Baumketner 2006 - Folding Landscapes Abeta 12-28

8/6/2019 Baumketner 2006 - Folding Landscapes Abeta 12-28

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Folding Landscapes of the AlzheimerAmyloid-(12-28) Peptide

Andrij Baumketner and Joan-Emma Shea

Department of Chemistry andBiochemistry, University ofCalifornia Santa Barbara, SantaBarbara, CA 93106, USA

The energy landscape for folding of the 12-28 fragment of the Alzheimeramyloid (A) peptide is characterized using replica-exchange moleculardynamics simulations with an all-atom peptide model and explicit solvent.At physiological temperatures, the peptide exists mostly as a collapsedrandom coil, populating a small fraction (less than 10%) of hairpins with a-turn at position V18F19, with another 10% of hairpin-like conformationspossessing a bend rather than a turn in the central VFFA positions. A smallfraction of the populated states, 14%, adopt polyproline II (PPII)conformations. Folding of the structured hairpin states proceeds throughthe assembly of two locally stable segments, VFFAE and EDVGS. Theinteractions stabilizing these locally folded structural motifs are in conflictwith those stabilizing the global fold of A12-28, a signature of underlyingresidual frustration in this peptide. At increased temperature, thepopulation of both -strand and PPII conformations diminishes in favorof -turn and random-coil states. On the basis of the conformationalpreferences of A 12-28 monomers, two models for the molecular structureof amyloid fibrils formed by this peptide are proposed.

2006 Elsevier Ltd. All rights reserved.

*Corresponding authorKeywords: protein folding; conformational space sampling; replica ex-change molecular dynamics simulations; Alzheimer amyloid- peptide

Introduction

Alzheimer's disease belongs to a class of neuro-degenerative disorders1 characterized by the pre-sence of amyloid deposits in the brain.1,2 In the caseof Alzheimer's disease, these deposits consist ofaggregates of the 39 to 42-residues long amyloid (A) peptide.3 While it is well established that fibrils

possess a large -sheet content,4

the nature of themonomeric species in solution remains poorlycharacterized. Determining the molecular structureof A monomers poses a serious experimentalchallenge, in large part because of the readiness ofthese peptides to aggregate. However, an under-standing of the mechanisms of peptide self-assem-

bly necessitates a structural characterization of themonomeric form of the peptide. Different mono-meric conformations can give rise to differentintermediates on (or off) pathway to aggregation,

and hence to different aggregate end-products andfibril morphologies. Because investigations of theclinically relevant A peptides are prohibitive,5

model systems consisting of fragments of thesepeptides are emerging as viable tools for elucidatingthe general principles underlying protein aggrega-tion. The 12-28 fragment, A12-28 (sequenceVHHQKLVFFAEDVGSNK), the focus of this work,

is a particularly attractive system, as it is toxic to thecell, forms amyloid fibrils and populates on-path-way aggregation intermediates (ADDLs, paranuclei,etc.) similar to those found for the full-lengthpeptide.6

Recent work by Grslund and co-workers hasbegun to shed important new light on the nature ofthe conformations populated by the A12-28 pep-tide in aqueous solutions.79 Using a combination ofspectroscopic probes, including circular dichroismand NMR, those authors showed that the peptideco-exists in a temperature-dependent equilibrium

between polyproline II (PPII), -strand and random-coil conformations. From a theoretical perspective,

the nature of the transition from the -helicalstructure populated in apolar media to a -richstructure in water was probed by Tiana and co-workers through 100 ns simulations.1012

Abbreviations used: PPII, polyproline II; SASA, solvent-accessible surface area.E-mail address of the corresponding author:

[email protected]

doi:10.1016/j.jmb.2006.07.032 J. Mol. Biol. (2006) 362, 567579

0022-2836/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
mailto:[email protected]:[email protected]


