Molecular Dynamics Study To Investigate the Effect of Chemical Substitutions ofMethionine 35 on the Secondary Structure of the Amyloidâ (Aâ(1-42)) Monomer inAqueous 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 investigateconformational changes in the secondary structure of the Aâ(1-42) monomer associated with the substitutionof 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 inthree distinct regions (the central hydrophobic core (CHC) region 17-21 (LVFFA), stable turn segment 24-27 (VGSN), and second hydrophobic region 29-35 (GAIIGLM)) of all monomers have been analyzed indetail, and results are compared with experiments. Our 20 ns simulations indicate that the most significantchanges 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 wild-type (WT)-Aâ(1-42) monomer, for Met35(O) the second hydrophobic region is characterized by the formationof internalâ-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 computeddipole moment and solvent accessible surface area (SASA) per residue of these substituents. These structuralmodifications can affect interaction between monomers, which in turn may influence the oligomerizationprocess involved in Alzheimer’s disease (AD).

I. Introduction

Alzheimer’s disease (AD) is characterized by the abundanceof intraneuronal neurofibrillary tangles and the extracellulardeposition of the amyloidâ (Aâ) peptide as amyloid plaques.1

Normally, Aâ is a soluble 4.8 kDa peptide that is producedubiquitously in the human body throughout life by a proteolyticcleavage of the transmembrane region of the 110-135 kDaamyloid precursor protein (AâPP).1 The two predominant formsof the Aâ-peptide are produced in vivosAâ(1-40) and Aâ-(1-42)swith the following primary structure:

DAEFR5HDSGY10EVHHQ15KLV -FF20AEDVG25SNKGA30-IILGLM 35VGGV V40IA42

Among these two forms, Aâ(1-42) has been known to bemore neurotoxic than Aâ(1-40) and is observed to be a majorcomponent in amyloid plaques that typify AD.2-5 Experimentalstructural studies have shown that, in vitro, the early assemblyof the Aâ(1-42) peptide involves the formation of pentamer/hexamer units termed as paranuclei.6,7 These paranuclei sub-sequently self-associate into large oligomers, which appear togenerate protofibrils.6 The fibril structures of Aâ(1-40) andAâ(1-42) aggregates have been determined by various experi-mental techniques such as electron microscopy,8-10 X-raydiffraction,8,11 electron paramagnetic resonance (EPR) spec-troscopy,12 and solid-state nuclear magnetic resonance (NMR)spectroscopy.13-17

The recent solid-state NMR experiments16,18-20 indicate thatthe most common structure of an Aâ monomer in fibrils consistsof parallel cross-â sheets with dynamic regions in the N- and

C-terminus, a central hydrophobic core (CHC) in the 17-21region (LVFFA), and a turn region between residues 24-27(VGSN). Esler et al.21 observed that a disruption of the CHCregion correlates to the diminished ability of the Phe19f Tyrmutant of Aâ(1-42) to bind to well-formed amyloid deposits.Interestingly, several mutants (e.g., Flemish (Ala21f Gly),Arctic (Glu22f Gly), Italian (Glu22f Lys), Iowa (Asp23fAsn), and Dutch (Glu22f Gln)) of this peptide exist eitherwithin or in the vicinity of the CHC.22-26 In structure-activitystudies on Aâ(25-35) and Aâ(1-42), the turn in the 24-27(VGSN) region and the second hydrophobic domain between29 and 35 (GAIIGLM) residues were reported to be essentialfor the aggregation.27

In addition, the redox state of Met35 amino acid residue ofAâ(1-42) has also been suggested to be critical in theaggregation and biological activity of amyloids, yet the mech-anism by which it exerts its influence is not well understood.6,28

In recent studies, it has been reported that the oxidation of Met35side chain of Aâ(1-42) to methionine sulfoxide (Met35(O))diminishes both aggregation and toxicity6,29,30which contradictssome earlier held views.7,31,32 For instance, Hou et al.29

demonstrated that the oxidation of Met35 to Met35(O) signifi-cantly hinders the rate of fibril formation of Aâ(1-42) at thephysiological pH. It was reported that the oxidized form, Met35-(O), alters the morphology of Aâ(1-42) oligomers and preventsthe formation of protofibrils. Recently, Johansson et al.30

examined the effect of the Met35 oxidation in two extremelyaggregation-prone peptides, wild-type (WT)-Aâ(1-42) andArctic-Aâ(1-40), for oligomer and protofibril formation. Theyconcluded that it significantly attenuates the aggregation of thesetwo forms and thereby reduces neurotoxicity.

