kromasil column (4.6× 250 mm, 5 µm) and 30 min linear gradient of 0-80%acetonitrile in 0.1% aqueous trifluoroacetic acid as a mobilephase. Analytical data were as follows. hAâ‚28: Rt ) 14.3 min,MS ) 3262.2 [M + H]+, Mcalc ) 3261.5. Ac-hAâ28: Rt )14.7 min,MS ) 3304.6 [M+ H]+, Mcalc ) 3303.5. rAâ‚28: Rt

) 15.2 min,MS ) 3164.6 [M + H]+, Mcalc ) 3163.4. Ac-rAâ28: Rt ) 15.5 min,MS ) 3207.2 [M+ H]+, Mcalc ) 3206.5.The peptides were first dissolved in H2O or D2O, followed bythe addition of SDS-d25 (Sigma Chemical Company). The SDS-d25 concentration was kept high relative to the peptide concen-tration, so that it was well above 8 mM (the critical micelleconcentration)47 and also above the average aggregation number.The pH values were measured at room temperature, and theywere adjusted at desired values with either DCl or NaODsolutions. The desired concentration of zinc ions was achievedby using a stock solution of zinc chloride (Sigma ChemicalCompany) either in water or deuterium oxide. TSP-d4, 3-(tri-methylsilyl)-[2,2,3,3-d4] propansulphonate, sodium salt, wasused as internal reference standard.

CD Measurements.CD spectra were obtained with a JascoJ-815 spectropolarimeter, and the temperature was controlledwith a PTC-423S temperature controller. A quartz cell with 1cm optical path was used. The spectra range was 190-250 nmwith a resolution of 0.2 nm and a bandwidth of 1 nm. A scanspeed of 20 nm/min with 1 s response time was employed. Thebackground spectrum was subtracted, and the results wereexpressed as mean residue molar ellipticity [Θ].

NMR Measurements. Solutions of Aâ28 were prepared atconcentrations varying from 0.3 to 1.0 mM. NMR measurementswere performed either at 14.1 T with a Bruker Avance 600 MHzor at 9.4 T with a Bruker AMX 400 MHz spectrometers atcontrolled temperatures ((0.2 K) using a TBI (triple broadbandinverse) or BBI (broadband inverse) probe. Water suppressionwas achieved either by presaturation or by the excitationsculpting method.48 Proton resonance assignment was ac-complished through total correlation spectroscopy (TOCSY),nuclear Overhauser effect specctroscopy (NOESY), and rotating-frame Overhauser spectroscopy (ROESY) standard experiments.TOCSY spectra were obtained using the MLEV-17 pulsesequence with a mixing time of 75 ms. NOESY and ROESYspectra were obtained at different values of the mixing time tooptimize the best one. Spectra processing was performed on aSilicon Graphics O2 workstation using the XWINNMR 2.6software.

Structure Determination and Molecular Dynamics Simu-lations. For all of the investigated peptides, the intensities ofNOESY cross-peaks, referenced to cross-peaks involving protonpairs at fixed distances, were converted into proton-protondistance constraints; the constraints were used to build apseudopotential energy for a restrained simulated annealing (SA)calculation in torsional angle space. In particular, we performedthe calculation with the program DYANA,49 with 300 randomstarting structures of the peptide and of its metal complex and10 000 steps of SA. Since only one molecule can be given asinput in the program, the peptide was linked to Zn2+ through along chain of linkers, that is, residues made by atoms withoutvan der Waals radius, which could freely rotate around theirbonds, without causing steric repulsions and thus enabling oneto sample a large number of relative positions of the ligandwith respect to the metal ion before the minimization step. Themodel for the Zn2+-hAâ28 complex was generated with thesame program, but using as constraints only the binding of Zn2+

to its coordinating atoms and the conservation of theR-helicalregion.

On the best structures of the free peptides and of zinccomplexes, an energy minimization followed by a moleculardynamics (MD) simulation in explicit water was performedusing the program GROMACS50,51with the OPLS force field.52

First, the structures were energy minimized in vacuo, then theywere solvated using a parallelepiped box of water with periodicboundary conditions, with a minimum distance between anyatom of the peptide and the box edge of 1.5 nm, in the presenceof a pre-equilibrated SDS micelle and of a number of Na+

counterions to balance the negative charge of the micelle. Thepeptide was positioned so as to place its C-terminalR-helicalregion near the external surface of the micelle, in agreementwith previous studies showing that the “whole” Aâ40/42peptideinteracts with SDS micelles at their surface, rather than withintheir hydrophobic core. The resulting systems were again energyminimized and subsequently brought to the temperature of 298K through six MD runs of 10 ps each, in which the temperaturewas progressively raised. Then MD simulations of severalhundred picoseconds at constant temperature (T ) 298 K) wereperformed. During the simulations, peptide, micelle, and solventseparately were weakly coupled to a temperature bath at thechosen temperature and to a pressure bath at 1 atm, withrelaxation timesτT ) 0.1 ps andτP ) 0.5 ps, respectively,using Berendsen’s weak coupling algorithm;53 bond lengths wereconstrained to equilibrium values using the SHAKE procedure,54

with a geometric tolerance of 10-4, and the time step was setto 2 fs. Nonbonded interactions were treated using a twin range

SCHEME 1: List of the Used Peptide Sequences

Human and Rat Zn2+ â-Amyloid (1-28) Complexes J. Phys. Chem. B, Vol. 112, No. 1, 2008101

method:55 within a short-range cutoff of 0.8 nm, all interactionswere determined at every time step, while longer rangecontributions within a cutoff of 1.4 nm were evaluated eachtime the pair list was generated. For the free peptides, norestraints were used, while for the zinc complexes, distancerestraints of 0.2 nm between the metal ion and its coordinatingatoms were imposed.