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In the present work, we use enhanced samplingsimulation techniques to further probe the nature ofthe monomeric species populated by the A12-28peptide over a range of temperatures.1318 The useof efficient sampling techniques is essential in orderto obtain a correct statistical description of con-formational space for peptides with energy land-scapes dominated by high barriers and deepminima. Simulations are an invaluable complementto experiment, capable of providing a detailedatomistic picture of the folding process. In contrastto most experimental approaches that can probeonly the highest populated states of a molecule,these simulations provide direct access to the entireconformational space sampled under a given set ofconditions. This is especially critical for the study ofthe monomeric state of this A peptide, whichappears to be mostly unstructured under normal

physiological conditions, populating only a smallfraction of ordered states. Although not detectable by ensemble-averaging experimental techniquessuch as NMR, structured conformational ensembleswith low population may nonetheless play a majorrole in initiating protein aggregation.

We model the A12-28 peptide using an all-atomforce field,19 and an explicit water model,20 and per-form our simulations using the replica-exchangemolecular dynamics method.14,1618Oursimulationsreveal, in agreement with experiment, that thepeptide exists predominantly in a collapsed, mostlyunstructured state under physiological conditions.We also uncover the presence of structured hairpin-

like conformations, co-existing with less structuredstates. While such states were not discussed inexperimental studies, their presence is consistentwith the experimental observations reported on thispeptide, as well as with other theoretical investiga-tions. Emerging from our studies is the interestingobservation that the principle of minimal frustra-tion is violated in the folding of these peptides.Frustration is present both in the hydrophobicforces and in the formation of local structuredelements. The folding mechanism of these hairpins,their temperature-dependence, and their role ininitiating aggregation into fibrils is discussed.

Results

Conformations populated by the A12-28peptide

We first turn to an analysis of the conformationspopulated during the replica exchange moleculardynamics simulations at low temperatures.

Secondary structure analysis: co-existence of PPII,random coil, -strand and-hairpin conformations

The secondary structure of the conformationssampled in our simulations was assigned using thePROSS protocol.21 This protocol distinguishes

between -helices, -strands, -turns and random

coils based on and angles. The results of ouranalysis at 280 K are shown in Figure 1 for all 17residues of the peptide. A12-28 is seen to populatemostly random-coil states, that co-exist with PPII, -strand and -turn structures. The highest popula-tion of PPII states (26%) is observed in the centraland C-terminal regions of the peptide, for residuesA21E22 and S26N27, respectively. Residues A21E22were seen in the experiments reported by Grslundet al.9 to populate more PPII structure than any otherresidue in the central part of the sequence, while noexperimental information is available for the sec-ondary structure of the C-terminal part. The otherresidues of the peptide (with the exception of F19and G25) exhibit non-vanishing amounts of PPII. Ahigh propensity to form -strand is observed forresidues F20V24 (populated 20% of the time) andH14-L17 (15% populated). These two -strand

regions are connected by a -turn at V18F19,suggesting the presence of a -hairpin conforma-tion. A non-vanishing propensity to turn formationis seen for the G25N27 and H14Q15 segments. No-turn structure was reported in the experimentsreported by Grslund et al.,9 as this secondarystructure element was not included in the analysis.Our observation of-turns, however, is consistentwith other simulations for A12-28,1012 as well asexperiments for a number of other A peptides.2225A detailed comparison with experimental findingson this peptide is given in a later section.

Nature of structured states:-hairpin formationIn order to further characterize the structured

conformational states of A12-28, we performed aclustering analysis of all conformations visited bythe peptide at 280 K. Our analysis (described inMethods) reveals two main families of structures,

Figure 1. Residue-specific secondary structure ofA12-28 at 280 K. The analysis is carried out accordingto the PROSS protocol,21 which distinguishes five differenttypes of secondary structure: -helix, PPII, -strand, -turn and random coil. The population of -helices isvanishing. All other secondary structure types aredistributed non-uniformly along the sequence.