* To whom correspondence should be addressed. E-mail: rpr@miami.edu.Tel.: 305-284-9372. Fax: 305-284-4571.

2159J. Phys. Chem. B2008,112,2159-2167

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

In order to examine possible electronic and steric effects ofthe Met35 residue on the morphology and size distribution ofAâ(1-42) oligomers, Bitan et al.6 used four different Cγ-methylene chemical substitutions of Met35 (e.g., methioninesulfoxide (Met35(O)), methionine sulfone (Met35(O2)), nor-leucine (Met35(CH2)), and homoleucine (Met35(CHCH3))) andcompared the results with the WT-Aâ(1-40) and WT-Aâ(1-42) peptides. It was observed that the oxidation of Met35 toMet35(O) and Met35(O2), in the WT-Aâ(1-42) peptide,blocked the paranucleus formation and produced oligomersindistinguishable in size and morphology from those producedby WT-Aâ(1-40).6 Furthermore, it was suggested that theprimary effect controlling the oligomerization pathway is of anelectronic nature that arises from the chemical alterations ofthe Cγ-center of Met35. These alterations increase the Met35solvation free energy, thereby disfavoring the burial of theMet35 side chain in the apolar core of an Aâ aggregate.6

In addition to the aforementioned experimental studies,molecular dynamics (MD) simulations have also been performedto investigate the structure and dynamics of the selected smallfragments33 and low-order oligomers and fibrils.34-36 Massi etal.37 suggested that the CHC (LVFFA) region and the turn(VGSN) are particularly stable and could play an important rolein the initial deposition of the peptide monomer on the fibrilsurface. Luttmann and Fels38 performed 10 ns simulations usingdifferent NMR structures of the entire Aâ(1-40) and Aâ(1-42) peptides (PDB ID 1BA4, 1AML, and 1IYT39) deposited inthe protein data bank (PDB)40 and predicted high stability inthe CHC region.

The aforementioned experimental and theoretical resultsindicate that the structure and dynamics of the Aâ(1-42)monomer can play a central role in the formation of paranucleiand their further aggregation into fibrils. In order to investigatethe conformational changes in the secondary structure of theAâ(1-42) monomer associated with the alterations in electroniccharacter of the chemical groups attached to the Cγ-methyleneof Met35, we performed 20 ns MD simulations on the solvatedWT-Aâ(1-42) monomer and its chemically substituted forms(Met35 f Met35(O), Met35f Met35(O2), and Met35fMet35(CH2)) in aqueous solution. The available experimentaland theoretical information provided an ideal background toinitiate this study.

II. Methods

All MD simulations were performed using GROMACSsoftware package,41,42 using the GROMOS force field 53A5.43

The starting structure of the Aâ(1-42) monomer was extractedfrom the NMR structure (structure 10, PDB ID 1IYT39)deposited in the PDB.40 In a previous MD simulation study (10ns), this particular structure was reported to successfullyreproduce the most important conformational changes observedexperimentally.38 Nonpolar hydrogen atoms in the system weretreated implicitly by the united atom approach, where they arecollapsed into the connected heavy atom and thus not treatedexplicitly in the simulations. Both N- and C-terminals containpositive and negative charge, respectively. Aâ(1-42) and thechemically substituted peptides were placed in the center of abox with dimensions 0.50× 0.40 × 0.60 nm3, where thedistance to the edge of the box from Aâ(1-42) was chosen tobe 2.0 nm. These boundaries rule out unwanted effects fromthe applied periodic boundary conditions (PBC). The boxcontains over 3700 single point charge (SPC) water molecules.44

Some water molecules were replaced by sodium and chlorideions to neutralize and simulate the experimentally used 150 mMion concentration.