In order to build the micelle, first, we added the single SDSmolecule topology to the chosen force field; then, we arranged50 SDS molecules in a roughly spherical shape; this initialmicelle was subsequently equilibrated through an energyminimization and a molecular dynamics simulation in explicitwater in the presence of 50 Na+ ions, imposing distanceconstraints between each of the terminal carbons of thehydrophobic tails and a dummy atom placed at the center ofthe micelle.

Results and Discussion

The1H NMR assignment of human and rat amyloid fragmentsin H2O/SDS at pH 7.5 and 298 K has been obtained by meansof one- and two-dimensional (1D and 2D) NMR experimentsand agrees with previous reports.56 The choice of the H2O/SDSmedium was determined for several reasons: (i) the C-terminalregion of Aâ40 is inserted in the membrane while incorporatedwithin APP,57 and micelles have been widely and successfullyused for mimicking the membrane environment for manypeptides and proteins;58,59 (ii) A â is known to bind varioushydrophobic macromolecules,60,61 and interactions of Aâ pep-tides with lipid bilayers or neuronal membranes have a numberof physiological effects, including the increase of intracellularcalcium levels and the formation of voltage-dependent ionchannels;62,63(iii) the presence of SDS micelles at 6< pH < 8tends to favorR-helix formation at the C terminus of Aâ28,preventing the precipitation that occurs at the used NMR sampleconcentrations.56-59 In particular, it was found that the Aâpeptide remains on the micelle surface and does not becomeimbedded into the hydrophobic interior.56

rA â(1-28) and Ac-rA â(1-28). The NOESY spectra of therat peptide show HR(i)-Hâ(i + 3) and HN(i)-HN(i + 1)connectivities among residues in the C-terminal region sug-gesting the presence of anR-helical conformation in agreementwith other studies of human Aâ peptides in membrane mimick-ing environments.56-59

The NOESY cross-peaks were converted into intra-residue,sequential, and medium range distance restraints and used forstructure calculation. On the best structures, an energy mini-mization and molecular dynamics simulation were performedin water in the presence of an SDS micelle, in order to reproduceexperimental conditions and to evaluate the effect of the micelleon the peptide secondary structure. The best 10 obtainedstructures, shown in Figure 1A, are well superimposed on thestretch of residues from 16 to 24, with a backbone root meansquare deviation (rmsd) of 0.02 nm and a low number ofconstraint violations, all within 0.03 nm.

Occurrence of theR-helix is apparent, as verified by theobserved NOESY connectivities, as it is also evident that theN-terminal region retains conformational flexibility, as suggestedby the absence of nontrivial connectivities.

The occurrence of anR-helix structure was also ratified bythe two negative bands at 222 and 208 nm in CD spectra (datanot shown); moreover, the observed [Θ]222 value is consistentwith the occurrence of approximately 15% ofR helix,64 inagreement with the structures obtained by NMR, where onlyresidues from 20 to 24 adopt a well definedR-helical conforma-

tion. The CD experiments, performed also at higher tempera-tures, confirmed the conservation of the secondary structure(data not shown) at all the investigated temperatures.

Addition of Zn2+ to rAâ‚ 28, at pH 7.5 and 298 K, causedbroadening of NMR signals, especially on the aromatic protonsof histidines (Figure 2A) on Glu-3, Phe-4, Gly-5, Asp-7, Ser-8, Phe-10, Glu-11, Leu-17, Val-18, and Glu-22 amide protonsand on Glu-11 and Gln-15 HR, Hâ, and Hγ (Figure 3A).Moreover the Asp-1 and Val-12 HR were largely shifted upfield (-0.14 and-0.08 ppm, respectively) upon metal addition(Figure 3B).

At T > 310 K, most of the proton resonances, particularlythose of the His aromatic protons, which were very broad atT) 298 K, returned to be detectable; 1D and 2D spectra wereconsequently recorded also at this temperature in order to get abetter description of the zinc binding domain.

The Zn-induced changes in chemical shift experienced byrAâ28 at T ) 318 K are reported in Figure 4. The most affectedsignals are within the flexible N-terminal region; in particular,the largest shifts are on the N-terminal Asp-1, His-6, His-14,and Glu-11; less marked effects are evident also on Glu-3, Val-12, and Arg-13. This pattern of chemical shift variations clearlyindicates Zn2+ coordination to His-6 and His-14 imidazoles,Asp-1 N-terminus, and Glu-11 side-chain carboxylate. Similarshifts were also observed for Ac-rAâ28 with the trivialexception of the effects recorded on Asp-1 HR and Hâ, sinceacetylating prevents metal binding to the terminal amino group.Moreover, the two peptides show almost identical shifts forHis-6 aromatic protons, being the His-6 Hε shift much morepronounced than the His-6 Hδ one and suggesting metal bindingof His-6 via Nδ in both peptides. On the contrary, a diversebehavior of His-14 was found. The His-14 aromatic protons, infact, are almost equally shifted (∆δ ) -0.11 ppm for Hδ and-0.14 ppm for Hε) in the case of rAâ28, while for thecorresponding protected peptide, His-14 Hδ is less shifted thanHε (∆δ ) -0.1 and-0.20 ppm), suggesting a diverse metalbinding of His-14. In particular, the obtained results indicatezinc coordination to His-14 Nε for the unprotected fragmentand to His-14 Nδ for the protected one.