568 Alzheimer Amyloid-(12-28) Peptide


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which we refer to as cluster 1 (C1) and cluster 2(C2), that were sampled more frequently than otherconformations. The most representative conforma-tions belonging to these clusters (the centroids)are shown in Figure 2. Conformations belonging to

both centroids have an overall -hairpin topology, but they differ in the nature of the turn region.Conformations belonging to cluster C1 show a -turn at position V18F19, with strands extendingover residues F20D23 and H14L17. The C-terminal D23K28 segment is mostly unstructured.Cluster C1 is stabilized by a number of hydrogen

bonds and salt-bridges, including a hydrogen bondbetween L17 and F20 (defining the turn region) andsalt-bridges D23N terminus, D23K28 and K16E22. Conformations of cluster C2 show a bend atresidues L17F19, with strands formed by residuesF20D23 and H13K16. Hydrogen bonds stabiliz-

ing these structures include interactions H14E22,H13E22, V18K16, F20V18 as well as D23G25,and S26N terminus. A salt-bridge K16E22 ispresent, as was the case for the C1 structures.

In order to probe the relative population of theconformational states sampled by the peptide, wegenerated a free energy surface for A12-28 as afunction of the first two principal components at280 K. (Figure 3(a)). The centroid of cluster C1 waschosen as the reference state for building theprincipal components, and is the lowest free energy

state on the folding map. The second most popu-lated ensemble corresponds to conformations

belonging to C2. Clusters C1 and C2 have apopulation of approximately 10%. Two other clus-ters, noted C3 and C4 in Figure 3 have populations

below 5%. Due to the low population of theseclusters, we focus the analysis presented here on thestructured clusters C1 and C2.

Residual frustration in folding

Frustration in hydrophobic forces

The possible contribution of hydrophobic forcesto the stability of the folded states is investigated

by considering the solvent-accessible surface area(SASA) of the peptide as a measure of the strengthof the hydrophobic forces.26 Hydrophobic forces

are known to drive the folding of globularproteins,27 and may play a role in the folding ofA12-28. The mean SASA, averaged over allrecorded conformations at 280 K, is plotted as afunction of the first two principal components PC1and PC2 in Figure 3(b). All clusters identified havelow SASA values, with cluster C1 displaying thelowest value. The low SASA regions correlate wellwith the highly populated regions in the freeenergy map shown in Figure 3(a), implying animportant role for the hydrophobic effect in

Figure 2. Most representative conformations (centroids) of the two most populated clusters (a) C1 and (b) C2, foundin the present simulations of A12-28 at 280 K.

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folding. Interestingly, however, not all conforma-tions with a small exposed surface area are highlypopulated. For instance, the conformations withsmall surface area located at approximately(PC1=1.1; PC2=0.5) delineate the free energy

barrier linking the conformations of cluster C3 withthe rest of the states visited and are, as a result, nothighly populated. The fact that conformations withlow SASA values exist that are not populatedsignificantly, implies that the folding landscapes of

A12-28 are not ideally smooth. Further evidencethat the hydrophobic interactions are not welloptimized for the most populated states observedin our simulations can be seen from an analysis ofthe specific hydrophobic interactions in the struc-tured conformations of the peptide. Specifically,hydrophobic residues V12 and V24 form a contactin the conformations belonging to cluster C1,thereby reducing the exposure of hydrophobicsurface to the solvent. In contrast, in conformationsof the equally populated cluster C2, both residuesV12 and V24 fully expose their hydrophobic side-chains to water. The presence of residual frustra-tion in the hydrophobic interactions in A12-28was observed in earlier simulations.10

Our results are at odds with the principle ofminimal frustration,2830 in that we observe adiscrepancy between the states favored by the

hydrophobic forces and those actually populatedin the simulations. The residual sequence frustra-t ion observed here may be a hallmark of amyloidogenic peptides that sets these systemsapart from more foldable sequences.31 We notethat it is possible that current force fields over-estimate the degree of frustration that proteinsreally possess.

