Molecular Dynamics Study of the Beta Amyloid Peptide of Alzheimer's

Mar 22, 2007 - Metal Binding to Amyloid-β1–42: A Ligand Field Molecular Dynamics Study. Shaun T. MutterMatthew TurnerRobert J. DeethJames A. Platts...
0 downloads 0 Views 603KB Size
Download PDF
J. Phys. Chem. B 2007, 111, 3789-3799

3789

Molecular Dynamics Study of the Beta Amyloid Peptide of Alzheimer’s Disease and Its Divalent Copper Complexes Duilio F. Raffa and Arvi Rauk* Department of Chemistry, UniVersity of Calgary, 2500 UniVersity DriVe NW, Calgary, Alberta, Canada T2N 1N4 ReceiVed: December 27, 2006; In Final Form: February 15, 2007

The Aβ1-42 monomer structure was assessed with a 790 ns molecular dynamics (MD) simulation, and the results were compared with the NMR experiment on Aβ10-35 and Aβ1-40. Previous theoretical work in a model of the His13-His14 region of Aβ defined the possible Cu(II) binding geometries at this site (Raffa et al. J. Biol. Inorg. Chem. 2005, 10, 887-902). MD simulations totalling almost 2 µs were also carried out on Cu(II)/Aβ1-42 systems, using the ab initio structures as templates for the copper binding site. This work finds that the copper-free Aβ1-42 system may stabilize after ∼350 ns into a collapsed coil conformation, and we find good agreement with some, but not all, of the structural features determined experimentally for the Aβ10-35 and Aβ1-40 peptides. The results of the Cu(II)/Aβ1-42 systems are compared to the Cu(II)-free Aβ1-42 simulation.

Introduction The Aβ peptide is a normally soluble 4.3 kDa peptide found in all biological fluids, but it accumulates as the major constituent of the extracellular deposits that are the pathological hallmark of Alzheimer’s disease (AD).1,2 Aβ is generated as a mixture of polypeptides manifesting carboxyl-terminal heterogeneity. The two main isoforms are Aβ1-40 and Aβ1-42. The Aβ1-40 isoform is the predominant soluble species,3,4 whereas the more neurotoxic form,5 Aβ1-42, is the predominant species found in senile plaque (SP) deposits (see Chart 1).6 Little is known about the structure of Aβ1-42 in aqueous solution. Stable seedless solutions of Aβ1-42 have been shown to be monomeric,7 the CD structure of which is consistent with a predominantly random coil peptide.8,9 No NMR data are available for aqueous Aβ1-42, but complete assignment and implied structures are available for the more soluble Aβ10-35 and Aβ1-40.10 NMR data for Aβ1-40 at pH 7.4 shows NOE cross-peaks among the central 26 residues, indicating a conformation that is stable on the millisecond NMR time scale. No medium or long-range crosspeaks were detected within the N-terminal domain (residues 1-9) and the C-terminal domain (residues 36-40), indicating that these residues of Aβ1-40 are not as well structured as the central 26 residues. The NMR spectra of the 10-35 region of Aβ1-40 and its HR chemical shifts are nearly identical to those of Aβ10-35, suggesting that this 26 residue central portion adopts the same conformation in both Aβ10-35 and Aβ140.10 The structure of Aβ10-35 deduced from the NMR NOE constraints and presumably that of the core of the Aβ1-40 peptide is collapsed into a compact series of loops, strands, and turns and has no R-helical or β-sheet secondary structure.10 Molecular dynamics (MD) studies, similar to those of the present work, on a monomer of Aβ1-40 in aqueous solution show that the peptide adopts a predominantly random coil conformation,11 with a small amount of R-helical and β-sheet structure. * To whom correspondence [email protected].

should

be

addressed.

E-mail:

As part of the present investigation, we report below the results of a long-term (790 ns) MD simulation of Aβ1-42 beginning from an initial structure whose core is modeled by the NMR structure of Aβ10-35. Our primary interest is in the effect of Cu(II) on the solution structure of Aβ1-42. The β-sheet content of a phosphate buffer solution of Aβ1-40 rises with increasing Cu(II) concentration, as judged by CD spectroscopy.17,20 Trace amounts of Cu(II) are sufficient to induce Aβ1-40 and Aβ1-42 nucleation, which causes the remainder of the Aβ solution to precipitate by a seeding mechanism.12,13 In human blood plasma, more than 98% of the amino-acid-bound Cu(II) occurs in histidine (His) complexes.14,15 Aβ has three His residues and binds Cu(II) with high affinity12 in a 1:1 ratio.16 1H NMR spectra,17 Raman,18 and selective oxidation19 experiments indicate that His13 and His14 are the two most firmly established ligands in the squareplanar17,20 coordination sphere of Cu(II) bound to Aβ. On the basis of spectroscopic studies on the shorter, more soluble Aβ128, His6 has also been postulated as another probable ligand for Cu(II),17,20 as has the N-terminus.17,21 In this paper, we report the results of MD simulations on the aqueous Cu(II)/Aβ1-42 system, using structures A, B, and C (Figure 1) as templates for the copper binding site and the collapsed-coil structure of the copper-free system as the initial geometry. Previous ab initio calculations on a model of the His13-His14 region of Aβ found that species A and C2 are approximately equally populated at pH ) 7.22 In A, the oxygen of the backbone carbonyl (Oc) is involved in the Cu(II) coordination sphere along with the Nπ of both imidazoles. A water molecule was used to fill the fourth coordination site of the Cu(II). Τhe coordination of His6 was modeled by replacing the water molecule of A with 4-methylimidazole. Thus, species B (Figure 1) models the involvement of His6 in addition to His13 and His14 in the Cu(II) coordination sphere. Structure B is the most stable by roughly 14 kJ mol-1.23 In C, the Cu(II) is coordinated to the deprotonated nitrogen of the backbone.

10.1021/jp0689621 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/22/2007

3790 J. Phys. Chem. B, Vol. 111, No. 14, 2007

Raffa and Rauk

CHART 1

Methods Structural Models. Aβ1-42 Monomer. Our initial Aβ142 structure, shown in Figure 2, was built starting from the Aβ10-35 random coil structure, PDB entry 1HZ3. The 1-9 and the 36-40 portions in coil conformation were added by

Figure 1. B3LYP/6-31G(d)-optimized geometries. Distances in Å. We will designate the Cu(II) geometry in compound A as Cu(II)O, the geometry in compound B as Cu(II)OHis, and the geometry in compound C as Cu(II)N.