Figure 1. Best ten structures of the free Aâ28 from rat (A) and fromhuman (B) obtained through restrained simulated annealing based onthe experimental data. (C) Superimposition of the first structure fromrat (blue) and human (red) Aâ28. The figure was created with MOLMOL2K.1.0.

102 J. Phys. Chem. B, Vol. 112, No. 1, 2008 Gaggelli et al.

The metal concentration dependence of chemical shiftsallowed determining the dissociation constantKd and thecomplex stoichiometry,65,66by titrating the Hε protons of His-6and His-14 (Figure 1s, Supporting Information). Standardregression analysis was used to fit the obtained exponentialcurves to the data, obtainingKd in the range 200-400µM at T) 318 K.

NOESY spectra recorded at 318 K provided additionalinformation on the Zn2+ binding domain: long-range NOE werein fact detected among residues located at or very close to themetal binding site. In particular, Figure 5 shows the cross-peaksbetween His-14/Glu-11, His-14/Phe-10, His-6/Glu-11, and Asp-1/Glu-11. All of the NOEs were converted into distanceconstraints which were used for structure calculation. On thebest structures, an energy minimization and molecular dynamicssimulation were performed in water in the presence of an SDS

micelle, in order to reproduce experimental conditions. Theobtained structures (Figure 6) show two different well super-imposed regions: (i) the C-terminal one with a rmsd calculatedfrom Lys-16 to Ser-26 at 0.076( 0.032 nm for the backboneand 0.135( 0.053 nm for the heavy atoms and (ii) theN-terminus region including the metal binding site, with a rmsdcalculated from Asp-1 to His-14 at 0.064( 0.019 nm for thebackbone and 0.116( 0.043 nm for the heavy atoms. TheR-helical structure observed for the free peptide betweenresidues 20 and 24 is conserved also in the presence of the metalion, suggesting that the C-terminus region is still interactingwith the micelle and it is not perturbed by the metal ion, inagreement also with the negligible shift variations measured forthat peptide region (Figure 4). Moreover, the CD experimentsperformed at temperatures in the range 298-328 K, in thepresence of Zn2+, revealed that theR helix is not perturbed bythe metal ion binding (data not shown).

Figure 2. Aromatic region of1H 1D spectra of the (A) rAâ28 0.9 mM in the presence of 1.0 Zn2+ equiv; (B) hAâ28 0.4 mM in the presence of 1.0Zn2+ equiv; in H2O, 100 mM SDS at pH 7.5 at different temperatures.

Figure 3. Selected regions of the TOCSY spectrum of rAâ28 0.9 mM, 100 mM SDS at pH 7.5 andT ) 298 K: in the absence (black) and in thepresence of 1.0 Zn2+ equiv (magenta). The broadening of selected amide protons (A) and the chemical shift variation (B) upon metal addition areshown.


hAâ(1-28) and Ac-hAâ(1-28).Both peptides were solublein aqueous SDS micelles. The solutions remained stable overseveral weeks, without any evidence of aggregation or precipita-tion. NMR experiments demonstrated an helical conformationin the C-terminal region of both peptides, in agreement withprevious investigations.56-59 From NOESY experiments, afamily of structures was obtained (Figure 1B): the N-terminalregion is completely disordered and flexible, while a wellorganizedR-helical domain encompasses residues 16 to 24. Theconformations determined for the human fragments are verysimilar to those obtained for the rat peptides; Figure 1Ccompares the two structures which are quite well superimposed

in the R-helical domain. As in the case of the rat peptides, theCD experiments showed absorptions typical ofR-helicalconformation (data not shown). The two (rat and human)amyloid CD spectra were very similar to each other revealinga similar interaction with the micellar surface, with a consequentstructuring of the C-terminal region, identical for both peptides.

Upon Zn2+ addition, several proton resonances were broad-ened at room temperature: the metal-induced effects weresimilar to those detected for the rat fragment (vide supra): (i)the TOCSY fingerprint region showed the disappearance of thesame amide protons (Figure 2s), (ii) the His aromatic protonscompletely disappeared (Figure 2B), (iii) the Arg-5, Glu-11,

Figure 4. Histogram showing all of the obtained chemical shift variations (∆δ) for protons of rAâ28 (white) and Ac-rAâ28 (gray) 0.9 mM, 100 mMSDS at pH 7.5 andT ) 318 K in the absence and in the presence of 1.0 Zn2+ equiv.

Figure 5. Selected regions of the NOESY spectrum of rAâ28 0.9 mM, 100 mM SDS at pH 7.5 in the presence of 1.0 Zn2+ equiv. The long-rangeNOEs between residues located in the proximity of the binding site are circled.


and Gln-15 spin-systems were completely lost (Figure 7), and(iv) a shift of -0.04 and 0.02 ppm was measured for Asp-1and Val-12 HR, respectively (Figure 7). These findings are inagreement with what had been previously observed with thehuman full length Aâ40,35 where the His broadenings and theAsp-1 and Val-12 shifts were monitored by titrating the1H-13C HSQC spectra with Zn2+.

1H NMR experiments on metal complexes of hAâ28 and Ac-hAâ28 were also performed at higher and lower temperaturesin order to reduce the Zn2+-induced line broadening. Whilelowering the temperature to 278 K did not yield better spectra,raising the temperature to 328 K allowed detection of all threeHis aromatic protons (Figure 2B).