All the starting structures were subsequently energy mini-mized with the steepest descent method for 2000 steps. Theresults of these minimizations produced the initial structure forthe MD simulations. The MD simulations were carried out witha constant number of particles (N), pressure (P), and temperature(T), i.e., NPT ensemble. The SETTLE algorithm was used toconstrain the bond length and angle of the water molecules,45

while the LINCS algorithm was utilized to constrain the bondlength of the peptide.46 The long-range electrostatic interactionswere calculated by the particle-mesh Ewald (PME) method.47,48

A constant pressure of 1 bar was applied with a couplingconstant of 1.0 ps; peptide, water molecules and ions werecoupled separately to a bath at 300 K with a coupling constantof 0.1 ps. The PBC were applied and the equation of motionwas integrated at time steps of 2 fs. For Met(O), the parametersproposed by Strader and Feller49 for dimethyl sulfoxide wereused. For the sulfur and sulfur oxygen in sulfone (Met(O2)),DFT/6-31G(d) calculated partial charges and geometric param-eters for dimethyl sulfone were used.

All the structures in aqueous solution are characterized bythe secondary structure analysis, performed using the definitionof secondary structure of proteins (DSSP) protocol,50 contactmaps, and similarity factors of the most representative structuresobtained from the cluster analysis. In the cluster analysis, thetrajectories are analyzed by grouping structurally similar frames(root-mean-square-deviation (rmsd) cutoff) 0.30 nm),51 theframe with the least rmsd deviation from its neighbors is denotedas a “middle” structure, which is used to represent that particularcluster. The electronic changes are characterized in terms ofdipole moment of the Cγ-substituted groups of Met35. Thesolvent accessible surface area (SASA) per residue is alsocomputed to assess the hydrophobic character of differentregions of the monomers. The VMD suite of software was usedfor visualizations and for the preparation of structural diagramspresented in this study.52

III. Results and Discussion

In this study, we performed 20 ns simulations of the solvatedWT-Aâ(1-42) and Met35-substituted methionine sulfoxide(Met35(O)), methionine sulfone (Met35(O2)), and norleucine(Met35(CH2)) monomers to obtain a dynamical picture of theconformational changes that occur in aqueous solution. The mainemphasis of these simulations is to explore the conformationalalterations that take place in the secondary structure of the CHCregion 17-21 (LVFFA), the stable turn segment 24-27(VGSN), and the second 29-35 (GAIIGLM) hydrophobicregion. These regions have been experimentally proposed toplay critical roles in the aggregation process.21,37,53The rmsdvalues for all four simulations are depicted in Figure 1, andthey clearly indicate metastable conformations after 6 ns ofsimulation for all (Aâ(1-42), Met35(O), Met35(O2), andMet35(CH2)) trajectories. Only the Met35(O2) trajectory showslarge fluctuations, but after 10 ns it also stabilizes to a ratherstable conformation. In the thermodynamically equilibratedregion, these four trajectories do not show any significantchanges and the overall rmsd deviations remain below 0.30 nm.

III.a. Wild-Type (WT)-A â(1-42). The NMR structure ofthe monomeric form of WT-Aâ(1-42) reveals that it exists ina loosely formed collapsed coil state in aqueous solution.53 Inthis medium, it appears to adopt a well-defined helical confor-mation within its central hydrophobic core (CHC) in the 17-21 (LVFFA) region and a dominant turn in the 24-27 (VGSN)region. The flanking regions, N- and C-terminus, are proposedto be more dynamic and partially unstructured.

