Molecular Dynamics Study To Investigate the Effect of Chemical

Jan 26, 2008 - ... Marvin, J.; Padilla, D.; Ravichandran, V.; Schneider, Thanki, B. N.; Weissig, H.; Westbrook, J. D.; Zardecki, C. Crystallography 20...
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J. Phys. Chem. B 2008, 112, 2159-2167

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Molecular Dynamics Study To Investigate the Effect of Chemical Substitutions of Methionine 35 on the Secondary Structure of the Amyloid β (Aβ(1-42)) Monomer in Aqueous Solution Luciano Triguero, Rajiv Singh, and Rajeev Prabhakar* Department of Chemistry, UniVersity of Miami, 1301 Memorial DriVe, Coral Gables, Florida 33146 ReceiVed: September 6, 2007; In Final Form: NoVember 19, 2007

In this study, all-atom molecular dynamics simulations in the explicit water solvent are performed to investigate conformational changes in the secondary structure of the Aβ(1-42) monomer associated with the substitution of the Cγ-methylene position of the Met35 amino acid residue by sulfoxide (Met35(O)), sulfone (Met35(O2)), and norleucine (Met35(CH2)). The effects of these substitutions on the structural changes that occur in three distinct regions (the central hydrophobic core (CHC) region 17-21 (LVFFA), stable turn segment 2427 (VGSN), and second hydrophobic region 29-35 (GAIIGLM)) of all monomers have been analyzed in detail, and results are compared with experiments. Our 20 ns simulations indicate that the most significant changes take place in the second hydrophobic region of the Met35(O) and Met35(O2) monomers. However, for the Met35(CH2) monomer, this region does not exhibit significant deviations. In comparison to the wildtype (WT)-Aβ(1-42) monomer, for Met35(O) the second hydrophobic region is characterized by the formation of internal β-sheets separated by stable turns, whereas for Met35(O2) it exhibits a more helical conformation. These substantial changes in the secondary structure can be explained in terms of an increase in the computed dipole moment and solvent accessible surface area (SASA) per residue of these substituents. These structural modifications can affect interaction between monomers, which in turn may influence the oligomerization process involved in Alzheimer’s disease (AD).

I. Introduction Alzheimer’s disease (AD) is characterized by the abundance of intraneuronal neurofibrillary tangles and the extracellular deposition of the amyloid β (Aβ) peptide as amyloid plaques.1 Normally, Aβ is a soluble 4.8 kDa peptide that is produced ubiquitously in the human body throughout life by a proteolytic cleavage of the transmembrane region of the 110-135 kDa amyloid precursor protein (AβPP).1 The two predominant forms of the Aβ-peptide are produced in vivosAβ(1-40) and Aβ(1-42)swith the following primary structure: DAEFR5HDSGY10EVHHQ15KLV-FF20AEDVG25SNKGA30IILGLM35VGGV V40IA42 Among these two forms, Aβ(1-42) has been known to be more neurotoxic than Aβ(1-40) and is observed to be a major component in amyloid plaques that typify AD.2-5 Experimental structural studies have shown that, in vitro, the early assembly of the Aβ(1-42) peptide involves the formation of pentamer/ hexamer units termed as paranuclei.6,7 These paranuclei subsequently self-associate into large oligomers, which appear to generate protofibrils.6 The fibril structures of Aβ(1-40) and Aβ(1-42) aggregates have been determined by various experimental techniques such as electron microscopy,8-10 X-ray diffraction,8,11 electron paramagnetic resonance (EPR) spectroscopy,12 and solid-state nuclear magnetic resonance (NMR) spectroscopy.13-17 The recent solid-state NMR experiments16,18-20 indicate that the most common structure of an Aβ monomer in fibrils consists of parallel cross-β sheets with dynamic regions in the N- and * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: 305-284-9372. Fax: 305-284-4571.