Figure 2. a. Starting Aβ1-42 structure. Portions 1-9 and 36-40 taken from structure PDB entry 1AML are shown in red. The 10-35 portion was taken from structure PDB entry 1HZ3. Added residues Ile41 and Ala42. b. Peptide in the box used for the MD simulation and its dimensions.

homology modeling from the Aβ1-40 trifluoroethanol/water structure, PDB entry 1AML,24 with the addition of the last two aminoacids, Ile41-Ala42. Cu(II)/Aβ1-42 Complexes. Three separate Cu(II)/Aβ1-42 complexes were considered. They differ in the Cu(II) binding site, which was inserted in the Aβ1-42 structure using structures A, B, and C (Figure 1) as templates. Cu(II) Parametrization. The Cu(II) geometry in compound A22 is designated as Cu(II)O, and the geometry in compound C22, as Cu(II)N. Using these structures as templates, new extended structures that include two more glycine residues were optimized without constraints at the ab initio B3LYP/6-31G(d) level. The resulting geometries are denominated Cu(II)Otetrapeptide and Cu(II)N-tetrapeptide, respectively, and are shown in Figure 3a and b. These two structures are the benchmarks for the addition of the Cu(II) parameters to the Gromacs25 force field of the GROMACS26 suite of software, as described below. Bonds. Rupture of the bonds to the Cu(II) is not probable, given the high copper binding affinity,12,22 so these bonds are described in the usual harmonic approximation.26 This hypothesis will be checked in the Binding Site Stability section, with a shorter simulation where bonds are described by a Morse potential, suitable to permit bond rupture. The force constants necessary to describe the Cu(II)-peptide bonds are derived from ab initio calculations on the Cu(II)-O and the Cu(II)-N bonds of two small model systems, [4-methylimidazole-cis(NH3)2H2O-Cu(II)]2+ and [4-methylimidazole-NH3-trans(H2O)2Cu(II)]2+. Comparison of the ab initio force constants of the bonds to Cu and Gromacs force constants of a N-H bond of an amide and a C-C bond of an imidazole ring leads to a compromise value for the Cu(II)-peptide force constant of 144 300 kJ mol-1 nm-2. This value will be assigned to all the bonds in which copper takes part. The Gromacs force field was applied for the peptidic part of the molecule. Angles. The ab initio geometry of the Cu(II) coordination sphere is distorted from square planar to some extent due to

Figure 3. Ab initio B3LYP/6-31G(d)-optimized geometries; distances in Å. a. Cu(N)-tetrapeptide b. Cu(O)-tetrapeptide; MM optimized geometries; distances in Å; charges in bold. c. Cu(N)-tetrapeptide geometry. d. Cu(O)-tetrapeptide geometry.

MD Study of Alzheimer’s Beta Amyloid Peptide Jahn-Teller effects. In the empirical force field, the copper reference geometry is taken to be square planar. It is assumed that the asymmetric environment will distort the square plane in the direction dictated by steric effects. Additional angles were defined to keep copper on the bisector of the imidazole C-Nπ-C angle. The force constants from the Gromacs force field for the N-H angles were also used for the N-Cu(II) angles. In the Gromacs force field, improper dihedrals are used to prevent cis/trans isomerization where needed, for example, in the amide group to prevent rotation around the CdN bond. In the same spirit, improper dihedral angles were defined to prevent interchange of the copper ligands and to keep the copper approximately in the imidazole ring plane. Again, the Gromacs N-H force constants were applied. Charges. Natural bond orbital (NBO) analysis on the Cu(II)O-tetrapeptide and on the Cu(II)N-tetrapeptide provide guidance for the assignment of the charge on Cu(II) in different binding environments. The NBO charges on Cu(II), +1.41 and +1.32, respectively, were judged to be too high and were adjusted to +1.3 and +1.2, respectively. The total local charge in the Cu(II)O case is +2, whereas in the Cu(II)N geometry, it is +1. In each case, the excess charge after accounting for the charge on Cu(II) is distributed among the residues interacting with the copper cation, namely His13; His14 residues; and the water molecule, as specified in Figure 3. Lennard-Jones Interactions. In the Gromacs force field, these interactions for metals are described by the repulsive part of a Lennard-Jones 6-12 function. The force field does not have parameters to describe the Cu(II) nonbonded interactions. The Gromacs Zn(II) parameters were used in their place. These parameters and charges lead to the molecular mechanics (MM) optimized structures shown in Figure 3c and d. In this figure it can also be appreciated that the Gromacs force field treats hydrogen atoms attached to carbon in the united atom representation. The root-mean-square deviations (RMSD) between the ab initio optimized and the MM optimized structures for the Cu(II)O-tetrapeptide and the Cu(II)N-tetrapeptide are 0.76 and 0.26 Å, respectively. The main differences come from a small rotation of the water. The same RMSDs without taking into account the water molecule are 0.30 and 0.18 Å. Molecular Dynamics Simulation of Aβ1-42 and Cu(II)/Aβ142 Complexes. The studied molecules were placed in the center of a 60 × 40 × 40 Å box, Figure 2, with enough sodium cations to ensure electroneutrality, and the box was filled with ∼3000 SPC water molecules. MM energy minimizations provided the initial structures for the MD simulations. The MD simulations were carried out with constant number, pressure, temperature, and periodic boundary conditions. The LINCS method was used to constrain bond lengths. Electrostatic interactions were calculated using the Particle-Mesh Ewald algorithm. A constant pressure of 1 bar was applied with a coupling constant of 1.0 ps; peptide, water, and sodium were coupled separately to a temperature bath at 300 K with a coupling constant of 0.1 ps. A time step of 3 fs was used. Measurement of the radius of gyration, distances, and counts of hydrogen bonds and salt bridges were performed every 72 ps by using facilities within the GROMACS package. The RMSD, calculated with the Profit27 suite of software and applied to the atoms of the backbone, was used as a measure of similarity of structures. The trajectories were analyzed grouping structurally similar frames by cluster analysis (RMSD cutoff ) 3.0 Å).28 To reduce the computational effort, the number of similar conformations is counted for every seventh frame, yielding a time step of 504