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The time evolution of the secondary structure (Figure 2a) inour 20 ns simulations of the WT-Aâ(1-42) monomer showsthat the first four residues (DAEF) adopt a coil conformationthroughout the simulation. The segment 5-15 (RHDSGYEVH-HQ) evolves into a stable helical structure. This is also reflectedin the analysis of the contact map (e.g., contacts of the sidechain of a residue with its neighbors) depicted in Figure 3a

(lower triangle). As shown in this figure, there are many contactsin the two parallel rows along the diagonal in the 5-15 region.

The CHC region (17-21, LVFFA) has been proposed to playan important role in the initial deposition of the peptidemonomer on the fibril surface.21,37 During the simulation, thisregion undergoes two distinct transformations. The first trans-formation occurs at 2 ns, when the initialR-helix conformation

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

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

Secondary Structure of the Amyloidâ Monomer J. Phys. Chem. B, Vol. 112, No. 7, 20082161

starts changing toward a more open 5-helix structure (Figure2a). This metastable conformation fluctuates for about 8 ns andfinally transforms back to the initialR-helix conformation andremains in this stable conformation for the last 10 ns of thesimulation. In this region, similar fluctuations were also observedin the 10 ns simulations performed by Luttmann and Fels,38

but due to the shorter simulation time the stabilization of theR-helix that occurs after 10 ns could not be observed. It isnoteworthy that both studies demonstrate the presence of ahelical region formed between residues 5 (R) and 21 (A).

The dominant curve in the 24-27 region predicted fromNMR studies in soluble WT-Aâ(1-42) is also observed in oursimulation (Figures 2a and 4a). In Figure 2a, the presence of aturnlike structure is depicted by yellow color. The contact map(Figure 3a, lower triangle) also shows an ordered structure inthis 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 largedynamical transformations between turn and bend. Thesetransformations are also reflected in Figure 5a, where changesin the angle between short fragments (24-25 and 27-38) ofthe peptide on both sides of the turnlike conformation, repre-sented by a vector pointing from the N-terminus to theC-terminus, are depicted. A turn or bend structure show largeangles, and ideally, for a turn conformation the angle shouldbe close to 180°. During the simulation this angle increases andfluctuates significantly around 110°.

The C-terminal region containing residues 29-40 (G-V)exhibits a very dynamic character as indicated by variousexperiments.54-57 The initial helical conformation is found tofluctuate between turns, bend, and coil conformations indicatinga rather irregular region. However, after 14 ns a stable bendstructure is observed in the second hydrophobic segmentbetween residues 29-35 (G-M). This trajectory also indicates

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

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

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a mixture of turn,â-sheet, and bend contents (green, yellow,and red, respectively) close to the C-terminus of WT-Aâ(1-42), Figure 2a. The calculated dipole moment of the Cγ-substituted group in WT-Aâ(1-42) is 1.76 D (Table 1). In orderto characterize the hydrophobicity, we calculated the SASA forall 42 residues of Aâ(1-42) monomer (Figure 6a-c), and forMet35 this value is calculated to be 0.42 nm2 (Table 1 andFigure 6c). The last two residues (IA) retain a coil-likeconformation throughout the simulation.

III.b. Met35(O)-A â(1-42). The oxidation of methionine(Met35) to methionine sulfoxide (Met35(O)) is achieved byadding a double-bond oxygen atom to the sulfur atom of Met35in the initial PDB structure (1IYT-10). The secondary structureanalysis and contact map of the Met35(O)-Aâ(1-42) monomerare shown in Figures 2b and 3a (upper triangle). Throughoutthe simulation, similar to WT-Aâ(1-42), the first three residues(DAE) adopt a coil structure (Figure 2b). However, with respectto WT-Aâ(1-42), an important difference is observed in thelength of the helical conformation in the N-terminus segment.In WT-Aâ(1-42), this helical region starts from residue 5 (R),whereas in Met35(O)-Aâ(1-42) it starts from residue 9 (G). Acomparison of the contact maps between WT-Aâ(1-42) andMet35(O)-Aâ(1-42) in Figure 3a clearly shows this difference.