Frustration in local structured elements: implicationsfor the folding mechanism

The clustering and secondary structure analysispresented in the preceding section revealed that the

amount of structured states populated by A12-28is low. We probe here whether shorter segmentswithin this peptide can exhibit a greater extent ofstructuring than the peptide as a whole, andwhether they may play a role in folding thehairpins. We divided the peptide into segments oflength N=414 residues and the resulting confor-mations were clustered based on mutual RMSDamong C atoms. The result of this analysisrevealed two segments, each of five residues, withenhanced propensity for structuring: the C-terminalE22-S26 (EDVGS) segment and the V18-E22(VFFAE) segment located within the central hydro-

phobic cluster.Representative structures of the most highlypopulated conformations of both segments areshown in Figure 4(a). Both the VFFAE andEDVGS segments adopt a bend structure as theirmost highly populated state. Interestingly, theEDVGS bend is identical with the structureadopted by this sequence in the context of theA21-30 peptide.32 The A21-30 shows a bendlocated at residues 2428, stabilized partly byCoulombic interactions between residues 22 and28. This peptide is resistant to proteolysis and is

Figure 3. (a) Free energy map (in units ofkBT) of theA12-28 peptide at 280 K projected onto the first twoprincipal components PC1 and PC2. The centroid ofcluster C1 was used as the reference state to compute theprincipal components. (b) Mean solvent-accessible surfacearea (in nm2), averaged over all recorded conformations at280 K. The data are shown as a function of the first twoprincipal components PC1 and PC2. The location of thefour most populated clusters is shown by contour lines.

Figure 4. Free energy map (in units of kBT) of A12-28 as a function of C

RMS deviation over fragment VFFAE,RMSD1, and C

RMS deviation over fragment EDVGS, RMSD2, at (a) 280 K and (b) 376 K. VFFAE and EDVGS fragmentswere seen in our simulations to be most structured. Unfolding of clusters C1 and C2 is seen readily at the highertemperature.



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Figure 4 (legend on previous page)



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believed to nucleate folding of the full-length,A40/42 peptide.33 The most highly populatedVFFAE conformations (18%) are present in bothclusters C1 and C2, while those of EDVGS (13%)are present in C2 (as well as in some other lesshighly populated A12-28 clusters), but not incluster C1. In fact, the EDVGS bend appears to beincompatible with the structures belonging to C1.Indeed, the bend requires the folding of the V24N27 backbone onto the side-chain of D23, whichwould disrupt the C1 -hairpins, due to stericclashes between C-terminal and N-terminal atoms.Energetically, the disruption of -hairpins is veryunfavorable, as it entails breaking some of theinter-strand hydrogen bonds, in particular the

bond between the carboxy oxygen atoms of D23and the amino group of the N terminus. Thus, itappears that the interactions that stabilize local

structured elements in A12-28 are in discord withthose that are needed to stabilize global foldsassembled from these local structures, anothermanifestation of the residual frustration present inA12-28. It is interesting to note that foldableproteins can satisfy the principle of minimalfrustration, even though their fragments maypossess thermodynamically stable traps. Amyloi-dogenic sequences, on the other hand, can display

both local and global frustration.The structured segments VFFAE and EDVGS can

provide insights into the folding mechanism of theC1 and C2 structures. The free energy map forfolding of the A12-28 peptide as a function of C

RMS deviation over VFFAE and EDVGS residuesfrom the most stable conformations adopted bythese segments is shown in Figure 4(a). The foldingof C1 conformations follows a primary foldingpathway that does not involve the formation of theEDVGS motif. A second, less-populated pathwayleads to C1 conformations indirectly, by passingthrough C2 states. Two major pathways exist forthe folding of C2 conformations as well. In the first,the EDVGS motif is formed first and then thecentral loop is assembled. Alternatively, a restruc-turing of C1 conformations leads to C2 statesthrough the breaking of the central -turn accom-

panied by the formation of the EDVGS motif. It isclear from the non-diagonal shape of Figure 4(a)that the EDVGS and VFFAE motifs do not foldconcurrently.