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3791 ps. This is an appropriate time frame, considering that the RMSD difference between frames 504 ps apart is always below 3.0 Å, the value used to discriminate structures. The frame with the largest number of neighbors is denoted the “middle” structure and is the structure representing that cluster. The largest cluster with all its members is eliminated from the pool of structures. The analysis is repeated for the remaining frames until all of them have been classified. The VMD suite of software29 was extensively used for visualization, secondary structure analysis by the STRIDE method,30 and preparation of structural diagrams. Experimental and theoretical approaches predict an estimate of the time scale, t, in ns, for folding a single domain small protein ( 350 ns) the sidechains of the Lys28 and the Asp23 residues also form a salt bridge, which is randomly broken and remade. The NH3+ and the COO- groups are within 3.5 Å 22% of the time. The 14-17 loop observed in the NMR structure is also observed in Figure 4c; however, the 27-30 and 30-33 turns of Figure 4d are assigned (by STRIDE) in part as coil conformation. The RMSD between the NMR and the MD for the 29-35 C-terminal region is 3.9 Å, almost within our criterion for being the same conformation. For comparison to experimental NOE-derived values, some of the distances were calculated33,40 as -1/6, where the notation indicates an average, for the time comprising the most populated cluster (t > 350 ns, Figure 4a). Only those distances larger than the corresponding NOE upper boundary41-44 by 0.5 Å are considered NOE violations.45 We find agreement in some cases, but not in others. The upper NOE boundary between the amide hydrogens of Phe19 and Phe20 is 2.50 Å, to be compared with the MD average of 2.81 Å. For the Leu17Val18 residues, the NOE distance is 2.75 Å and 2.68 Å for the MD; for the Glu22-Val24 separation, we have 3.00 and 2.61 Å, respectively; for the Val18-Met35(methyl) residues, we have 6.50 for the NOE distance and 3.91 Å for the MD; and for the Ala21 and Ser26 residues, 6.00 and 5.09 Å, respectively. However, for the Asn27-Ala30, we have 5.00 and 5.87 Å; for the Lys28-Met35, we have 5.50 and 6.88 Å; for the His14Glu22 residues, we have 7.00 and 9.55 Å. In each of these cases, the average distance is well above the NOE restraint showing differences between our simulation of Aβ1-42 and the NMR structure of the Aβ10-35 peptide. In summary, we find in our Aβ1-42 MD trajectory the same radius of gyration of Aβ1-40,34,35 but a different collapsed coil conformation.10 Notably, three regions (3-5, 7-11, and 3337) have paired in an antiparallel β-sheet fashion after ∼350 ns of simulation, whereas the lack of cross-correlation peaks in the NMR spectrum of Aβ1-40 implies that this region is

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3793

Figure 6. a. Cluster analysis profile for trajectory AbCuN (RMSD 3.0 Å). b. Secondary structure profile, as assigned by STRIDE, for trajectory AbCuN. c. Middle structure of the most populated cluster of trajectory AbCuN. Hydrogen bonds represented by dotted lines. Labeled as “a” between the carboxylate (COO) of Asp23 and H Leu17; “b” Oc Val18 and H Gly29; “c” Oc Arg5 and H Glu11; “d” Oc Phe19 and H Ser26; “e” Oc Leu34 and H Ile31; “f” Oc Val36 and H Val39; “g” Oc Val39 and H Ala30. The residues that limit the regions described by NMR are shown explicitly.

disordered in the shorter peptide. The 10-15 and the 17-21 portions do have conformations that are similar to the Aβ1035 NMR experiment,10 but the conformations of the 16-28 and the 29-35 segments are different. The 14-17, 22-25, and 2427 turns and the contribution to the polar surface of the Glu22Lys28 section experimentally observed for Aβ10-3510 are also found in the MD of Aβ1-42. Two regions (22-25 and 2427) of the NMR structure described as turns by STRIDE have paired in a π-helix in the MD simulation of Aβ1-42. Conformation of the Cu(II)N/Aβ1-42 Complex Monomer in Water (Trajectory AbCuN). The cluster analysis with a RMSD cutoff of 3.0 Å is presented in Figure 6a. The clusters show increasingly larger populations with time, with one predominant conformation after the initial ∼160 ns. Thus, the AbCuN trajectory has apparently equilibrated twice as fast as the Ab trajectory. Proceeding as in the Ab case, we analyze the last ∼340 ns of the trajectory represented by the middle structure shown in Figure 6c. The secondary structure profile by STRIDE analysis for trajectory AbCuN is shown in Figure 6b. The Cu(II)-containing peptide conformation is clearly coil (not R-helix or β-sheet) with a very slight increase in the β-strand conformation after ∼250 ns. The hydrogen bonds network of the AbCuN trajectory is different from that of the Cu(II)-free trajectory, Ab (compare Figures 4c and 6c). The average radius of gyration is 9.0 Å, essentially the same as that of the most populated cluster of the Cu(II)-free system.

3794 J. Phys. Chem. B, Vol. 111, No. 14, 2007

Raffa and Rauk

Figure 7. a. Cluster analysis profile for trajectory AbCuN (RMSD 3.0 Å). b. Secondary structure profile, as assigned by STRIDE, for trajectory AbCuN. c. Middle structure of the most stable cluster of trajectory AbCuN, hydrogen bonds represented by dotted lines. Labeled as “a” between the Oc His6 and H His13; “b” COO Asp23 and H Leu17; “c” Oc Asp23 and H Glu15; “d” Oc Glu22 and H Arg5; “e” salt bridge between COO Glu22 and H (amine) Arg5; “f” Oc Phe19 and H Lys28; “g” Oc Ala42 and H Ala21. d. Superimposition of the middle structure of the most populated cluster and the conformation present after ∼420 ns (also within the most populated cluster).

In the most stable cluster, the carboxylate group of Glu22 has moved into the coordination sphere of the copper. Ab initio calculations on model copper complexes with similar ligands show that the displacement of the water molecule by a carboxylate group is roughly isoergonic.46 Given that the electrostatic term has shown to be enough to keep the carboxylate in the surroundings sharing the coordination sphere with the water molecule, we believe that in this case, the force field is representing properly this interaction and have not defined an explicit bond between the copper and the carboxylate. Coordination of the copper to the carboxylate group of Glu22 improves the stability of the 17-21 hydrophobic core, making possible hydrogen bonds a and b. Hydrogen bond e keeps the 31-34 region as a turn, and f pairs two β-bridge portions through a 36-39 loop in an incipient β-sheet. The hydrogen bond g keeps the 31-34 and the 36-39 loops connected. Neither Lys16 nor Lys28 are involved in any salt bridge. Conformation of the Cu(II)O/Aβ1-42 Complex Monomer in Water (Trajectory AbCuO). The cluster analysis with a RMSD cutoff of 3.0 Å is shown in Figure 7a. We find one predominant conformation after the initial ∼50 ns. The average radius of gyration is 9.2 Å (same as the Cu(II)-free system). The secondary structure profile for trajectory AbCuO is shown in Figure 7b. The peptide conformation is mostly coil with β-sheet near the C-terminus. In the AbCuO site, the Cu(II) interacts with the carboxylate of Glu3 rather than Glu22. The copper carboxylate interaction together with hydrogen bond “a” restricts the movement of this portion of the molecule. Hydrogen bond f binds the C-terminus