In order to measure the Zn2+-induced changes in chemicalshift, NOESY and TOCSY spectra were recorded also atT )328 K. At this temperature, all amide protons disappeared (alsoin the absence of the metal ion) because of the exchange withthe solvent, and therefore no structure analysis could be

performed. The changes observed in the presence of 1.0 Zn2+

equivalents atT ) 328 K are reported in Figure 8. The mostaffected residues are His-6, Tyr-10, Glu-11, His-13, and His-14; less pronounced but still significant effects are seen for Asp-1, Ala-2, Glu-3, and Arg-5 resonances. The large His aromaticshifts strongly suggest the imidazole as the metal binding group,as previously found in other studies,23,29,31-33,35 indicating thatthe micelle-amyloid interactions do not perturb the metalbinding ability. A different behavior was observed for His-6with respect to the His-13-His-14 pair. His-13 and His-14 Hδand Hε protons were approximately equally shifted, consistentlywith Nε as the donor atom in both residues, whereas the His-6Hε (∆δ ) -0.22 ppm) was much more affected than Hδ (∆δ) -0.10 ppm), indicating the involvement of Nδ rather thanNε in Zn2+ coordination.

The behavior of unprotected hAâ28 and that of N-protectedhAâ28 are compared in Figure 8: the largest deviations occur,as expected, at the N terminus, while residues between His-6

Figure 6. Snapshots from the MD simulation of Zn2+-rAâ28 complex, superimposed with the structure obtained from experimental data. Twodifferent regions are obtained: (left) the C-terminal one showing helical structural elements among 16-24 residues; (right) the N-terminal oneillustrating the metal binding domain with Zn2+ tetrahedrally coordinated to Asp-1 NH2, His-6 Nδ, Glu-11 COO- and His-14 Nε. The figure wascreated with MOLMOL 2K.1.0.

Figure 7. Selected regions of the TOCSY spectrum of hAâ28 0.4 mM, 100 mM SDS at pH 7.5 andT ) 298 K: in the absence (black) and in thepresence of 1.0 Zn2+ equiv. (magenta). The broadening of selected cross-peaks and the chemical shift variations upon metal addition are shown.


and His-14 exhibit almost identical changes suggesting thepresence of a single Zn2+ binding site. However, the samecomparison reveals some peculiarity concerning the His resi-dues. In fact, in the case of the acetylated fragment, His-6, His-13, and His-14 display similar shifts on the aromatic protons,being Hε always more affected than Hδ, such that the involve-ment of different imidazole nitrogens (Nδ instead of Nε) wasfound for His-13 and His-14 for Ac-hAâ28.

The shifts experienced by His Hε allow estimatingKd alsofor Zn2+-hAâ28. As in the case of the rat peptide, a metaltitration was performed (Figure 3s), and aKd in the range 30-50 µM was found atT ) 328 K. Such a value is consistent

with previous results reporting a micromolar dissociationconstant calculated atT < 310 K.31,35,67,68

As already mentioned, the structure of Zn2+-hAâ28 at highertemperatures could not be obtained; however, the CD experi-ments, performed at room and at higher temperatures on theZn2+ complexes (Figure 4s), revealed that the metal ion doesnot interfere with theR-helical region which is present in themetal complexes. Moreover, the similarity between the CDspectra (in the presence and in the absence of the metal ion)led us to assume that the conformation of the C-terminal regionis practically conserved between the free and the metal boundstates. Therefore, a structural model (Figure 9) was built with

Figure 8. Histogram showing all of the obtained chemical shift variations (∆δ) for protons of hAâ28 (white) and Ac-hAâ28 (gray) 0.4 mM, 100mM SDS at pH 7.5 andT ) 328 K in the absence and in the presence of 1.0 Zn2+ equiv.

Figure 9. Snapshots from the MD simulation of Zn2+-hAâ28 complex, superimposed with the model based on experimental data. Global view ofthe peptide conformation (left); metal binding domain with Zn2+ coordinated to Asp-1 NH2, His-6 Nδ, Glu-11 COO-, His-13 Nε, and His-14 Nε(right). The figure was created with MOLMOL 2K.1.0.


Zn2+ ion bound to the imidazoles, the N terminus, and the Glu-11 carboxylate, while the C-terminus conformation was pre-served as that found for the free peptide.

Conclusions

Addition of Zn2+ to rat and human Aâ28 in H2O/SDSselectively broadens the1H NMR signals atT < 310 K, asalready observed in previous NMR investigations on humanAâ40, Aâ28, and Aâ16.26,33,35,37The strong broadening effects,observed in aqueous as well as in membrane-mimickingenvironments, suggest that metal binding is not affected by thepresence of SDS micelles: the SDS-Aâ interaction in factoccurs at the C terminus, leaving the N-terminal regioncompletely free to interact with Zn2+ (Figure 1).

The Zn2+-induced line broadening has alternatively beenattributed either to conformational exchange at a rate inintermediate regime with respect to the NMR time scale26,33,35,37

or to occurrence of aggregation phenomena.35 The similar effectsmonitored for the rat and human fragments led us to supportan intermediate exchange regime rather than aggregation, sinceit has been reported that the rat Aâ is less susceptible to Zn2+-induced aggregation than the human peptide.15 Moreover, whileaggregation has been shown to be favored at 318 K rather thanat 277 K,69,70 the line broadening is reduced by raising thetemperature.