Effects of temperature on the structure ofA12-28

Temperature-dependence of the secondarystructure

The total amounts of secondary structure as afunction of temperature are summarized in Table 1.The content of-strand and PPII structure is seen to

decrease gradually from 280 K to 376 K, (consistentwith CD experiments,7,9 and the fact that PPIIstructures are stabilized by energetic rather thanentropic terms34), while the content of-turn and

random coil increases. The change in secondarystructure content per residue upon raising thetemperature is presented in Figure 5(a). It is seenthat the general trend for PPII and -strandstructure in most residues is to decrease withtemperature. In particular, the -strands centeredaround segments H14K16 and F20V24 lose about10% in population as the temperature is raised from280 K to 376 K. Similarly, the PPII population ofsegment S15N16 and residues H13 and Q15 drops

by about 5% following this change in temperature.The highest increase in -turn structure is observedfor residues F19F20. Although the change insecondary structure with temperature is small, it isstatistically significant, as illustrated in the plots ofsecondary structure as a function of temperature inFigure 5(b).

Nature of the hairpin conformations at hightemperatures

We now probe more specifically the effect oftemperature on the conformations belonging to C1and C2. At elevated temperatures, structuredconformations of both clusters C1 and C2 breakup, gradually taking on more flexible states. As thetemperature is raised, the population of theseclusters drops dramatically from about 10% at280 K to less than 1% at 376 K. The molecular sizeof A12-28 is not affected strongly by temperature.

The radius of gyration computed over C

atomsremains at 0.7-0.8 nm at both high and lowtemperature. The high-temperature ensembles arenot structurally homogeneous and cannot beclustered efficiently. Hence, to gain further insightinto the nature of the high-temperature structuresof A12-28, we examine the temperature behaviorof the local structural motifs VFFAE and EDVGS.The free energy surface as a function of the CRMS deviation over fragment VFFAE (denotedRMSD1), and the C RMS deviation over fragmentEDVGS (denoted RMSD2), at 376 K is shown inFigure 4(b). The VFFAE motif does not surviveheat-denaturation, its population dropping from

18% at 280 K to 6% at 376 K. In contrast, theconformations of the EDVGS segment are almostunaffected by temperature. Their relative popula-tion of 13% at 280 K remains at the same level at

Table 1. Secondary structure (SS) content of A12-28 as afunction of temperature

T (K)

SS content (%)

PPII -Strand -Turn Coil

280 13.7(2) 10.9(4) 9.2(4) 66.4(4)297 13.3(2) 9.2(2) 10.4(2) 67.2(3)315 13.0(2) 8.7(3) 10.8(2) 67.5(4)333 13.1(2) 8.6(2) 10.9(2) 67.3(3)354 13.0(2) 7.1(2) 11.5(2) 68.2(3)376 12.3(2) 6.3(2) 11.8(2) 69.4(3)

Estimated errors in the first digit after the decimal point areshown in parentheses.



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376 K. This difference in temperature behaviormay be related to the different nature of forcesstabilizing the VFFAE and EDVGS segments.

The hairpin topology is retained at hightemperatures

The nature of the conformations populated at hightemperatures are further explored through freeenergy maps (Figure 6) at 280 K and 376 K plottedas a function of two parameters D1 and D2quantifying the degree of structuring present in thecentral -hairpin. The parameter D1 denotes thedistance between C atoms of L17 and F20, andcharacterizes the degree of formation of the L17F20(LVFF) -turn. The parameter D2 denotes thedistance between the C atoms of A21 and K16,

and is a measure of the extent to which structure ispreserved further away from the turn. At 280 K fourmajor minima in the D1D2 free energy map areobserved. Two of them, located at D1 0.5, D2 0.55