The secondary structure analysis (Figure 2b) shows that forMet35(O)-Aâ(1-42), the CHC (17-21) region adopts a moreopen 5-helix conformation. The representative structures ob-tained from a cluster analysis over the last 10 ns trajectoriesare superimposed to best overlap the CHC region. Thesuperposition of WT-Aâ(1-42) and Met35(O)-Aâ(1-42),Figure 4a, also indicates some noticeable differences in the CHCregion, particularly in the last three residues (FFA). In com-parison to WT-Aâ(1-42), significant conformational changesare observed in the region 22-35 (E-G). The secondarystructure analysis (Figure 2b) indicates the formation of sporadic,

therefore metastable,â-sheet conformations between residues23-24 (D-V) and 27-28 (N-K), which are nonexistent inWT-Aâ(1-42). The curved structure in the 24-27 (V-N)region of WT-Aâ(1-42) now becomes more pronounced asindicated by the angle of 150° between the vectors representingthe 24-25 and 27-28 fragments on both sides of the turnlikeconformation (Figure 5b). In comparison to WT-Aâ(1-42) theaverage value of this angle has now increased by ca. 40°. It isnoticeable that this angle undergoes large fluctuations duringthe first 10 ns and after that they decrease significantly andeventually become smaller than the corresponding fluctuationsin WT-Aâ(1-42). These results indicate that the Met35fMet35(O) substitution stabilizes the bendlike structure observedin WT-Aâ(1-42) to a more turnlike structure.

Like WT-Aâ(1-42), the C-terminus segment has beenobserved to undergo large dynamical rearrangements (Figure2, parts a and b). Another noteworthy difference has beenobserved in the segment composed of residues 28-35 (KGAI-IGLM). The contact map in this segment (Figure 3a) shows adistinct difference in the conformation between Met35(O)-Aâ-(1-42) (upper triangle) and WT-Aâ(1-42) (lower triangle). ForWT-Aâ(1-42), this region is shown to have a random distribu-tion of points, which is characteristic of a region with irregularstructure. It also shows that residues 30 (A) and 31 (I) are incontact with residues 16 (K) and 17 (L). The contact map(Figure 3a, lower triangle) provides contact distances of 0.3-0.4 nm between these residues. However, in the case of Met35-(O)-Aâ(1-42), the conformation in this region is more ordered(Figure 3a, upper triangle) which is also confirmed in the ribbonrepresentation depicted in Figure 4a (WT; yellow color andMet35(O); blue color). This segment, 28-35 (KGAIIGLM), inMet35(O)-Aâ(1-42) monomer conforms a helical structure withclose contact between neighboring residues and not as a resultof interactions with N-terminus residues.

These conformational changes observed in the C-terminusregion between WT-Aâ(1-42) and Met35(O)-Aâ(1-42) arecaused by the electronic change introduced by the oxidation ofMet35. Met35 is a hydrophobic residue, and in WT-Aâ(1-42),due to a specific orientation, the side chain of this residue istotally buried (Figure 4a). The oxidation of Met35 to Met35-(O) alters this orientation by increasing the dipole moment ofthe Cγ-substituted group by a factor of 2 (Table 1) and makingMet35 accessible to interaction with water molecules. Theseinteractions, in turn, destroy the contacts between the C- andN-terminus residues. These conformational changes are sup-ported by the SASA value of Met35 in Met35(O)-Aâ(1-42)of 0.87 nm2, which is greater than the corresponding value of0.41 nm2 for WT-Aâ(1-42) (Table 1 and Figure 6c).

The last C-terminus segment, between residues 36-42(VGGVVIA), follows a dynamics similar to WT-Aâ(1-42).This segment is characterized by two smallâ-sheet conforma-tions connected through a bend (Figure 2b). It was found thatthe length ofâ-sheet in the oxidized form (Met35(O)) is slightlysmaller than what was observed in WT-Aâ(1-42).