The intermediate regime with respect to the NMR time scalemay arise from (i) exchange between the free and the boundstates, (ii) exchange between two or more binding sites, or (iii)exchange between different bound conformations.

The first hypothesis was immediately ruled out by the factthat proton signals are still broadened after saturation of the ratand human Aâ28 zinc binding sites obtained with approximatelytwo and one metal equivalents, respectively. Further metaladditions yield negligible chemical shift variations at theinvestigated higher temperature (Figures 1s and 3s, SupportingInformation), indicating the exclusive presence of the boundpeptides.

The presence of multiple Zn2+ binding modes was alsoexcluded since the absence of metal binding donors such asthe N-terminal (Ac-hAâ28, Ac-rAâ28) and the His-13 imidazole(rAâ28) did not affect the signal line broadening, suggesting thepresence of a unique Zn2+ binding site as suggested earlier.22,26-38

The reduction of the NMR signal intensities induced by Zn2+

at room temperature was therefore ascribed to the presence of

different bound conformations. The largest effects were detected,not only for the residues involved in metal binding (His-6, Glu-11, His-13, His-14) or those nearby (Arg-13, Gln-15), but alsofor many amide protons (Figure 2A and 3s). The full disap-pearance of amide protons of residues far away from the Zn2+

binding site (Leu-17, Val-18, Phe-19 for both peptides and Phe-20 and Ala-21 for hAâ28) suggests that they experience two ormore conformations since the amide protons are prone to largechemical shift variations depending on their chemical environ-ment.

The metal induced broadenings verified with either rat orhuman fragments largely limit the extraction of precise informa-tion on the Zn2+ binding sites, since most of the residues in theN-terminal region are vanished out by metal additions, and itmay also explain why different results have previously beenreported on those systems; however, the observed chemical shiftvariations atT ) 318 K can solve the ambiguity detected atroom temperature.

Raising the temperature up to 328 K yielded faster chemicalexchange rates, and therefore, sharper NMR lines for Zn-amyloid peptide complexes were observed. Monitoring allproton signals at higher temperatures allowed delineating themetal effects on the investigated systems: in particular, thechemical shift variation analysis was performed atT ) 318 Kand T ) 328 K for rat and human peptides, respectively. Infact, only atT ) 328 K, His-6, His-13, and His-14 Hε (in hAâ28)could be unambiguously assigned in the1H NMR spectra.

The addition of 1.0 Zn2+ equivalents resulted in largechemical shift variations on Asp-1, His-6, Glu-11, and His-14for the rat peptide (Figure 4) and Asp-1, His-6, Tyr-10, Glu-11, His-13, and His-14 for the human peptide (Figure 8).Moreover, for both systems, an upfield shift was detected forHis aromatic protons and Asp-1 HR, while a downfield shiftwas found for Tyr-10 HR, Hâ and Glu-11 HR, Hâ, and Hγ.Such behavior can be accounted for by considering that, in thezinc histidine complexes, the carboxyl carbons shift downfieldwhile the amino and imidazole nitrogens shift upfield.71 As aconsequence, the upfield shift of Asp-1, His-6, His-13, and His-14 can be attributed to Zn2+ binding at the terminal aminonitrogen and imidazole nitrogens, respectively, while the down-field shift observed on Glu-11 supports the participation of itscarboxylate in the metal ion coordination. The large downfieldchanges observed for Tyr-10 can be ascribed to conformationalchanges caused by metal ion binding, rather than to direct

Figure 10. Comparison of the chemical shift variations of hAâ28 (red) and rAâ28 (black) protons caused by the addition of 1.0 Zn2+ equiv at pH7.5, T ) 328 K (human) andT ) 318 K (rat).


involvement of phenolate in coordination. This is supported byvery small effects observed on the Tyr-10 Hε and Hδ.Furthermore, the shifts experienced by His aromatic protonsallowed determining which imidazole nitrogen is bound to themetal ion; that is, His-6 coordinates via Nδ, and His-13 (whenpresent) and His-14 coordinate through Nε.

Acetylating of the N terminus yields the expected negligibleeffects observed for Asp-1. This clearly supports the involve-ment of the amino group in metal binding for both rat (Figure4) and human (Figure 8) Aâ28. Previous reports had alreadysuggested Zn2+ coordination to N terminus in the case of thehuman peptide,26,35whereas NMR studies on Zn2+-rat peptideindicated the Arg-13 side chain NH2 as the additional bindingdonor, besides those of His-6 and His-14.43 This latter study,however, was performed in different experimental conditions(DMSO), which could give a strong impact on the obtainedresults.

All four investigated systems display strong Zn2+-inducedchanges on Glu-11 (Figure 4 and 8): in particular, the Glu-11Hγ’s are shifted downfield upon metal addition. These resultsstrongly suggest binding of the Glu-11 carboxylate in both ratand human peptides, in agreement with previous NMR studieson hAâ16.33

Finally, a different Zn2+-imidazole coordination has beenshown for His-13 and His-14 (Figure 4 and 8) for N-protectedversus unprotected peptides: coordination of the terminal aminogroup favors the His-13 and His-14 metal binding via Nε ratherthan Nδ donor.