to the hydrophobic core (17-21). In the C-terminal region, two short β strands, Met35-Val36 and Val39-Val40, are paired by hydrogen bonds g and h. After the first ∼50 ns, as in the Cu(II)-free system, the sidechains of the Lys28 and the Asp23 residues make a salt bridge, which is broken and remade repeatedly. In this case, 66% of the time they are within 3.5 Å of each other. The increased presence of this salt bridge as a result of the Cu(II) binding is consistent with the solid-state, fibril NMR structure.38 However, the salt bridge between Glu22 and the amine group of Arg5, present 46% of the time, would disrupt the ability of the Cu(II)/Aβ1-42 monomer to adopt the fibril structure. Changes in the conformation are noticed in the STRIDE analysis of residue Phe20 and the Leu34-Val39 region at ∼420 ns (Figure 7b). We will briefly examine this structure, despite the fact that the analysis did not identify it as a different cluster, because it may represent an incipient new conformation not fully developed in the time of this simulation. The new secondary structure arises after a change in the ψ angle of Met35. This generates a slightly different backbone conformation in which the Met35-Ala42 segment has changed its orientation. The two conformations are superimposed in Figure 7d. Cu(II)OHis6/Aβ1-42 Complex in Water (Trajectory AbCuOHis). According to an ab initio study, an additional imidazole will displace the Cu(II)-bound water in an exothermic process.46 The addition of a His from a second Aβ has been proposed to be the mechanism of Cu(II)-induced aggregation.18,20 His6 has also been shown as a ligand for Cu(II) in aqueous Aβ1-28, creating a large cycle.17,20 We restrict this

MD Study of Alzheimer’s Beta Amyloid Peptide

Figure 8. a. Cluster analysis profile for trajectory AbCuO3His (RMSD 3.0 Å). b. Secondary structure profile, as assigned by STRIDE, for trajectory AbCuO3His. c. Middle structure of the most populated cluster of trajectory AbCuO3His. Hydrogen bonds represented by dotted lines. Labeled as “a” between Oc Asp23 and H Gln15; “b” COO Asp23 and H Lys16; “c” COO Asp23 and H Vall8; “d” Oc Ala2 and H(amine) Lys28; “e” Oc Phe20 and H lle31; Oc Val24 and H Asn27; “g” Oc lle41 and H Met35.

study to the Aβ monomer in which all three His residues are bound to the Cu(II), modeled by structure B (Figure 1), in which Cu(II) is also bound to the Oc.47 In this binding site, denoted Cu(II)OHis, the Cu(II) is bound to the oxygen of the backbone and His6, His13, and His14, and the Cu(II) coordination is 3N1O, as in experiments with Aβ1-16, Aβ1-28, Aβ1-40, and Aβ1-42.17,21 The cyclization of proteins is known to increase the folding rate due to the reduction of the conformational space.48 Since the conformation of His13, His14, and the cyclized segment to His6 is relatively restricted due to the Cu(II) bonding, the longest portion free to fold comprises the last 28 residues, leading to a minimum folding time of 280 ns (N ) 28 in eq 1). As a result, the 350 ns of this trajectory is approximately equivalent to the 500 ns of the previous Cu(II)-containing systems (N ) 42). The cluster analysis with a cutoff of 3.0 Å, Figure 8a, shows one predominant conformation after the initial ∼130 ns. The radius of gyration for this most populated cluster is 8.9 Å. The secondary structure profile for trajectory AbCuOHis is shown in Figure 8b. The peptide conformation is predominantly coil. The appearance of the π-helix after ∼270 ns is a

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3795 consequence of a slight tightening of the Val18-Asp23 turns. This is not enough of a change to register as a different conformation by our RMSD ) 3.0 Å criterion. The importance of the N-terminus as a ligand for the Cu(II) has been pointed out in the literature, on the basis of CD and EPR evidence.17,21 Our results find evidence of the direct involvement of the carboxylate of Asp1 in the coordination sphere of the Cu(II) together with the three His residues. The approach of the carboxylate of Asp1 to the Cu(II) is a secondary consequence of tying His6 to the metal and is due to the electrostatic attraction between the positively charged Cu(II) and the negatively charged carboxylate. We take it as evidence that the N-terminus could be a Cu(II) ligand, although our simulation does not permit this, since the N-terminus is protonated. Ab initio results on small models of the Cu(II)OHis geometry show that the involvement of the three His residues and the N-terminus (modeled by a methylamine molecule) is not stable with respect to the shedding of ligands by the Cu(II) (see data on structures B, S1, and S2 in the Supporting Information). As a result, both the N-terminus and His6 would not be ligands to Cu(II) at the same time. The short β-bridge portions seen in Figure 8b correspond to the Ala21 and Ala30 residues paired by the hydrogen bond e and may indicate incipient development of a short β-sheet structure, although this is not seen in the time of this simulation. Hydrogen bond e, together with f, keeps the 24-27 region in turn conformation. In the C-terminal region, g keeps the Gly37Gly38 loop in position. Binding Site Stability and Water Replacement. The energy minimizations that provide the initial structures for the MD runs also provide some evidence of the effect of the backbone on the geometry of the Cu(II) binding site. In every case, the resulting geometries of the different binding sites have only slight distortions from the reference ab initio geometries shown in Figure 1. In an aqueous environment, strain introduced by the entire peptide chain may result in the replacement of a histidine ligand by a water molecule. The stability of the Cu(II) binding site can be assessed through a simulation in which Morse potentials are defined for the Cu(II)-Nπ(His) bonds. This would allow bond rupture in the case that the stress exerted by the backbone on the binding site is higher than the energy required to replace any of the Cu(II)-peptide bonds with a copper-water bond. The Cu(II)O geometry (Figure 1) is chosen to analyze its behavior when the Cu(II) bonds to His13 and His14 are represented by Morse potentials. Therefore, the initial conformation of the AbCuOmorse simulation is the last frame (at 500 ns) of the trajectory AbCuO with the bound water removed. The Cu(II)-His Morse potential has the well depth set to 70.0 kJ mol-1. This value is the lowest ∆H°g(298K) for the replacement of a single histidine residue by water in different models of the binding site studied.22,46 The curvature at the minimum corresponds to the same harmonic force constant used for the AbCuO trajectory. These parameters are applied to the Cu(II)-Nπ bonds, and no interaction is defined between the copper and the backbone carbonyl oxygen, letting the electrostatic attraction mimic the behavior of this bond. The trajectory is denoted AbCuOmorse. The removal of the water molecule attached to the Cu(II) created a space for any other group to get close to the copper atom. As was mentioned before, Glu3 and Glu22 in the AbCuO and AbCuN trajectories, respectively, were able to approach the Cu(II) closely enough to coordinate even with the water present. The AbCuOmorse simulation is a 64 ns extension of the AbCuO trajectory. The cluster analysis with a RMSD cutoff of