and D1 0.8, D2 0.55 and denoted by I and II,correspond to clusters C1 and C2, respectively(recall that the LVFF turn is missing from C2conformations). The third minimum (III) is char-acterized by both large values of D1 and D2 anddenotes all the conformations that do not formL17F20 and K16A21 contacts. The fourth minimum(IV) denotes conformations in which the LVFF turnis present but the entire hairpin is not welldeveloped. The different conformations that canpossibly be classified according to two distances, D1and D2, are shown schematically in Figure 6(a), nextto the free energy minimum with which theycorrespond. Increasing temperature results in astrong depopulation of states II and enhancedpopulation of the fully unfolded states (III).More conformations are seen to occupy the half-

open hairpin states (IV) at high temperature, whilethe total number of conformations with both L17F20and K16A21 contacts present does not change muchwith temperature. Taken together, these results

Figure 5. Change in secondarystructure content (%) of A12-28 (a)for each residue, as the temperatureisraisedfrom280Kto376Kand(b)as a function of temperature.



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indicate that the hairpin-like structures populated atlow temperatures do not disappear from theconformational ensembles completely. Rather, theytransform into a looser or swollen conformationsthat still maintain the topology of a hairpin. This isparticularly true for the central -hairpin LVFFwhose population is not affected by temperature.The presence of hairpin-like conformations popu-

lated at high temperature is consistent with theconclusions illustrated by Figure 5 for the tempera-ture-dependence of the secondary structure.

Comparison with experiment

Grslund et al. have analyzed residue-specificchanges of secondary structure content in A12-28using 1H NMR.9 Dihedral angles evaluated fromJ-coupling measurements were used to distinguishbetween different secondary elements, leading tothe conclusion that residues V18F20 and V24populate substantial amounts of-strand structure

at 280 K. In our simulations, on the other hand, wefind that residues V24 and F20 adopt predomi-nantly -strand conformations at 280 K, whileresidues V18 and F19 populate mostly -turnsand that, as a result, hairpin structures are seen atlow temperatures. The apparent discrepancy in theexperimental and computational results (i.e. thepresence or the absence of hairpins) can bereconciled as follows. The -turn motif, present inour simulation (and missing from the analysispresented by Grslund et al.8) is characterized bya wide range of angles, from 140 to 50,35 andthus can easily be mistaken for either PPII or -strand states. Dihedral angles alone cannot be

used as a reliable descriptor of secondary structure,as there is significant overlap in the angles ofdifferent structural elements. Theoretical anglesfor residues V18F20 and V24, calculated from our

simulation data are compared to the experimentalvalues shown in Figure 7. In general, we observe agood agreement between theoretical and experi-mental results. In particular, the experimental andtheoretical values coincide within statistical errorsfor residues V18 and F19 while for residues F20 andV24 our simulations appear to slightly overestimatethe -strand content. Whether the structure popu-

lates a strand or a -turn may appear to bemerely a matter of semantics, we believe that ourfinding of a -turn in the monomeric state of A12-28 may have important implications for theaggregation of this peptide into fibrils. Indeed, ithas been noted recently that the amount of-turncontent present in other A peptides is positivelycorrelated with the ability of these peptides toaggregate.36 In Discussion and Conclusions, wepropose a fibril model based on the hairpinstructures seen in our simulations.

The differences in temperature-dependence seenexperimentally and computationally can also be

attributed to insensitivity of the

angle overlap asa probe of secondary structure. Experimentally,residues V18F20 and V24 (determined to be -strands at low temperature) undergo a structuraltransition into random-coil states at high tempera-ture. In our simulations, we find that at hightemperatures, only residues V18 and V24 undergoa transition to random-coil states, while residueF19 actually becomes more structured, depopulat-ing coil states in favor of PPII, -strand and -turnconformations. For F20, the population of random-coil structures remains constant, with -strandconformations transformed into PPII and -turns.Overall, in the central (F19-F20) part of the

sequence, we observe an enhanced propensity for-turn formation. An increase in the -turnstructure observed in our simulations for F19F20,produces temperature changes in angles