Rat and human amyloid Aâ28 exhibit very similar effects inthe presence of Zn2+, with the trivial difference of the His-13missing in the rat peptide sequence. The additional involvementof His-13, in human peptide, in the metal coordination sphere,determines a distinctly more stable Zn2+ complex, which showsa dissociation constant at least 1 order of magnitude lower thanthe other. The comparison of the chemical shift variations ofthe rat and human peptides (Figure 10) has shown that the largestdifferences (besides those already mentioned on His-13) concernresidues located at positions 5 and 10, which are the other twomutations found in the rat amyloid sequence, and these findingsare revealing for the strong conformational changes occurringat these positions upon Zn2+ binding. The interaction with His-13, in fact, is apparently determining a strong conformationalrearrangement which involves Tyr-10. This may explain whymany previous investigations had indicated Tyr-10 as a metalbinding residue. Figure 10 also shows that, for hAâ28, bothAsp-1 HR and Glu-11 Hγ exhibit smaller effects than those forrAâ28. Such a difference could be explained assuming that forthe human peptides three and not two imidazole nitrogensparticipated in the metal coordination and the N-terminal aminogroup and Glu-11 carboxylate could play a minor role in thestabilization of the metal complex.

The diverse temperature dependence of His-6 aromaticprotons of human and rat peptides (Figure 2) suggests that,besides the larger affinity of human peptide-Zn2+ complex,there are also different kinetic processes possible. In particular,at T ) 310 K, the His-6 aromatic protons are in the fastexchange region of the NMR time scale only for the rat peptide,while such resonances are in the coalescence region for thehuman variant.

On the basis of the obtained results, a structural model hasbeen built for the human Zn2+ complex, while a structure ofthe Zn2+-rAâ28 complex has been determined by NMR. Theobtained structures indicate the presence of two distinctdomains: the N-terminal one where the metal binding site is

located, and the C-terminal one, which is interacting with themicelle surface. The results, obtained atT > 310 K, describethe averaged structure of the two or more metal boundconformations which are in intermediate exchange regimerespect to the NMR time scale. Interestingly, rat and humanAâ28 show different kinetic features, entering the exchangeprocess the fast regime at physiological temperature (310 K)only for the rat peptide. The large broadening of Val-18, Phe-19, Phe-20, Ala-21 amide protons which constitute theâ1 sheetof the Aâ42 fibrils,72 suggest a significant conformational changein this hydrophobic region upon Zn2+ binding, which cantherefore promote theâ-sheet formation. The observed differ-ence on kinetic and thermodynamic features of human and ratZn2+ complexes could therefore be the key to understand thebiophysical difference of the two peptide sequence. As pointedout in recent studies,73,74 the propensity to form aggregates isstrongly influenced by the nature of the amino acid side chain,and in the case of Aâ-Zn2+ complexes, we found that the H13Rmutation results in subtle affinity and kinetic differences betweenhuman and rat peptide suggesting its determinant role in theaggregation process of Aâ.

Acknowledgment. We acknowledge the CIRMMP (Con-sorzio Interuniversitario Risonanze Magnetiche di Metallopro-teine Paramagnetiche) and the MIUR (FIRB RBNE03PX83_003)for financial support.

Supporting Information Available: 1H chemical shift ofHis-6 and His-14 Hε of rAâ28 as a function of zinc concentra-tion. Finger print region of the TOCSY spectrum of hAâ28 0.4mM, 100 mM SDS at pH 7.5 andT ) 298 K, in the absenceand in the presence of 1.0 Zn2+ equiv, showing the broadeningof selected amide protons upon metal addition.1H chemical shiftof His-6, His-13, and His-14 Hε of hAâ28 as a function of zincconcentration. CD spectra of 3µM hAâ28 in presence of 1.0Zn2+ equiv in H2O/SDS solutions, pH 7.5 at 298 (lower trace),310, 318, and 328 K (upper trace). This material is availablefree of charge via the Internet at http://pubs.acs.org.

References and Notes

(1) Selkoe, D. J.Physiol. ReV. 2001, 81, 741.(2) Barrow, C. J.; Zagorski, M. G.Science1991, 253, 179.(3) Fraser, P. E.; Nguyen, J. T.; Surewicz, W. K.; Kirschner, D. A.

Biophys. J.1991, 60, 1190.(4) Hilbich, C.; Kisters-Woike, B.; Reed, J.; Masters, C. L.; Beyreuther

K. J. Mol. Biol. 1991, 218, 149.(5) Burdick, D.; Soreghan, B.; Kwon, M.; Kosmoski, J.; Knauer, M.;

Henschen, A.; Yates, J.; Cotman, C.; Glabe, C.J. Biol. Chem.1992, 267,546.

(6) Otvos, L., Jr.; Szendrei, G. I.; Lee, V. M.; Mantsch, H. H.Eur. J.Biochem.1993, 211, 249.

(7) Talafous, J.; Marcinowski, K. J.; Klopman, J.; Zagorski, M. G.Biochemistry1994, 33, 7788.

(8) Hou, L.; Shao, H.; Zhang, Y.; Li, H.; Menon, N. K.; Neuhaus, E.B.; Brewer, J. M.; Byeon, I. J.; Ray, D. G.; Vitek, M. P.; Iwashita, T.;Makula, R. A.; Przybyla, A. B.; Zagorski, M. G.J. Am. Chem. Soc.2004,126, 1992.

(9) Kirschner, D. A.; Inouye, H.; Duffy, L. K.; Sinclair, A.; Lind, M.;Selkoe, D. J.Proc. Natl. Acad. Sci. U.S.A.1987, 84, 6953.