C-terminus, a central hydrophobic core (CHC) in the 17-21 region (LVFFA), and a turn region between residues 24-27 (VGSN). Esler et al.21 observed that a disruption of the CHC region correlates to the diminished ability of the Phe19 f Tyr mutant of Aβ(1-42) to bind to well-formed amyloid deposits. Interestingly, several mutants (e.g., Flemish (Ala21 f Gly), Arctic (Glu22 f Gly), Italian (Glu22 f Lys), Iowa (Asp23 f Asn), and Dutch (Glu22 f Gln)) of this peptide exist either within or in the vicinity of the CHC.22-26 In structure-activity studies on Aβ(25-35) and Aβ(1-42), the turn in the 24-27 (VGSN) region and the second hydrophobic domain between 29 and 35 (GAIIGLM) residues were reported to be essential for the aggregation.27 In addition, the redox state of Met35 amino acid residue of Aβ(1-42) has also been suggested to be critical in the aggregation and biological activity of amyloids, yet the mechanism by which it exerts its influence is not well understood.6,28 In recent studies, it has been reported that the oxidation of Met35 side chain of Aβ(1-42) to methionine sulfoxide (Met35(O)) diminishes both aggregation and toxicity6,29,30 which contradicts some earlier held views.7,31,32 For instance, Hou et al.29 demonstrated that the oxidation of Met35 to Met35(O) significantly hinders the rate of fibril formation of Aβ(1-42) at the physiological pH. It was reported that the oxidized form, Met35(O), alters the morphology of Aβ(1-42) oligomers and prevents the formation of protofibrils. Recently, Johansson et al.30 examined the effect of the Met35 oxidation in two extremely aggregation-prone peptides, wild-type (WT)-Aβ(1-42) and Arctic-Aβ(1-40), for oligomer and protofibril formation. They concluded that it significantly attenuates the aggregation of these two forms and thereby reduces neurotoxicity.

10.1021/jp0771872 CCC: $40.75 © 2008 American Chemical Society Published on Web 01/26/2008

2160 J. Phys. Chem. B, Vol. 112, No. 7, 2008 In order to examine possible electronic and steric effects of the Met35 residue on the morphology and size distribution of Aβ(1-42) oligomers, Bitan et al.6 used four different Cγmethylene chemical substitutions of Met35 (e.g., methionine sulfoxide (Met35(O)), methionine sulfone (Met35(O2)), norleucine (Met35(CH2)), and homoleucine (Met35(CHCH3))) and compared the results with the WT-Aβ(1-40) and WT-Aβ(142) peptides. It was observed that the oxidation of Met35 to Met35(O) and Met35(O2), in the WT-Aβ(1-42) peptide, blocked the paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by WT-Aβ(1-40).6 Furthermore, it was suggested that the primary effect controlling the oligomerization pathway is of an electronic nature that arises from the chemical alterations of the Cγ-center of Met35. These alterations increase the Met35 solvation free energy, thereby disfavoring the burial of the Met35 side chain in the apolar core of an Aβ aggregate.6 In addition to the aforementioned experimental studies, molecular dynamics (MD) simulations have also been performed to investigate the structure and dynamics of the selected small fragments33 and low-order oligomers and fibrils.34-36 Massi et al.37 suggested that the CHC (LVFFA) region and the turn (VGSN) are particularly stable and could play an important role in the initial deposition of the peptide monomer on the fibril surface. Luttmann and Fels38 performed 10 ns simulations using different NMR structures of the entire Aβ(1-40) and Aβ(142) peptides (PDB ID 1BA4, 1AML, and 1IYT39) deposited in the protein data bank (PDB)40 and predicted high stability in the CHC region. The aforementioned experimental and theoretical results indicate that the structure and dynamics of the Aβ(1-42) monomer can play a central role in the formation of paranuclei and their further aggregation into fibrils. In order to investigate the conformational changes in the secondary structure of the Aβ(1-42) monomer associated with the alterations in electronic character of the chemical groups attached to the Cγ-methylene of Met35, we performed 20 ns MD simulations on the solvated WT-Aβ(1-42) monomer and its chemically substituted forms (Met35 f Met35(O), Met35 f Met35(O2), and Met35 f Met35(CH2)) in aqueous solution. The available experimental and theoretical information provided an ideal background to initiate this study. II. Methods All MD simulations were performed using GROMACS software package,41,42 using the GROMOS force field 53A5.43 The starting structure of the Aβ(1-42) monomer was extracted from the NMR structure (structure 10, PDB ID 1IYT39) deposited in the PDB.40 In a previous MD simulation study (10 ns), this particular structure was reported to successfully reproduce the most important conformational changes observed experimentally.38 Nonpolar hydrogen atoms in the system were treated implicitly by the united atom approach, where they are collapsed into the connected heavy atom and thus not treated explicitly in the simulations. Both N- and C-terminals contain positive and negative charge, respectively. Aβ(1-42) and the chemically substituted peptides were placed in the center of a box with dimensions 0.50 × 0.40 × 0.60 nm3, where the distance to the edge of the box from Aβ(1-42) was chosen to be 2.0 nm. These boundaries rule out unwanted effects from the applied periodic boundary conditions (PBC). The box contains over 3700 single point charge (SPC) water molecules.44 Some water molecules were replaced by sodium and chloride ions to neutralize and simulate the experimentally used 150 mM ion concentration.