Figure 6. Free energy map (in units ofkBT) of the A12-28 peptide at (a) 280 K and (b) 376 K, projected onto D1, the

distance between C

of F20 and L17, and D2, the distance between C

of A21 and K16. D1characterizes structuralcompleteness of the LVFF turn while D2indicates how much hairpin-like structure is maintained further away from theturn. The drawings show four different conformations, (I), (II), (III) and (IV), that can be populated by the central KLVFFAsegment of A12-28.



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consistent with a structural shift toward disor-dered random-coil states.

Discussion and Conclusions

It is well established that conformations of theAlzheimer amyloid peptides, when incorporatedinto amyloid fibrils, are rich in -structural motifssuch as -strands or -hairpins.4 However the na-ture of the monomeric forms of these peptides insolution is poorly characterized at an atomicallydetailed level. Most spectroscopic probes point to apredominantly random-coil conformation in aqu-eous solutions at a low concentration ofpeptide andnormal temperature and pressure,3740 and the re-sults of our simulations largely support this view.Our simulations indicate that the A12-28 fragment

populates a structurally diverse ensemble of confor-mations, co-existing with one another in a dynamicequilibrium. The conformations populated underphysiological temperatures are mostly collapsedcoils, with radius of gyration at least half that of afully extended polypeptide chain. The molecularsize of the populated states is largely unaffected bytemperature, a result that is in-line with the findingreported by Grslund et al., who concluded that thehydrodynamic radius of A12-28 increases by lessthan 4% as the temperature is raised from 0 C to25 C.8

Approximately 20% of the conformations sampledby the peptide at 280 K are seen to populate struc-

tures with a hairpin topology. Half of these possess aturn located at L17F20 and constitute true hairpins, while the other half possess a bend inthis region and correspond to hairpin-like loops. The

conformations belonging to these two classes arevery similar in terms of their size, as evaluated byradius of gyration and end-to-end distance. Experi-mentally, the monomeric conformations of A12-28in aqueous solution were characterized by Grslundet al. using NMR and CD spectroscopy.7

9 Focusingprimarily on the secondary structure of the peptide,these authors found that A12-28 monomers exist inequilibrium between -strand, PPII and random-coil conformations. At increased temperature, thetotal amounts of-strand and PPII structure wereseen to decrease in favor of random-coil states.9 Oursimulations, on the other hand, report the presenceof a hairpin at low temperatures, with increased -turn structuring of the F19 residue. Our results areconsistent with those reported by Grslund et al.:thedifference in secondary structure assignment resultsfrom differences in resolution of the secondary

structural probes used. (The angles used in theexperimental assignment do not distinguishbetween -strands and -turns.)

Our observation of -strands and -turns is inagreement with previous studies on various Apeptides.22,23,41,24,25 In particular, -hairpins wereseen in a number of earlier simulations onA12-28.1012 A transition from an -helical state ofA12-28, the native state of this peptide in apolarmedium, to a -hairpin was followed in 100 nsconstant-temperature molecular dynamics simula-tions by Daidone et al.,10 and by Simona et al.11 Themost frequently sampled conformation in the work

by Daidone et al. was a -hairpin characterized by a

type II turn located at residues F19F20A21E22.10

Inaddition to the hairpin, a compact bend intermediatewas found, stabilized by K16E22 and K16D23 salt-

bridges and close-packing of the 1721 (LVFFA)

Figure 7. Dihedral -angles for residues V18F20 and V24, calculated in our simulations and those obtainedexperimentally.9 Estimated errors of the theoretical values are shown.