(10) Gorevic, P. D.; Castano, E. M.; Sarma, R.; Frangione, B.Biochem.Biophys. Res. Commun.1987, 147, 854.

(11) Lovell, M. A.; Robertson, J. D.; Teesdale, W. J.; Campbell, J. L.;Markesbery, W. R.J. Neurol. Sci.1998, 158, 47.

(12) Maynard, C. J.; Bush, A. I.; Masters, C. L.; Cappai, R.; Li, Q. X.Int. J. Exp. Path.2005, 86, 147.

(13) Kozlowski, H.; Brown, D. R.; Valensin, G.Metallo Chemistry ofNeurodegeneration Biological, Chemical and Genetic Aspects; RSCPublishing: Piccadilly, London, 2006.

(14) Religa, D.; Strozik, D.; Cherny, R. A.; Volitakis, I.; Haroutunian,V.; Winblad, B.; Naslund, J.; Bush, A. I.Neurobiology2006, 67, 69.


(15) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; Paradis, M. D.;Vonsattel, J. P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi, R. E.Science1994, 265, 1464.

(16) Bush, A. I.; Moir, R. D.; Rosenkranz, K. M.; Tanzi, R. E.Science1995, 268, 1921.

(17) Huang, X.; Atwood, C. S.; Moir, R. D.; Hartshorn, M. A.; Vonsatell,J. P.; Tanzi, R. E.; Bush, A. I.J. Biol. Chem.1997, 272, 26464.

(18) Raman, B.; Ban, T.; Yamaguchi, K.; Sakai, M.; Kawai, T.; Naiki,H.; Goto, Y.J. Biol. Chem2005, 280, 16157.

(19) Karr, J. W.; Kaupp, L. J.; Szalai, V. A.J. Am. Chem. Soc.2004,126, 13534.

(20) Esler, W. P.; Stimson, E. R.; Jennings, J. M.; Ghilardi, J. R.;Mantyh, P. W.; Maggio, J. E.J. Neurochem.1996, 66, 723.

(21) Morgan, D. M.; Dong, J.; Jacob, J.; Lu, K.; Apkarian, R. P.;Thiyagarajan, P.; Lynn, D. G.J. Am. Chem. Soc.2002, 124, 12644.

(22) Syme, C. D.; Nadal, R. C.; Rigby, S. E. J.; Viles, J. H.J. Biol.Chem.2004, 279, 18169.

(23) Garai, K.; Sengupta, P.; Sahoo, B.; Maiti, S.Biochem. Biophys.Res. Commun.2006, 345, 210.

(24) Cardoso, S. M.; Rego, A. C.; Pereira. C.; Oliveira, C. R.Neurotox.Res.2005, 7, 273.

(25) Talmard, C.; Guilloreau, L.; Coppel, Y.; Mazarguil, H.; Faller, P.ChemBioChem2007, 8, 163.

(26) Syme, C. D.; Viles, J. H.Biochim. Biophys. Acta2006, 1764, 246.(27) Kowalik-Jankowska, T.; Ruta, M.; Wisniewska, K.; Lankiewicz,

L. J. Inorg. Biochem.2003, 95, 270.(28) Liu, S.; Howlett, G.; Barrow, C. J.Biochemistry1999, 38, 9373.(29) Karr, J. W.; Akintoye, H.; Kaupp, L. J.; Szalai, V. A.Biochemistry

2005, 44, 5478.(30) Tickler, A. K.; Smith, D. G.; Ciccotosto, G. D.; Tew, D. J.; Curtain,

C. C.; Carrington, D.; Masters, C. L.; Bush, A. I.; Cherny, R. A.; Cappai,R.; Wade, J. D.; Barnham, K. J.J. Biol. Chem.2005, 280, 13355.

(31) Mekmouche, Y.; Coppel, Y.; Hochgrafe, K.; Guilloreau, L.;Talmard, C.; Mazarguil, H.; Faller, P.ChemBiochem.2005, 6, 1663.

(32) Zirah, S.; Stefanescu, R.; Manea, M.; Tian, X.; Cecal, R.; Kozin,S. A.; Debey, P.; Rebuffat, S.; Przybylski, M.Biochem. Biophys. Res.Commun.2004, 321, 324.

(33) Zirah, S.; Kozin, S. A.; Mazur, A. K.; Blond, A.; Cheminant, M.;Segalas-Milazzo, I.; Debey, P.; Rebuffat, S.J. Biol. Chem.2006, 281, 2151.

(34) Ma, Q. F.; Hu, J.; Wu, W. H.; Liu, H. D.; Du, J. T.; Fu, Y.; Wu,Y. W.; Lei, P.; Zhao, Y. F.; Li, Y. M.Biopolymers2006, 83, 20.

(35) Danielsson, J.; Pierattelli, R.; Banci, L.; Graslund, A.FEBS J.2007,274, 46.

(36) Stellato, F.; Menestrina, G.; Serra, M. D.; Potrich, C.; Tomazzolli,R.; Meyer-Klaucke, W.; Morante, S.Eur. Biophys. J.2006, 35, 340.

(37) Curtain, 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.

(38) Miura. T.; Suzuki, K.; Kohata, N.; Takeuchi, H.Biochemistry2000,39, 7024.

(39) Shivers, B. D.; Hilbich, C.; Multhaup, G.; Salbaum, M.; Beyreuther,K.; Seeburg, P. H.EMBO J.1988, 7, 1365.

(40) Vaughan, D. W.; Peters, A.J. Neuropathol. Exp. Neurol.1981,40, 472.