Triguero et al. All the starting structures were subsequently energy minimized with the steepest descent method for 2000 steps. The results of these minimizations produced the initial structure for the MD simulations. The MD simulations were carried out with a constant number of particles (N), pressure (P), and temperature (T), i.e., NPT ensemble. The SETTLE algorithm was used to constrain the bond length and angle of the water molecules,45 while the LINCS algorithm was utilized to constrain the bond length of the peptide.46 The long-range electrostatic interactions were calculated by the particle-mesh Ewald (PME) method.47,48 A constant pressure of 1 bar was applied with a coupling constant of 1.0 ps; peptide, water molecules and ions were coupled separately to a bath at 300 K with a coupling constant of 0.1 ps. The PBC were applied and the equation of motion was integrated at time steps of 2 fs. For Met(O), the parameters proposed by Strader and Feller49 for dimethyl sulfoxide were used. For the sulfur and sulfur oxygen in sulfone (Met(O2)), DFT/6-31G(d) calculated partial charges and geometric parameters for dimethyl sulfone were used. All the structures in aqueous solution are characterized by the secondary structure analysis, performed using the definition of secondary structure of proteins (DSSP) protocol,50 contact maps, and similarity factors of the most representative structures obtained from the cluster analysis. In the cluster analysis, the trajectories are analyzed by grouping structurally similar frames (root-mean-square-deviation (rmsd) cutoff ) 0.30 nm),51 the frame with the least rmsd deviation from its neighbors is denoted as a “middle” structure, which is used to represent that particular cluster. The electronic changes are characterized in terms of dipole moment of the Cγ-substituted groups of Met35. The solvent accessible surface area (SASA) per residue is also computed to assess the hydrophobic character of different regions of the monomers. The VMD suite of software was used for visualizations and for the preparation of structural diagrams presented in this study.52 III. Results and Discussion In this study, we performed 20 ns simulations of the solvated WT-Aβ(1-42) and Met35-substituted methionine sulfoxide (Met35(O)), methionine sulfone (Met35(O2)), and norleucine (Met35(CH2)) monomers to obtain a dynamical picture of the conformational changes that occur in aqueous solution. The main emphasis of these simulations is to explore the conformational alterations that take place in the secondary structure of the CHC region 17-21 (LVFFA), the stable turn segment 24-27 (VGSN), and the second 29-35 (GAIIGLM) hydrophobic region. These regions have been experimentally proposed to play critical roles in the aggregation process.21,37,53 The rmsd values for all four simulations are depicted in Figure 1, and they clearly indicate metastable conformations after 6 ns of simulation for all (Aβ(1-42), Met35(O), Met35(O2), and Met35(CH2)) trajectories. Only the Met35(O2) trajectory shows large fluctuations, but after 10 ns it also stabilizes to a rather stable conformation. In the thermodynamically equilibrated region, these four trajectories do not show any significant changes and the overall rmsd deviations remain below 0.30 nm. III.a. Wild-Type (WT)-Aβ(1-42). The NMR structure of the monomeric form of WT-Aβ(1-42) reveals that it exists in a loosely formed collapsed coil state in aqueous solution.53 In this medium, it appears to adopt a well-defined helical conformation within its central hydrophobic core (CHC) in the 1721 (LVFFA) region and a dominant turn in the 24-27 (VGSN) region. The flanking regions, N- and C-terminus, are proposed to be more dynamic and partially unstructured.

Secondary Structure of the Amyloid β Monomer

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Figure 1. rmsd plotted against time for all four different simulations: WT-Aβ(1-42), Met35(O)-Aβ(1-42), Met35(O2)-Aβ(1-42), and Met35(CH2)-Aβ(1-42).

Figure 2. Secondary structure assignment per residue plotted against time for all four trajectories: (a) WT-Aβ(1-42), (b) Met(O)-Aβ(1-42), (c) Met35(O2)-Aβ(1-42), and (d) Met35(CH2)-Aβ(1-42).