In order to obtain a measure of the similarity between differentstructures we also computed the rmsd values between thecorresponding representative structures in every trajectory. Thermsd value of 0.74 nm (Table 2) between the structuresrepresenting the WT-Aâ(1-42) and Met35(O)-Aâ(1-42) tra-jectories indicate significant differences between these twostructures. The major difference is located in the secondhydrophobic region (28-35), with an rmsd value of 0.53 nm.It is important to mention that the size of the helical segmentfor the two monomers in the N-terminus region is significantly

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

Secondary Structure of the Amyloidâ Monomer J. Phys. Chem. B, Vol. 112, No. 7, 20082163

different, which along with the conformational changes observedin the secondary structure of the 28-35 (KGAIIGLM) regionmay play a role in possible oligomerization or depositionmechanisms observed for these two monomers.6

The oxidation of Met35 to Met35(O) significantly augmentsits dipole moment by 1.96 D (Table 1). This increase inducesmarked changes in the SASA values of residues (in particularof Ile31, Lys34, and Met35) in the second hydrophobic region(28-35), Figure 6c. Our simulations predict that Ile31 andMet35 become more exposed to water molecules, whereasLys34 adopts more hydrophobic character. The Met35fMet35(O) substitution introduces only moderate structuralchanges in the CHC and turn (24-28) regions. The computed

SASA values in these two regions are also very similardemonstrating small differences in their hydrophobic characters(Figure 6, parts a and b).

III.c. Met35(O 2)-Aâ(1-42). The oxidation of methionine(Met35) to methionine sulfone (Met35(O2)) is achieved by theinclusion of two oxygen atoms to the sulfur atom of Met35.According to the secondary structural analysis (Figure 2c),similar to WT-Aâ(1-42) and Met35(O)-Aâ(1-42), the regioncomposed of 1-9 residues undergoes large dynamical changesand transforms in to a coil structure. The region 9-21 of Met35-(O2)-Aâ(1-42), which also includes the CHC segment, indicatessmall changes but retains the overall helical structure observedboth in the WT-Aâ(1-42) and Met35(O)-Aâ(1-42) monomers.

However, substantial differences were observed in the seg-ment comprise of residues 22-35, which is located just abovethe CHC region (toward the C-terminus). These differences areshown in the secondary structural analysis and contact mapdepicted in Figures 2c and 3b (upper triangle), respectively. Thissegment is now characterized by a more ordered secondarystructure, containing stableâ-sheets connected by turns formedat residues 25-27 and 30-32, respectively. The distancebetween the C- and N-terminus is significantly increased, whichindicates a decrease in interaction between them (Figure 4b).This change is also shown in the time dependence values ofthe angle quantifying the turnlike conformation in the 24-27region (Figure 5c). For Met35(O2)-Aâ(1-42), this value fluctu-ates around 110°, but the amplitudes of fluctuations aredecreased, indicating a more stable bend conformation than theone observed in both WT-Aâ(1-42) and Met35(O)-Aâ(1-42).

These changes in the secondary structure of the Met35(O2)monomer can be explained in terms of increase in the dipolemoment of the Cγ-substituted sulfone group. The Met35fMet35(O2) chemical substitution increases both the size and thepolarity of the Cγ-methylene group of Met35. The dipolemoment of this side chain in Met35(O2)-Aâ(1-42) is 4.30 D,which is 2.54 and 0.58 D larger than the dipole moment of thecorresponding side chains in WT-Aâ(1-42) and Met35(O)-Aâ-(1-42), respectively (Table 1). In the sulfone form, Met35-(O2), this residue acts as a strong hydrogen bond acceptor andtends to form hydrogen bond with the amide backbone of theneighboring residue. In our simulations, we found an intermo-

TABLE 1: Calculated Dipole Moments and Solubility Areas for Different Substitutions of Met35 in the Aâ(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 centralhydrophobic core (17-21), (b) turn (24-28), and (c) the secondhydrophobic region (28-35).

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lecular hydrogen bond between Met35(O2) and Ile41. The SASAvalues of Ile31, Lys34, and Met35 are enhanced, in particularfor Met35 by a factor of 3 (0.42-1.25 nm2), therefore exposingall these residues to greater interactions with the surroundingwater molecules.