(41) Atwood, C. S.; Moir, R. D.; Huang, X.; Scarpa, R. C.; Bacarra, N.M.; Romano, D. M.; Hartshorn, M. A.; Tanzi, R. E.; Bush, A. I.J. Biol.Chem.1998, 273, 12817.

(42) Kang, J.; Lemaire, H. G.; Unterbeck, A.; Salbaum, J. M.; Masters,C. L.; Grzeschik, K. H.; Multhaup, G.; Beyreuther, K.; Mu¨ller-Hill, B.Nature1987, 325, 733.

(43) Huang, J.; Yao, Y.; Lin, J.; Ye, Y. H.; Sun, W. Y.; Tang, W. X.J.Biol. Inorg. Chem.2004, 9, 627.

(44) Steward, J. M.; Young, J. D.Solid Phase Peptide Synthesis;PierceChemical Company: Rockford, IL, 1993.

(45) The Millipore 9050 Plus PepSynthesizer Operator’s; MilliporeCorporation: Billerica, MA, 1992.

(46) Sole, N.; Barany, G.J. Org. Chem.1992, 57, 5399.(47) Henry, G. D.; Sykes, B. D.Methods Enzymol.1994, 239, 520.(48) Hwang, T. L.; Shaka, A. J.J. Magn. Reson. A1995, 112, 275.(49) Guntert, P.; Mumenthaler, C.; Wu¨thrich, K. J. Mol. Biol. 1997,

273, 283.(50) Lindahl, E.; Hess, B.; van der Spoel, D.J. Mol. Model.2001, 7,

306.(51) Berendsen, H.; van der Spoel, D.; van Drunen, R.Comput. Phys.

Commun.1995, 91, 43.(52) Jorgensen, W. L.; Tirado-Rives, J.J. Am. Chem. Soc.1988, 110,

1657.(53) Berendsen, H.; Postma, J.; van Gunsteren, W. F.; Di Nola, A.; Haak,

J. J. Chem. Phys.1984, 81, 3684.(54) Ryckaert, J.; Ciccotti, G.; Berendsen, H.J. Comput. Phys.1977,

23, 327.(55) Berendsen, H.Treatment of long range forces in Molecular

Dynamics. In Molecular Dynamics and Protein Structure; Hermans, J., Ed.;Polycrystal Book Service: Western Springs, IL, 1995; p 18.

(56) Shao, H.; Jao, S.; Ma, K.; Zagorski, M. G.J. Mol. Biol.1999, 285,755.

(57) Poulsen, S. A.; Watson, A. A.; Fairlie, D. P.; Craik, D. J.J. Struct.Biol. 2000, 130, 142.

(58) Coles, M.; Bicknell, W.; Watson, A. A.; Fairlie, D. P.; Craik, D. J.Biochemistry1998, 37, 11064.

(59) Fletcher, T. G.; Keire, D. A.Protein Sci.1997, 6, 666.(60) McDonnell, P. A.; Opella, S. J.J. Magn. Reson. B1993, 102, 120.(61) Henry, G. D.; Sykes, B. D.Methods Enzymol.1994, 239, 520.(62) Du, Y.; Bales, K. R.; Dodel, R. C.; Liu, X.; Glinn, M. A.; Horn, J.

W.; Little, S. P.; Paul, S. M.J. Neurochem.1998, 70, 1182.(63) Matsubara, E.; Soto, C.; Governale, S.; Frangione, B.; Ghiso, J.

Biochem. J.1996, 316, 671.(64) Chen, Y. H.; Yang, J. T.; Chau, K. H.Biochemistry1974, 13, 3350.(65) Reuben, J.J. Am. Chem. Soc.1973, 95, 3534.(66) Vishwanath, C. K.; Easwaran, K. R.Biochemisty1981, 20, 2018.(67) Garzon-Rodriguez, W.; Yatsimirsky, A. K.; Glabe, C. G.Bioorg.

Med. Chem. Lett.1999, 9, 2243.(68) Clements, A.; Allsop, D.; Walsh, D. M.; Williams, C. H. J.

Neurochem. 1996, 66, 740.(69) Kusumoto, Y.; Lomakin, A.; Teplow, D. B.; Benedek, G. B.Proc.

Natl. Acad. Sci. U.S.A.1998, 95, 12277.(70) Yang, W. Y.; Larios, E.; Gruebele, M.J. Am. Chem. Soc.2003,

125, 16220.(71) Kidambi, S. S.; Lee, D. K.; Ramamoorthy, A.Inorg. Chem. 2003,

42, 3142.(72) Luhrs, T.; Ritter, C.; Adrian, M.; Riek-Loher, D.; Bohrmann, B.;

Dobeli, H.; Schubert, D.; Riek, R.Proc. Natl. Acad. Sci. U.S.A.2005, 102,17342.

(73) Meinhardt, J.; Tartaglia, G. G.; Pawar, A.; Christopeit, T.;Hortschansky, P.; Schroeckh, V.; Dobson, C. M.; Vendruscolo, M.;Fandrich, M.Protein Sci.2007, 16, 1214.

(74) Chiti, F.; Stefani, M.; Taddei, N.; Ramponi, G.; Dobson, C. M.Nature2003, 424, 805.


NMR Studies of the Zn 2+ Interactions with Rat and Human β-Amyloid...

Documents

Transcript of NMR Studies of the Zn 2+ Interactions with Rat and Human β-Amyloid...