The time evolution of the secondary structure (Figure 2a) in our 20 ns simulations of the WT-Aβ(1-42) monomer shows that the first four residues (DAEF) adopt a coil conformation throughout the simulation. The segment 5-15 (RHDSGYEVHHQ) evolves into a stable helical structure. This is also reflected in the analysis of the contact map (e.g., contacts of the side chain of a residue with its neighbors) depicted in Figure 3a

(lower triangle). As shown in this figure, there are many contacts in the two parallel rows along the diagonal in the 5-15 region. The CHC region (17-21, LVFFA) has been proposed to play an important role in the initial deposition of the peptide monomer on the fibril surface.21,37 During the simulation, this region undergoes two distinct transformations. The first transformation occurs at 2 ns, when the initial R-helix conformation

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Figure 3. Contact maps for (a) Met35(O)-Aβ(1-42)/WT-Aβ(1-42), (b) Met35(O2)-Aβ(1-42)/WT-Aβ(1-42), and (c) Met35(CH2)-Aβ(1-42)/ WT-Aβ(1-42). Each square provides the mean average distance (intensity coded) of heavy atoms for the side chains of the residues, which are less than 0.5 nm apart.

Figure 4. Ribbon presentations of the representative conformers obtained by cluster analysis performed on all 20 ns simulations trajectories. The peptide configurations are superimposed to best overlap the CHC (LVFFA) region: (a) superposition between WT-Aβ(1-42) and Met(O)-Aβ(1-42), (b) superposition between WT-Aβ(1-42) and Met35(O2)-Aβ(1-42), and (c) superposition between WT-Aβ(1-42) and Met35(CH2)-Aβ(1-42).

starts changing toward a more open 5-helix structure (Figure 2a). This metastable conformation fluctuates for about 8 ns and finally transforms back to the initial R-helix conformation and remains in this stable conformation for the last 10 ns of the simulation. In this region, similar fluctuations were also observed in the 10 ns simulations performed by Luttmann and Fels,38 but due to the shorter simulation time the stabilization of the R-helix that occurs after 10 ns could not be observed. It is noteworthy that both studies demonstrate the presence of a helical region formed between residues 5 (R) and 21 (A). The dominant curve in the 24-27 region predicted from NMR studies in soluble WT-Aβ(1-42) is also observed in our simulation (Figures 2a and 4a). In Figure 2a, the presence of a turnlike structure is depicted by yellow color. The contact map (Figure 3a, lower triangle) also shows an ordered structure in this segment with a mean average distance between CR-CR+3 below 0.5 nm. Despite being dominated by a turnlike conforma-

tion this region (24-27) has been observed to undergo large dynamical transformations between turn and bend. These transformations are also reflected in Figure 5a, where changes in the angle between short fragments (24-25 and 27-38) of the peptide on both sides of the turnlike conformation, represented by a vector pointing from the N-terminus to the C-terminus, are depicted. A turn or bend structure show large angles, and ideally, for a turn conformation the angle should be close to 180°. During the simulation this angle increases and fluctuates significantly around 110°. The C-terminal region containing residues 29-40 (G-V) exhibits a very dynamic character as indicated by various experiments.54-57 The initial helical conformation is found to fluctuate between turns, bend, and coil conformations indicating a rather irregular region. However, after 14 ns a stable bend structure is observed in the second hydrophobic segment between residues 29-35 (G-M). This trajectory also indicates

Secondary Structure of the Amyloid β Monomer

Figure 5. Angle parameter quantifying the turn in the 24-27 (VGSNK) region.

a mixture of turn, β-sheet, and bend contents (green, yellow, and red, respectively) close to the C-terminus of WT-Aβ(142), Figure 2a. The calculated dipole moment of the Cγsubstituted group in WT-Aβ(1-42) is 1.76 D (Table 1). In order to characterize the hydrophobicity, we calculated the SASA for all 42 residues of Aβ(1-42) monomer (Figure 6a-c), and for Met35 this value is calculated to be 0.42 nm2 (Table 1 and Figure 6c). The last two residues (IA) retain a coil-like conformation throughout the simulation. III.b. Met35(O)-Aβ(1-42). The oxidation of methionine (Met35) to methionine sulfoxide (Met35(O)) is achieved by adding a double-bond oxygen atom to the sulfur atom of Met35 in the initial PDB structure (1IYT-10). The secondary structure analysis and contact map of the Met35(O)-Aβ(1-42) monomer are shown in Figures 2b and 3a (upper triangle). Throughout the simulation, similar to WT-Aβ(1-42), the first three residues (DAE) adopt a coil structure (Figure 2b). However, with respect to WT-Aβ(1-42), an important difference is observed in the length of the helical conformation in the N-terminus segment. In WT-Aβ(1-42), this helical region starts from residue 5 (R), whereas in Met35(O)-Aβ(1-42) it starts from residue 9 (G). A comparison of the contact maps between WT-Aβ(1-42) and Met35(O)-Aβ(1-42) in Figure 3a clearly shows this difference. The secondary structure analysis (Figure 2b) shows that for Met35(O)-Aβ(1-42), the CHC (17-21) region adopts a more open 5-helix conformation. The representative structures obtained from a cluster analysis over the last 10 ns trajectories are superimposed to best overlap the CHC region. The superposition of WT-Aβ(1-42) and Met35(O)-Aβ(1-42), Figure 4a, also indicates some noticeable differences in the CHC region, particularly in the last three residues (FFA). In comparison to WT-Aβ(1-42), significant conformational changes are observed in the region 22-35 (E-G). The secondary structure analysis (Figure 2b) indicates the formation of sporadic,