3796 J. Phys. Chem. B, Vol. 111, No. 14, 2007

Raffa and Rauk

TABLE 1: RMSD in Å between the Most Populated Clusters of Every Run

TABLE 2: Interactions for the Most Populated Clusters of Each Runa

region

Ab vs AbCuN

AbCuN vs AbCuO

AbCuO vs Ab

Ab CuO vs AbCuOHis

1-42 1-9 17-21 36-42

8.5 3.4 2.3 2.4

7.7 3.6 2.0 1.6

9.1 5.2 1.4 2.8

8.0 3.7 1.3 1.3

3.0 Å shows only one cluster. The RMSD between this cluster and the most stable cluster of the AbCuO trajectory is 2.6 Å. This RMSD is below the 3.0 Å cutoff, meaning that both structures belong to the same cluster. The inclusion of the Morse description for the Cu(II)-peptide bonds and the removal of the water did not have any consequence for the conformation of the Aβ1-42 peptide, and the Cu(II) binding site was preserved within the time studied. However, some changes were noted. The time-averaged Cu(II)-Nπ His13 distance is 2.10 Å, longer than the initial 1.95 Å. For the Cu(II)-Nπ His14 distance, the time average is 2.20 Å, also longer than the initial 1.95 Å. The average Cu(II)-Oc His13 distance is 2.00 Å from the initial 1.88 Å. The longer Cu(II)-Nπ His14 distance denotes more tension on this residue, in agreement with its average position on a high energy area of the Ramachandran plot (φHis13 ) 14°, ψHis13 ) 77°, φHis14 ) 96°, ψHis14 ) 106°). Comparison of Structures. The RMSD between the most stable clusters of every trajectory is presented in Table 1. The four structures are all different from each other. The effect of the Cu(II) binding is appreciable in the 1-9 portion, where the highest RMSDs are found. Nevertheless, according to the 3.0 Å criterion used to discriminate conformations, the 17-21 and the 36-42 regions of the four systems are similar. The N-terminal region of the Ab cluster is hydrogen-bonded to the C-terminal portion. Each of the three Cu(II)-containing structures exhibit the N-terminal region folded in a series of turns and coil portions with a different “ring” or “loop”. In the AbCuN case, the His14-Cu(II)-Glu22 ring; in the AbCuO case, the Glu3-Cu(II)-His13 ring; in the AbCuOHis, the Asp1-His6Cu(II)-His13 ring show a different arrangement of hydrogen bonds and hydrophobic interactions. The conformation of the His13 residue is different in the starting AbCuN and AbCuO structures. The effect on the AbCuN trajectory is to initially bury the Cu(II), keeping it away from the solvent. It takes ∼34 ns until the Glu22 residue begins interacting with the Cu(II). This causes the conformational change that will ultimately expose the Cu(II) to the surface of the molecule, as in Figure 6a. In the AbCuO case, the Cu(II) binding creates a kink in that portion of the molecule, exposing the metal to the solvent from the beginning of the simulation. The N-terminal, the most flexible portion, takes ∼6 ns to fold back and begin to interact with the Cu(II) through the Glu3 residue, as in Figure 7c. The Cu(II) is exposed on the surface of the molecule in the AbCuN and the AbCuO structures. The molecular surface intersects the Cu(II) coordination plane at the His-Cu(II)-His axis. This surface around copper is very similar in both cases. It is composed of the two histidines trans to each other. In the AbCuN, the carboxylate group of the Glu22 occupies the apical position of a fifth ligand. The sixth ligand position is vacant, and in the pocket formed in that position, the only atom barely exposed below the Cu(II) is the Oc of His13. In the AbCuO case, the Glu3 is placed as fifth ligand on one side of the coordination plane and a pocket where the Oc of the His14 is scarcely exposed trans to it. There should be no difference in the reactivity of these sites on the basis of the similarity of the

interaction

Ab

AbCuN

AbCuO

AbCuOHis

salt bridge H bonds intrapeptide H bonds peptide-solvent total

2 25 84 111

5 23 80 108

8 21 85 114

7 23 77 107

a Species A is species 20x from reference 22; species C is species 35x from the same reference.

charge distribution in the surroundings of the copper cation. The Cu(II) is naturally more shielded in the AbCuOHis initial conformation. After ∼130 ns, the carboxylate of Asp1 starts interacting with the metal as the fifth apical ligand. This interaction definitely buries the Cu(II) in the interior of the molecule (Figure 8c). The φ and ψ angles for the His13 and His14 residues in the most populated cluster of trajectory Ab lie in the low-energy areas of the Ramachandran plot. His13 is at the end of a small coil portion in the β-sheet region of the Ramachandran plot, and His14 is in the R-helix region at the beginning of a three residue (His14-Leu17) turn. The effect of the Cu(II) binding on the peptide backbone is visible in the AbCuN (φHis13 ) 34°, ψHis13 ) -106°, φHis14 ) 55°, ψHis14 ) -73°), the AbCuO (φHis13 ) 14°, ψHis13 ) 77°, φHis14 ) 96°, ψHis14 ) 106°), and the AbCuOHis (φHis13 ) -6°, ψHis13 ) 102°, φHis14 ) 92°, ψHis14 ) 118°) trajectories, where these residues reside in very different and high-energy areas of the Ramachandran plot. The βC of the His13-His14 sidechains in the AbCuN middle structure (Figure 6c) lie on opposite sides of the backbone plane. The Cu(II) lies on the pro-R face of the backbone plane. Ab initio calculations previously predicted this conformation to be the most stable for this binding site.22 In the AbCuO case (Figure 7c), both βC’s lie on the same side of the backbone, and the Cu(II) lies on the pro-R face. This conformation is among the most stable ones for this binding site, as previously predicted by ab initio calculations.22 The Lys16-Phe20 region has been identified as the binding sequence during Aβ polymerization and fibril formation leading to the β-strand identified by NMR in the solid state. The Cu(II)-free system is the only structure with any β-bridge conformation in the Lys16-Phe20 region, assigned by STRIDE to only the Phe19 residue (Figure 4c). The π-helix region (Asp23-Asn27) observed in the Cu(II)free system (Figure 4c) is missing in all the Cu(II)-containing systems. In the Ab, AbCuN, and AbCuO cases, the C-terminal region has a portion with β bridge/strand character; however, the pairing of these portions into β-sheets is missing in the Cu(II)containing systems. In the Ab cluster, the C-terminal residues are hydrogen-bonded to the N-terminal section. In the AbCuN cluster, the Ile31-Leu34 and in the AbCuO case, the Val36Val39 loops remain close but not hydrogen-bonded as in a β-sheet. In the AbCuOHis case, the C-terminal domain is in coil conformation. Table 2 shows the average number of salt bridges and hydrogen bonds, as calculated by GROMACS, within the peptide and between the peptide and the solvent, for the time comprising the most populated cluster of the Cu(II)-containing systems. If one assumes that every hydrogen bond and every salt bridge contributes equally to the stability of the system, then the sum of the salt bridges and intrapeptide hydrogen bonds terms may be taken as a measure of the stability of each molecule in the absence of solvent. The sums for the Cu(II)containing structures are in the narrow range 28-30. Thus, the