In comparison to WT-Aâ(1-42), the formation of newhydrogen bonds along with the increased solvation of theC-terminus region induces large changes in the secondarystructure of Met35(O2)-Aâ(1-42). These changes are reflectedin the calculated similarity factor of 0.83 between the two mostrepresentative structures obtained from the trajectory analysis(Table 2). The superposition of these two structures is shownin Figure 4b. Noteworthy is the difference in the secondarystructure of the 22-35 region and a lack of contact betweenthe N- and C-terminus.

III.d. Met35(CH 2)-Aâ(1-42).The substitution of the sulfuratom in Met35 of the WT-Aâ(1-42) monomer by a methylene(-CH2) group leads to a norleucine (Met35(CH2)-Aâ(1-42))monomer. The first two residues of the N-terminus (DA)segment typify a coil structure followed by a helical conforma-tion involving R- and 5-helix structures in the 2-21 region(Figure 2d). This structure is similar to the one observed forthe WT-Aâ(1-42) monomer. An examination of the contact

map depicted in Figure 3c show this similarity, where manyinter-residue contacts are retained.

The superposition, alignment of the CHC region, of the mostrepresentative structures of the WT-Aâ(1-42) and Met35(CH2)-Aâ(1-42) trajectories is shown in Figure 4c. These twomonomers are very similar, even the turn conformation in the21-27 segment follows the same curvature (Figure 5, parts aand d). The SASA values of the 17-21 and 24-28 regions,Figure 6, parts a and b, predict the same trend with very similarvalues per residue.

The secondary structural analysis indicate that the C-terminusregion 28-35 adopts an irregular structure including coil, turn,and bend conformations. This dynamics is again very similarto the one found for WT-Aâ(1-42). The substitution of Met35by norleucine does not introduce any polarity in the Cγ-substituted side chain (Table 1); however the SASA values ofthis residue (Met35(CH2)) are slightly larger than Met35 (Figure6c). The two hydrogen atoms present in the methylene groupof Met35(CH2)-Aâ(1-42) tend to interact with surroundingwater molecules. These interactions induce moderate changesin the secondary structures in the vicinity of Met35 and exposeLys34 to the surrounding water molecules.

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

Secondary Structure of the Amyloidâ Monomer J. Phys. Chem. B, Vol. 112, No. 7, 20082165

Evidently, a detailed structural analysis indicates that theoverall secondary structure of Met35(CH2)-Aâ(1-42) is similarto the WT-Aâ(1-42) monomer (Figures 2d, 3c, 4c and Table2). It has been observed experimentally that the aggregation ofMet35(CH2)-Aâ(1-42) monomers yields a characteristic WT-Aâ(1-42) oligomer size distribution.6 This observation is ingeneral agreement with our MD simulations because monomerswith similar secondary structures are likely to produce equivalentoligomerization patterns.

IV. Summary and Conclusions

In this study, we performed 20 ns MD simulations on theWT-Aâ(1-42) monomer and its chemically substituted forms(Met35 f Met35(O), Met35f Met35(O2), and Met35fMet35(CH2)) in aqueous solution to investigate the conforma-tional changes associated with the electronic character of thechemical group attached to the Cγ-methylene of Met35. On thebasis of existing experimental and theoretical information,conformational changes that occur in three distinct regions (theCHC 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 resultsreported in this study could be further verified by advancedsampling enhanced methods such as generalized ensemble-basedtechniques (replica exchange methods, simulated scaling meth-ods, or accelerated MD methods).

For WT-Aâ(1-42), in the first 2 ns the initialR-helixconformation of the CHC region transforms to an open 5-helixstructure, where it fluctuates for about 8 ns and finally returnsto the initial stable helical form for the rest of the simulation(Figure 2a). The dominant curve in the region 24-27 undergoeslarge dynamical transformations between turn and bend (Figures2a 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 toa stable bend structure (Figure 2a). This region has experimen-tally been observed to be very dynamic.54-57 The dipole momentand SASA values of the Met35 residue in the WT-Aâ(1-42)monomer are 1.76 D and 0.42 nm2, respectively (Table 1 andFigure 6c).