J. Phys. Chem. B, Vol. 112, No. 7, 2008 2163 therefore metastable, β-sheet conformations between residues 23-24 (D-V) and 27-28 (N-K), which are nonexistent in WT-Aβ(1-42). The curved structure in the 24-27 (V-N) region of WT-Aβ(1-42) now becomes more pronounced as indicated by the angle of 150° between the vectors representing the 24-25 and 27-28 fragments on both sides of the turnlike conformation (Figure 5b). In comparison to WT-Aβ(1-42) the average value of this angle has now increased by ca. 40°. It is noticeable that this angle undergoes large fluctuations during the first 10 ns and after that they decrease significantly and eventually become smaller than the corresponding fluctuations in WT-Aβ(1-42). These results indicate that the Met35 f Met35(O) substitution stabilizes the bendlike structure observed in WT-Aβ(1-42) to a more turnlike structure. Like WT-Aβ(1-42), the C-terminus segment has been observed to undergo large dynamical rearrangements (Figure 2, parts a and b). Another noteworthy difference has been observed in the segment composed of residues 28-35 (KGAIIGLM). The contact map in this segment (Figure 3a) shows a distinct difference in the conformation between Met35(O)-Aβ(1-42) (upper triangle) and WT-Aβ(1-42) (lower triangle). For WT-Aβ(1-42), this region is shown to have a random distribution of points, which is characteristic of a region with irregular structure. It also shows that residues 30 (A) and 31 (I) are in contact with residues 16 (K) and 17 (L). The contact map (Figure 3a, lower triangle) provides contact distances of 0.30.4 nm between these residues. However, in the case of Met35(O)-Aβ(1-42), the conformation in this region is more ordered (Figure 3a, upper triangle) which is also confirmed in the ribbon representation depicted in Figure 4a (WT; yellow color and Met35(O); blue color). This segment, 28-35 (KGAIIGLM), in Met35(O)-Aβ(1-42) monomer conforms a helical structure with close contact between neighboring residues and not as a result of interactions with N-terminus residues. These conformational changes observed in the C-terminus region between WT-Aβ(1-42) and Met35(O)-Aβ(1-42) are caused by the electronic change introduced by the oxidation of Met35. Met35 is a hydrophobic residue, and in WT-Aβ(1-42), due to a specific orientation, the side chain of this residue is totally buried (Figure 4a). The oxidation of Met35 to Met35(O) alters this orientation by increasing the dipole moment of the Cγ-substituted group by a factor of 2 (Table 1) and making Met35 accessible to interaction with water molecules. These interactions, in turn, destroy the contacts between the C- and N-terminus residues. These conformational changes are supported by the SASA value of Met35 in Met35(O)-Aβ(1-42) of 0.87 nm2, which is greater than the corresponding value of 0.41 nm2 for WT-Aβ(1-42) (Table 1 and Figure 6c). The last C-terminus segment, between residues 36-42 (VGGVVIA), follows a dynamics similar to WT-Aβ(1-42). This segment is characterized by two small β-sheet conformations connected through a bend (Figure 2b). It was found that the length of β-sheet in the oxidized form (Met35(O)) is slightly smaller than what was observed in WT-Aβ(1-42). In order to obtain a measure of the similarity between different structures we also computed the rmsd values between the corresponding representative structures in every trajectory. The rmsd value of 0.74 nm (Table 2) between the structures representing the WT-Aβ(1-42) and Met35(O)-Aβ(1-42) trajectories indicate significant differences between these two structures. The major difference is located in the second hydrophobic region (28-35), with an rmsd value of 0.53 nm. It is important to mention that the size of the helical segment for the two monomers in the N-terminus region is significantly

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TABLE 1: Calculated Dipole Moments and Solubility Areas for Different Substitutions of Met35 in the Aβ(1-42) Peptidea

a

Dipole moments are calculated at the B3LYP/6-31G(d) level including continuum solvent model (PCM).