MD Study of Alzheimer’s Beta Amyloid Peptide

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3797

Figure 9. Filtered distance profile. The vertical red line shows the beginning of the most populated cluster. a. Trajectory Ab. Distance between the Nπ of His13 and the N-terminus (blue), the Nπ of His6 (red), the O of Tyr10 (green), and the S of Met35 (purple). For b, trajectory AbCuN; c, trajectory AbCuO; and d, trajectory AbCuO3His, distance between the Cu(II) and the N-terminus (blue), the Nπ of His6 (red), the O Tyr10 (green), and the S of Met35 (purple).

energetic differences among them are not significant. Structure Ab should not be directly compared to the Cu(II)-containing systems because the Cu(II) affinity is not taken into account in this analysis. The peptide-solvent hydrogen bonds term may be seen as the solvation energy. In this term, bigger differences are seen in the hydrogen bonds count. The AbCuO middle structure contains, on average, 85 hydrogen bonds to the solvent. This is 5 and 6 more than the AbCuN and the AbCuOHis cases, respectively, and approximately the same as the Cu(II)-free system. These differences represent a considerable amount of energy and suggest that the AbCuO molecule is the most stable due to higher “solvation” energy. Other Possible Cu(II) Ligands. The N-terminus and His6 have been proposed to take part in the copper coordination sphere together with His13 and His14. The coordination of Tyr10 and Met35 is less likely, but these residues have been implicated in electron transfer to the Cu(II). The analysis of the distance profile, presented in Figure 9, between the Nπ of His13 for the Cu(II)-free system and between the Cu(II) atom for the copper containing systems, for these four residues and for the four systems studied may provide valuable information. For the purpose of making a bond, a minimum distance of 4 Å will be assumed. This assumption is based in ab initio calculations in which the ligands at that distance usually wander away from the Cu(II) and hydrogen bond somewhere else on the peptidic part of the models. To evaluate the possibility of an electron transfer, the threshold is set to 8 Å, the minimum distance to obtain a minimum reaction rate in a thermodynamically favorable reaction.49 For clarity purposes, the distances plotted in Figure 9 are rolling averages of five adjacent frames to reduce scattering; the unfiltered distances are discussed below, and the relevant minimums are pointed out. The Cu(II)-free system, Ab, is described for comparison purposes, and the Nπ of His13 is used as a reference for the Cu(II) position. In this case, the distance is measured from the Nπ of His13 to the N-terminus, the Nπ of His6, the O of Tyr10, and the S of Met35 (Figure 9a). In the three Cu(II)-containing systems, the distance between the Cu(II) and the four residues is measured. For the most populated cluster (t > 350 ns) of the Ab trajectory, the His13-N-terminus and the His13-His6 distances oscillate between 15 and 20 Å, respectively. The His13-Tyr10

distance has a minimum of 5.4 Å at ∼420 ns. After that time, it rises above 17 Å. The His13-Met35 distance diminishes from ∼15 Å at ∼500 ns to 10 Å at ∼675 ns. If we consider the initial events of this trajectory, attention should be paid to the His13-N-terminus minimum distances of 5.8 Å at ∼95 ns and the His13-Tyr10 distance of 4.2 Å at ∼13 ns. The minimum His13-Met35 distance in this trajectory is 8.4 Å. A tentative conclusion is that the range of motion in the Cu(II)-free system, even after apparent equilibration, is sufficient to permit electron transfer from Tyr10 or Met35 to a Cu(II) atom, were it is bound to His13. For the most populated cluster of the AbCuN trajectory (t > 160 ns), the four distances lie above 10 Å (Figure 9b). Before that time, only the Cu(II)-N-terminus distance descends down to 4.5 Å in only one frame at ∼ 33 ns. Thus, in the AbCuN environment, Cu(II) is unlikely to accept additional ligands or participate in intramolecular redox chemistry. In the AbCuO case (Figure 9c) the Cu(II)-N-terminus distance oscillates between 6 and 12 Å along the time of the simulation. The remaining three distances are always above 7.7 Å. The Cu-His6 bond was restricted to 1.9 Å for the AbCuOHis simulation (Figure 9d). The Cu(II)-N-terminus distance reduces to 5.0 Å repeatedly along the time of the simulation as a consequence of the mentioned interaction between the Cu(II) and the carboxylate of the Asp1 residue. The Cu(II)-Tyr10 distance is 4.6 Å in three frames before 160 ns. After that time, when the most populated cluster arises, it always stays above 5.8 Å. The minimum NπHis13-NπHis14 distance in the Cu(II)-free system is 2.8 Å at ∼645 ns. Thus, a local geometry able to bind the Cu(II) may be spontaneously achieved within the most populated cluster. If His6 or the N-terminus is part of the Cu(II) coordination sphere of the AbCuN or the AbCuO systems, it may take a relatively long time until any of these groups gets close enough and remains close an appropriate amount of time to make possible the hydration water replacement to bind to copper. In these cases, the bond was not made in 500 ns. This “delay” may enhance the binding rate of copper to a histidine from a second Aβ molecule.18,50 The involvement of the N-terminus

3798 J. Phys. Chem. B, Vol. 111, No. 14, 2007 in the Cu(II) coordination sphere is possible in the system in which the three His residues are already involved as a consequence of the Asp1 carboxylate-Cu(II) interaction. On the basis of the long distance and the extremely short time spent by the Tyr10 close to the Cu(II) in the AbCuOHis system, we conclude that Tyr10 is not involved in the Cu(II) coordination sphere, in agreement with the experiment on Aβ128.17 However, the measured distance repeatedly below the 8 Å cutoff would permit a redox reaction’s taking place between the Cu(II) and the oxygen of the Tyr10 residue, as has been proposed in the literature.51,52 The long distances measured between the Cu(II) and the sulfur of the Met35 residue (the minimum distance is 10.4 Å in the AbCuOHis trajectory) do not support the involvement of Met35 in the copper coordination sphere of any of the coppercontaining systems, in agreement with the experimental nontoxicity of the Aβ1-42 monomer.50 Summary and Conclusions The Aβ1-42 monomer structure was assessed with a 790 ns simulation. Equilibrium is apparently reached after ∼350 ns. This MD simulation shows residues 1-9 and 36-40 of Aβ142 in a stable β-sheet conformation, unlike the case for Aβ140 monomer in which these regions are disordered. The NMR and MD conformations are locally similar for the 10-15 and 17-21 portions and different for the 16-28 portion. The 2225 and 24-27 turns identified by NMR are present in our Aβ142 simulation, but have rearranged in a π-helix conformation through a twist of Gly25. The polar atoms of this π-helix portionsGlu22, Asp23, Ser26, Asn27, and Lys28scontribute to the polar surface, as in the NMR model. The 14-17 loop observed in the NMR structure is also observed in the MD; however, the 27-30 and 30-33 turns of the NMR model are assigned in part coiled conformation in the MD. Only partial agreement was found between NOE-determined distances of the Aβ10-35 peptide and the corresponding values for the most populated cluster of Ab1-42 (t > 350 ns). Three Cu(II)/Aβ1-42 systems were studied, in which the Cu(II) binding was modeled by the structures of Figure 1 and 3. The Cu(II) binding disrupts the β-sheet structure between the N- and the C-terminal regions observed in the Cu(II)-free system. Substantial conformational differences were found in the 1-9 region (Table 1). In the most populated cluster of the AbCuN system ,the carboxyl group of Glu22 has moved into the coordination sphere of the copper. In the AbCuO configuration, the copper interacts with the carboxylate of Glu3 rather than Glu22. In this case, the sidechains of the Lys28 and the Asp23 residues make a salt bridge 66% of the time. In the AbCuOHis case, the copper interacts with the Asp1 residue together with the three His residues. In the AbCuN and AbCuO cases, the C-terminal region has a portion with β- character. In the AbCuOHis case, the C-terminal domain is in coil conformation. The Cu(II) is exposed on the surface of the complex in the AbCuN and the AbCuO structures. The molecular surface around these structures is very similar in both cases. The Cu(II) is buried in the interior in the AbCuOHis case, making it difficult for it to take part in electron-transfer chemistry. The inclusion of the Morse description for the Cu(II)-peptide bonds and the removal of the water in the AbCuO system did not have any conformational consequences, and the Cu(II) binding site was preserved within the 64 ns studied.