For Met35(O)-Aâ(1-42), the CHC (17-21) region adoptsa more open 5-helix conformation and the last three residues(FFA) of the CHC exhibit significant deviations from the WT-Aâ(1-42) monomer (Figures 2b and 4c). The curve regionbetween 24 and 27 (VGSN) residues becomes more dominant.In comparison to WT-Aâ(1-42), the angle between the vectorsrepresenting the 24-25 and 27-28 fragments on both sides ofthe turnlike conformation is now increased by ca. 40° (Figure5b). The 28-35 (KGAIIGLM) segment is also distinctlydifferent from the WT-Aâ(1-42) monomer. It adopts a moreordered helical-type structure due to the formation of closecontacts between neighboring residues and not as a result ofinteractions with N-terminus residues (Figures 2b and 3a). Theoxidation of Met35 to Met35(O) alters the orientation of Met35-(O) by increasing the dipole moment of the Cγ-substituted groupby 1.96 D and make it more accessible to interaction with watermolecules. This SASA value of 0.87 nm2 for Met35 in Met35-(O)-Aâ(1-42) is greater than the corresponding value of 0.41nm2 for WT-Aâ(1-42) (Table 1 and Figure 6c). The rmsd of0.74 nm between WT-Aâ(1-42) and Met35(O)-Aâ(1-42) alsosupports the major modifications in this region.

For Met35(O2)-Aâ(1-42), the CHC undergoes small changesbut maintains its overall helical structure found in the WT-Aâ-(1-42) and Met35(O)-Aâ(1-42) monomers (Figure 2c). In this

case, significant differences were observed in the 22-35 (E-M) region which lies adjacent to the CHC and includes boththe turn segment 24-27 (VGSN) and second hydrophobicregion 29-35 (GAIIGLM). This region now adopts a moreordered secondary structure, which consists of stableâ-sheetsconnected 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-Aâ(1-42) and Met35(O)-Aâ(1-42). This substitution, Met35f Met35(O2), furtherenhances the dipole moment and SASA values by 0.58 D and0.38 nm2, respectively (Table 1), and facilitates greater interac-tion of Met35 and neighboring residues with the surroundingwater molecules. The rmsd value between WT-Aâ(1-42) andMet35(O2)-Aâ(1-42) is 0.83, and contact between the N- andC-terminus residues is disrupted.

For Met35(CH2)-Aâ(1-42), the overall structure of themonomer (including the CHC region, stable turn, and secondhydrophobic region) is very similar to WT-Aâ(1-42). However,the SASA value of 0.65 nm2 for Met35(CH2) is slightly greaterthan 0.42 nm2 computed for Met35 in the WT-Aâ(1-42)peptide (Figure 6c and Table 1).

It has been experimentally indicated that the C-terminus ofthe WT-Aâ(1-42) monomer in aqueous solution does not forma well-defined secondary structure.7,58 Our simulations predictthat the oxidation of Met35 to Met35(O) and Met35(O2)diminishes this residue’s hydrophobicity, which subsequentlyinfluence its interactions with the neighboring residues (inparticular with Ile31, Lys34, and Ile41) and surrounding watermolecules. These electronic modifications induce significantchanges in the secondary structure of the C-terminus hydro-phobic region (28-35). These alterations in the secondarystructure along with the increased solvation energy may hinderthe intermolecular interactions between two monomers, whichare required for the formation of oligomers implicated in AD.

Acknowledgment. Financial support from the University ofMiami, Miami, Florida is acknowledged.

Supporting Information Available: Tables S1 and S2:optimized Cartesian coordinates (in angstroms) and charges formethionine sulfone (Met35(O2)). This material is available freeof charge via the Internet at http://pubs.acs.org.

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