Figure 6. Solubility area per residue (SASA) in nm2 for (a) the central hydrophobic core (17-21), (b) turn (24-28), and (c) the second hydrophobic region (28-35).

different, which along with the conformational changes observed in the secondary structure of the 28-35 (KGAIIGLM) region may play a role in possible oligomerization or deposition mechanisms observed for these two monomers.6 The oxidation of Met35 to Met35(O) significantly augments its dipole moment by 1.96 D (Table 1). This increase induces marked changes in the SASA values of residues (in particular of Ile31, Lys34, and Met35) in the second hydrophobic region (28-35), Figure 6c. Our simulations predict that Ile31 and Met35 become more exposed to water molecules, whereas Lys34 adopts more hydrophobic character. The Met35 f Met35(O) substitution introduces only moderate structural changes in the CHC and turn (24-28) regions. The computed

SASA values in these two regions are also very similar demonstrating small differences in their hydrophobic characters (Figure 6, parts a and b). III.c. Met35(O2)-Aβ(1-42). The oxidation of methionine (Met35) to methionine sulfone (Met35(O2)) is achieved by the inclusion of two oxygen atoms to the sulfur atom of Met35. According to the secondary structural analysis (Figure 2c), similar to WT-Aβ(1-42) and Met35(O)-Aβ(1-42), the region composed of 1-9 residues undergoes large dynamical changes and transforms in to a coil structure. The region 9-21 of Met35(O2)-Aβ(1-42), which also includes the CHC segment, indicates small changes but retains the overall helical structure observed both in the WT-Aβ(1-42) and Met35(O)-Aβ(1-42) monomers. However, substantial differences were observed in the segment comprise of residues 22-35, which is located just above the CHC region (toward the C-terminus). These differences are shown in the secondary structural analysis and contact map depicted in Figures 2c and 3b (upper triangle), respectively. This segment is now characterized by a more ordered secondary structure, containing stable β-sheets connected by turns formed at residues 25-27 and 30-32, respectively. The distance between the C- and N-terminus is significantly increased, which indicates a decrease in interaction between them (Figure 4b). This change is also shown in the time dependence values of the angle quantifying the turnlike conformation in the 24-27 region (Figure 5c). For Met35(O2)-Aβ(1-42), this value fluctuates around 110°, but the amplitudes of fluctuations are decreased, indicating a more stable bend conformation than the one observed in both WT-Aβ(1-42) and Met35(O)-Aβ(1-42). These changes in the secondary structure of the Met35(O2) monomer can be explained in terms of increase in the dipole moment of the Cγ-substituted sulfone group. The Met35 f Met35(O2) chemical substitution increases both the size and the polarity of the Cγ-methylene group of Met35. The dipole moment of this side chain in Met35(O2)-Aβ(1-42) is 4.30 D, which is 2.54 and 0.58 D larger than the dipole moment of the corresponding side chains in WT-Aβ(1-42) and Met35(O)-Aβ(1-42), respectively (Table 1). In the sulfone form, Met35(O2), this residue acts as a strong hydrogen bond acceptor and tends to form hydrogen bond with the amide backbone of the neighboring residue. In our simulations, we found an intermo-

Secondary Structure of the Amyloid β Monomer

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TABLE 2: rmsd Values (in nm) of the Substituted Monomers Relative to the WT-Aβ(1-42) Peptide Derived from the Representative Structures Obtained by the Cluster Analysis of the Simulated Trajectories

lecular hydrogen bond between Met35(O2) and Ile41. The SASA values of Ile31, Lys34, and Met35 are enhanced, in particular for Met35 by a factor of 3 (0.42-1.25 nm2), therefore exposing all these residues to greater interactions with the surrounding water molecules. In comparison to WT-Aβ(1-42), the formation of new hydrogen bonds along with the increased solvation of the C-terminus region induces large changes in the secondary structure of Met35(O2)-Aβ(1-42). These changes are reflected in the calculated similarity factor of 0.83 between the two most representative structures obtained from the trajectory analysis (Table 2). The superposition of these two structures is shown in Figure 4b. Noteworthy is the difference in the secondary structure of the 22-35 region and a lack of contact between the N- and C-terminus. III.d. Met35(CH2)-Aβ(1-42). The substitution of the sulfur atom in Met35 of the WT-Aβ(1-42) monomer by a methylene (-CH2) group leads to a norleucine (Met35(CH2)-Aβ(1-42)) monomer. The first two residues of the N-terminus (DA) segment typify a coil structure followed by a helical conformation involving R- and 5-helix structures in the 2-21 region (Figure 2d). This structure is similar to the one observed for the WT-Aβ(1-42) monomer. An examination of the contact