Raffa and Rauk The average radius of gyration of the four systems falls within the experimental range of 7.5-11.1 Å for Aβ1-42. The three Cu(II)-containing systems have predominantly coil conformation. According to the 3.0 Å criteria used to discriminate conformations, the 17-21 and 36-42 regions of the four studied systems are similar. Only the Phe19 residue, within the 17-21 region, of the Cu(II)-free system is assigned by STRIDE as a β-bridge conformation. As a crude measure of relative stability and solubility, we determined the average number of salt bridges and hydrogen bonds within the peptide and between the peptide and the solvent. Internal stability of the systems is similar, but the greater number of solute-solvent hydrogen bonds in the case of the AbCuO system suggests that it may be the most soluble due to higher “solvation” energy. The analysis of the distances shows that a local geometry favorable to the Cu(II) coordination may be spontaneously achieved by the Cu(II)-free system within the most populated cluster. The involvement of the N-terminus in the Cu(II) coordination sphere is possible as a consequence of the Asp1 carboxylate-Cu(II) interaction in the system in which the three His residues are already involved. It may take a relatively long time to establish His6 or the N-terminus as part of the Cu(II) coordination sphere of the AbCuN or the AbCuO systems. This “delay” may enhance the binding rate of copper to a histidine from a second Aβ molecule. The Met35 residue does not approach close enough to the Cu(II) binding site to make electron-transfer possible, but Tyr10 does. Aβ has been observed to reduce the Cu(II) ion.53 The present results imply that if the reduction of the Cu(II) to Cu(I) takes place in the Cu(II)-bound monomer, the reducing agent is most likely the Tyr10 residue. Supporting Information Available: Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald, B. L.; Beyreuther, K. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4245-4249. (2) Roher, A. E.; Chaney, M. O.; Kuo, Y. M.; Webster, S. D.; Stine, W. B.; Haverkamp, L. J.; Woods, A. S.; Cotter, R. J.; Tuohy, J. M.; Krafft, G. A.; Bonnell, B. S.; Emmerling, M. R. J. Biol. Chem. 1996, 271, 2063120635. (3) Shoji, M.; Golde, T. E.; Ghiso, J.; Cheung, T. T.; Estus, S.; Shaffer, L. M.; Cai, X. D.; McKay, D. M.; Tintner, R.; Frangione, B.; Younkin, S. G. Science 1992, 258, 126-129. (4) Vigo-Pelfrey, C.; Lee, D.; Keim, P.; Lieberburg, I.; Schenk, D. B. J. Neurochem. 1993, 61, 1965-1968. (5) Dore, S.; Kar, S.; Quirion, R. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4772-4777. (6) Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.; Ihara, Y. Neuron 1994, 13, 45-53. (7) Tseng, B. P.; Esler, W. P.; Clish, C. B.; Stimson, E. R.; Ghilardi, J. R.; Vinters, H. V.; Mantyh, P. W.; Lee, J. P.; Maggio, J. E. Biochemistry 1999, 38, 10424-10431. (8) Taylor, B. M.; Sarver, R. W.; Fici, G.; Poorman, R. A.; Lutzke, B. S.; Molinari, A.; Kawabe, T.; Kappenman, K.; Buhl, A. E.; Epps, D. E. J. Protein Chem. 2003, 22, 31-40. (9) Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330-335. (10) Zhang, S.; Iwata, K.; Lachenmann, M. J.; Peng, J. W.; Li, S.; Stimson, E. R.; Liu, Y. A.; Felix, A. M.; Maggio, J. E.; Lee, J. P. J. Struct. Biol. 2000, 130, 130-141. (11) Xu, Y.; Shen, J.; Luo, X.; Zhu, W.; Chen, K.; Ma, J.; Jiang, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5403-5407. (12) Atwood, C. S.; Scarpa, R. C.; Huang, X.; Moir, R. D.; Jones, W. D.; Fairlie, D. P.; Tanzi, R. E.; Bush, A. I. J. Neurochem. 2000, 75, 12191233.