map depicted in Figure 3c show this similarity, where many inter-residue contacts are retained. The superposition, alignment of the CHC region, of the most representative structures of the WT-Aβ(1-42) and Met35(CH2)Aβ(1-42) trajectories is shown in Figure 4c. These two monomers are very similar, even the turn conformation in the 21-27 segment follows the same curvature (Figure 5, parts a and d). The SASA values of the 17-21 and 24-28 regions, Figure 6, parts a and b, predict the same trend with very similar values per residue. The secondary structural analysis indicate that the C-terminus region 28-35 adopts an irregular structure including coil, turn, and bend conformations. This dynamics is again very similar to the one found for WT-Aβ(1-42). The substitution of Met35 by norleucine does not introduce any polarity in the Cγsubstituted side chain (Table 1); however the SASA values of this residue (Met35(CH2)) are slightly larger than Met35 (Figure 6c). The two hydrogen atoms present in the methylene group of Met35(CH2)-Aβ(1-42) tend to interact with surrounding water molecules. These interactions induce moderate changes in the secondary structures in the vicinity of Met35 and expose Lys34 to the surrounding water molecules.

2166 J. Phys. Chem. B, Vol. 112, No. 7, 2008 Evidently, a detailed structural analysis indicates that the overall secondary structure of Met35(CH2)-Aβ(1-42) is similar to the WT-Aβ(1-42) monomer (Figures 2d, 3c, 4c and Table 2). It has been observed experimentally that the aggregation of Met35(CH2)-Aβ(1-42) monomers yields a characteristic WTAβ(1-42) oligomer size distribution.6 This observation is in general agreement with our MD simulations because monomers with similar secondary structures are likely to produce equivalent oligomerization patterns. IV. Summary and Conclusions In this study, we performed 20 ns MD simulations on the WT-Aβ(1-42) monomer and its chemically substituted forms (Met35 f Met35(O), Met35 f Met35(O2), and Met35 f Met35(CH2)) in aqueous solution to investigate the conformational changes associated with the electronic character of the chemical group attached to the Cγ-methylene of Met35. On the basis of existing experimental and theoretical information, conformational changes that occur in three distinct regions (the CHC region 17-21 (LVFFA), stable turn segment 24-27 (VGSN), and second hydrophobic region 29-35 (GAIIGLM)) of all monomers have been analyzed in detail. The results reported in this study could be further verified by advanced sampling enhanced methods such as generalized ensemble-based techniques (replica exchange methods, simulated scaling methods, or accelerated MD methods). For WT-Aβ(1-42), in the first 2 ns the initial R-helix conformation of the CHC region transforms to an open 5-helix structure, where it fluctuates for about 8 ns and finally returns to the initial stable helical form for the rest of the simulation (Figure 2a). The dominant curve in the region 24-27 undergoes large dynamical transformations between turn and bend (Figures 2a and 4a). The second hydrophobic region 29-35 (GAIIGLM) initially exhibits large variations and fluctuates between turn, bends, and coils conformations, but after 14 ns it stabilizes to a stable bend structure (Figure 2a). This region has experimentally been observed to be very dynamic.54-57 The dipole moment and SASA values of the Met35 residue in the WT-Aβ(1-42) monomer are 1.76 D and 0.42 nm2, respectively (Table 1 and Figure 6c). For Met35(O)-Aβ(1-42), the CHC (17-21) region adopts a more open 5-helix conformation and the last three residues (FFA) of the CHC exhibit significant deviations from the WTAβ(1-42) monomer (Figures 2b and 4c). The curve region between 24 and 27 (VGSN) residues becomes more dominant. In comparison to WT-Aβ(1-42), the angle between the vectors representing the 24-25 and 27-28 fragments on both sides of the turnlike conformation is now increased by ca. 40° (Figure 5b). The 28-35 (KGAIIGLM) segment is also distinctly different from the WT-Aβ(1-42) monomer. It adopts a more ordered helical-type structure due to the formation of close contacts between neighboring residues and not as a result of interactions with N-terminus residues (Figures 2b and 3a). The oxidation of Met35 to Met35(O) alters the orientation of Met35(O) by increasing the dipole moment of the Cγ-substituted group by 1.96 D and make it more accessible to interaction with water molecules. This SASA value of 0.87 nm2 for Met35 in Met35(O)-Aβ(1-42) is greater than the corresponding value of 0.41 nm2 for WT-Aβ(1-42) (Table 1 and Figure 6c). The rmsd of 0.74 nm between WT-Aβ(1-42) and Met35(O)-Aβ(1-42) also supports the major modifications in this region. For Met35(O2)-Aβ(1-42), the CHC undergoes small changes but maintains its overall helical structure found in the WT-Aβ(1-42) and Met35(O)-Aβ(1-42) monomers (Figure 2c). In this