MD Study of Alzheimer’s Beta Amyloid Peptide (13) Atwood, C.; Moir, R. D.; Huang, X.; Scarpa, R. C.; Bacarra, N. M. E.; Romanos, D. M.; Hartshorn, M. A.; Tanzi, R. E.; Bush, A. I. J. Biol. Chem. 1998, 273, 12817-12826. (14) Hallman, P. S.; Perrin, D. D.; Watt, A. E. Biochem. J. 1971, 121, 549. (15) Neumann, P. Z.; Sass-Kortsak, A. J. Clin. InVest. Med. 1967, 46, 646. (16) Karr, J. W.; Kaupp, L. J.; Salı´a, V. A. J. Am. Chem. Soc. 2004, 126, 13534-13538. (17) Syme, C. D.; Nadal, R. C.; Rigby, S. E. J.; Viles, J. H. J. Biol. Chem. 2004, 279, 18169-18177. (18) Miura, T.; Suzuki, K.; Kohata, N.; Takeuchi, H. Biochemistry 2000, 39, 7024-7031. (19) Scho¨neich, C.; Williams, T. D. Chem. Res. Toxicol. 2002, 15, 717722. (20) Tickler, A. K.; Smith, D. G.; Ciccotosto, G. D.; Tew, D. J.; Curtain, C. C.; Carrington, D.; Masters, C. L.; Bush, A. I.; Cherney, R. A.; Cappai, R.; Wade, J. D.; Barnham, K. J. J. Biol. Chem. 2005, 280, 13355-13363. (21) Karr, J.; Akintoye, H.; Kaupp, L. J.; Szalai, V. A. Biochemistry 2005, 44, 5478-5487. (22) Raffa, D. F.; Go´mez-Balderas, R.; Brunelle, P.; Rickard, G. A.; Rauk, A. J. Biol. Inorg. Chem. 2005, 10, 887-902. (23) Raffa, D. F.; Rickard, G. A.; Rauk, A. J. Biol. Inorg. Chem. 2006, in press. (24) Sticht, H.; Bayer, P.; Willbold, D.; Dames, S.; Hilbich, C.; Beyreuther, K. Eur. J. Biochem. 1995, 233, 293-298. (25) van Gusteren, W. F.; Berendsen, H. J. C. Gromos-87 Manual; Biomos: Groningen, The Netherlands, 1987. (26) Van der Spoel, D.; van Buuren, A. R.; Apol, E.; Meulenhoff, P. J.; Tieleman, D. P.; Sijbers, A. L. T. M.; Hess, B.; Feenstra, K. A.; Lindhal, E.; van Drunen, R.; Berensden, H. J. C. Gromacs User Manual, version 3.1.1; Nijenborgh 4, 9747 AG Groningen: The Netherlands, 2002; www.gromacs.org. (27) Fitting was performed using the McLachlan algorithm (McLachlan, A. D. Rapid Comparison of Protein Structres. Acta Crystallogr. 1982, A38, 871-873) as implemented in the program ProFit (Martin, A. C. R.; http:// www.bioinf.org.uk/software/profit/). (28) Daura, X.; van Gunsteren, W. F.; Mark, A. E. Proteins: Struct. Funct. Genet. 1999, 34, 269-280. (29) Humphrey, W.; Dalke, A.; Schulten, K. VMD - Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33-38; . (30) Frishman, D.; Argos, P. Proteins: Struct. Funct. 1995, 23, 566579. (31) Kubleka, J.; Hofrichter, J.; Eaton, W. A. Curr. Opin. Struct. Biol. 2004, 14, 76-88. (32) Galzitskaya, O. V.; Ivankov, D. N.; Finkelstein, A. V. FEBS Lett. 2001, 489, 113-118. (33) Han, W.; Wu, Y. J. Am. Chem. Soc. 2005, 127, 15408-15416. (34) Walsh, D. M.; Lomakin, A.; Benedek, G. B.; Condron, M. M.; Teplow, D. B. J. Biol. Chem. 1997, 272, 22364-22372.

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3799 (35) Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330-335. (36) Tjernberg, L. O.; Naslund, J.; Lindqvist, F.; Johansson, J.; Karlstrom, A. R.; Thyberg, J.; Terenius, L.; Nordstedt, C. J. Biol. Chem. 1996, 271, 8545-8548. (37) Balbach, J. J.; Ishii, Y.; Antzutkin, O. N.; Leapman, R. D.; Rizzo, N. W.; Dyda, F.; Reed, J.; Tycko, R. Biochemistry 2000, 39, 13748-13759. (38) Petkova, A. T.; Yau, W.; Tycko, R. Biochemistry 2006, 45, 498512. (39) Sato, T.; Kienlen-Campard, P.; Ahmed, M.; Liu, W.; Li, H.; Elliott, J. I.; Aimoto, S.; Constantinescu, S. N.; Octave, J. N.; Smith, S. O. Biochemistry 2006, 45, 5503-5516. (40) Feenstraa, K. A.; Peterb, C.; Scheeka, R. M.; van Gunsteren, W. F.; Marka, A. E. J. Biomol. NMR 2002, 23, 181-194. (41) Doreleijers, J. F.; Mading, S.; Maziuk, D.; Sojourner, K.; Yin, L.; Zhu, J.; Markley, J. L.; Ulrich, E. L. J. Biomol. NMR 2003, 26, 139-146. (42) Doreleijers, J. F.; Nederveen, A. J.; Vranken, W.; Lin, J.; Bonvin, A. M.; Kaptein, R.; Markley, J. L.; Ulrich, E. L. BioMagResBank databases DOCR and FRED with converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures. J. Biomol. NMR 2005, 32, 1-12. (43) Nederveen, A. J.; Doreleijers, J. F.; Vranken, W.; Miller, Z.; Spronk, C. A.; Nabuurs, S. B.; Guntert, P.; Livny, M.; Markley, J. L.; Nilges, M.; Ulrich, E. L.; Kaptein, R.; Bonvin, A. M. RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank. Proteins 2005, 59, 662-672. (44) Nabuurs, S. B.; Nederveen, A. J.; Vranken, W.; Doreleijers, J. F.; Bonvin, A. M.; Vuister, G. W.; Vriend, G.; Spronk, C. A. DRESS: a database of refined solution NMR structures. Proteins 2004, 55, 483-486. (45) Monticelli, L.; Colombo, G. Theor. Chem. Acc. 2004, 112, 145157. (46) Rickard, G. A.; Gomez-Balderas, R.; Brunelle, P.; Raffa, D. F.; Rauk, A. J. Phys. Chem. A. 2005, 109, 8361-8370. (47) Ab initio structure B is 23.1 kJ mol-1 more stable than the structure resulting from binding of an imidazole to the Cu(II)N site at pH 7. (48) Otzen, D. E.; Fersht, A. R. Biochemistry 1998, 37, 8139-8149. (49) Gray, H. B.; Winkler, J. R. Annu. ReV. Biochem. 1996, 65, 537561. (50) Walsh, D. M.; Townsend, M.; Podlisny, M. B.; Shankar, G. M.; Fadeeva, J. V.; El Agnaf, O.; Hartley, D. M.; Selkoe, D. J. J. Neurosci. 2005, 25, 2455-2462. (51) Atwood, C. S.; Perry, G.; Zeng, H.; Kato, Y.; Jones, W. D.; Ling, K.; Huang, X.; Moir, R. D.; Wang, D.; Sayre, L. M.; Smith, M. A.; Chen, S. G.; Bush, A. I. Biochemistry 2004, 43, 560-568. (52) Ali, F. E. A.; Barnham, K. J.; Barrow, C. J.; Separovic, F. Aust. J. Chem. 2004, 57, 511-518. (53) Ciccotosto, G. D.; Tew, D.; Curtain, C. C.; Smith, D.; Carrington, D.; Masters, C. L.; Bush, A. I.; Cheny, R. A.; Cappai, R.; Barnham, K. J. J. Biol. Chem. 2004, 279, 42528 - 42534, and references therein.