Triguero et al. case, significant differences were observed in the 22-35 (EM) region which lies adjacent to the CHC and includes both the turn segment 24-27 (VGSN) and second hydrophobic region 29-35 (GAIIGLM). This region now adopts a more ordered secondary structure, which consists of stable β-sheets connected by turns involving residues 25-27 and 30-32, respectively (Figures 2c and 3b). In particular, the 24-27 (VGSN) region indicates a more stable bend conformation (Figure 5c) than observed in WT-Aβ(1-42) and Met35(O)Aβ(1-42). This substitution, Met35 f Met35(O2), further enhances the dipole moment and SASA values by 0.58 D and 0.38 nm2, respectively (Table 1), and facilitates greater interaction of Met35 and neighboring residues with the surrounding water molecules. The rmsd value between WT-Aβ(1-42) and Met35(O2)-Aβ(1-42) is 0.83, and contact between the N- and C-terminus residues is disrupted. For Met35(CH2)-Aβ(1-42), the overall structure of the monomer (including the CHC region, stable turn, and second hydrophobic region) is very similar to WT-Aβ(1-42). However, the SASA value of 0.65 nm2 for Met35(CH2) is slightly greater than 0.42 nm2 computed for Met35 in the WT-Aβ(1-42) peptide (Figure 6c and Table 1). It has been experimentally indicated that the C-terminus of the WT-Aβ(1-42) monomer in aqueous solution does not form a well-defined secondary structure.7,58 Our simulations predict that the oxidation of Met35 to Met35(O) and Met35(O2) diminishes this residue’s hydrophobicity, which subsequently influence its interactions with the neighboring residues (in particular with Ile31, Lys34, and Ile41) and surrounding water molecules. These electronic modifications induce significant changes in the secondary structure of the C-terminus hydrophobic region (28-35). These alterations in the secondary structure along with the increased solvation energy may hinder the intermolecular interactions between two monomers, which are required for the formation of oligomers implicated in AD. Acknowledgment. Financial support from the University of Miami, Miami, Florida is acknowledged. Supporting Information Available: Tables S1 and S2: optimized Cartesian coordinates (in angstroms) and charges for methionine sulfone (Met35(O2)). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Amyloid Proteins. The Beta Sheet Conformation and Disease; Sipe, J. D., Ed.; Wiley-VCH: Weinheim, Germany, 2005; Vol. 2. (2) Dahlgren, K. N.; Manelli, A. M.; Stine, W. B., Jr.; Baker, L. K.; Kraft, G. A.; LaDu, M. J. J. Biol. Chem. 2002, 277, 32046. (3) Pillot, T.; Drouet, B.; Queille, S.; Labeur, C.; Vandekerckhove, J.; Rosseneu, M.; Pincon-Raymond, M.; Chambaz, J. J. Neurochem. 1999, 73, 1626. (4) Suo, Z. M.; Humphrey, J.; Kundtz, A.; Sethi, F.; Placzek, A.; Crawford, F.; Mullan, M. Neurosci. Lett. 1998, 257, 77. (5) Younkin, S. G. Ann. Neurol. 1995, 37, 287. (6) Bitan, G.; Tarus, B.; Vollers, S. S.; Lashuel, H. A.; Condron, M. M.; Starub, J. E.; Teplow, D. B. J. Am. Chem. Soc. 2003, 125, 15359. (7) Riek, R.; Guntert, P.; Dobeli, H.; Wuthrich, K. Eur. J. Biochem. 2001, 268, 5930. (8) Malinchick, S. B.; Inouye, H.; Szumowski, K. E.; Kirschner, D. A. Biophys. J. 1998, 74, 537. (9) Tjernberg, L. O.; Callaway, D. J. E.; Tjernberg, A.; Hahne, S.; Lilliehook, L.; Terenius, L.; Thyberg, J.; Nordstedt, C. J. Biol. Chem. 1999, 274, 12619. (10) Tjernberg, L. O.; Tjernberg, A.; Bark, N.; Shi, Y.; Ruzsicska, B. P.; Bu, Z.; Thyberg, J.; Callaway, D. J. E. Biochem. J. 2002, 366, 343. (11) Serpell, C. C.; Blake, C. C. F.; Fraser, P. E. Biochemistry 2000, 39, 13269. (12) Torok, M.; Milton, S.; Kayed, R.; Wu, P.; McIntire, T.; Glabe, C. G.; Langen, R. J. Biol. Chem. 2002, 277, 40810.

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