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cluster of hydrophobic residues. While our resultsagree with the previous simulations,1012 in that -hairpins constitute a significantly populated confor-mational ensemble, we observe some discrepancyregarding details of the structural nature of A12-28.In particular, the -turn seen in the present work islocated at positions L17V18F19F20, which representsa two residue shift along the sequence toward the Nterminus with respect to the -turn of the earliersimulations.1012 We note that the-hairpins sampledin these simulations were visited in our simulations

(with a C

RMS2 from the most representativestate) but they were not populated significantly. Inaddition, the temperature-dependence of the hair-pins seen here differs from that seen by the sameauthors, who reported a non-monotonic tem-pera-ture-dependence.12 In their simulations, the hairpinpopulation, negligible at 273 K, rose to a maximumvalue of90% at 315 K, before decreasing to levelsof 60% as the temperature was raised further. Incontrast to these observations, our simulationsindicate clearly that the population of both -hairpins (C1) and hairpin-like (C2) structures inA12-28 decreases in a monotonous manner with

temperature, from 20% at 280 K, to less than 1% at376 K, without any increase in population at anintermediate temperature. The observed differences

between our results and those reported by Tiana etal.1012 are likely due to differences in simulationmethodologies (NVTensemble simulations1012 andthe replica exchange simulations performed here) aswell as to differences in force fields. Different forcefields have different propensities for the formationof secondary structure elements,42 a factor thatcould very well account for the discrepancies

between Tiana's results and ours.Finally, our simulations may provide new in-

sights into the internal organization of A12-28

fibrils, whose molecular structure is unknown.6

The structural transition from random-coil to -sheet forms the key element in many recenttheories of protein aggregation.4345 Growth of afibril from a pre-formed seed must involve theconversion of the unstructured coiled states popu-lated by monomeric A peptides into conforma-tions rich in -structure, as demonstrated by CDand NMR experiments.7,8,3740 What experimentsfail to tell us, however, is how these structuredseeds, which are required for fibrillization toproceed, are nucleated. The importance of the

nucleation event for the fibrillization process canbe appreciated from recent work by Tycko et al.,46

who showed that A40 peptides can grow intofibrils of differing morphologies, depending on thestructure of the particular seed that happened to

be present in the solution first. While fibrilnucleation that involves a major structural reorga-nization of its constituent monomeric units iscertainly a viable scenario for fibrillization, it isplausible also that the formation of the fibrillarseeds occurs from pre-formed monomers. In thislatter scenario, fibrillar seeds are nucleated fromthe structured states, rather than unstructured

coils, as this entails a less drastic conformationalentropy loss associated with the oligomerization.On the basis of the observation of two classes ofthe most structured monomeric species sampled inthe present simulations, we propose two modelsfor the molecular structure of A12-28 protofila-ments. The schematics of these models are shownin Figure 8. In the first model (Figure 8(a)), the -hairpin states of cluster C1 are assembled side-to-side to form anti-parallel -sheets. Several of these-sheets are required to form a protofilament. Theassembly of protofilaments into fibrils may occurdue to the favorable hydrophobic interactionsamong residues lined up at one side of the -

sheet. This model bears close resemblance to themodel of A11-25 fibrils proposed by Fraser et al.47

Structurally, the two models share the -hairpinmotif folded around the V18F19 -turn, but differG. Tiana, personal communication.

Figure 8. Two possible models of A12-28 protofilaments compatible with the results of the present simulations.Monomeric -hairpins (from cluster C1) align into larger anti-parallel -sheets in model (a), through side-to-side self-assembly. Several -sheets make up a protofilament. In model (b), loop-like A12-28 monomers (from cluster C2) line upon top of each other to form two parallel -sheets. Several of these -sheet complexes self-associate, possibly through theexposed hydrophobic residues, to form a protofilament. In both models, the inter-strand hydrogen bonds run parallel

with the fiber axis.



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in the conformational preferences of their C-terminal segments. While the model described

by Fraser et al. assumes that the entire A11-25peptide adopts a -hairpin conformation,47 in themodel proposed here, the five C-terminal residuesVGSNK are unstructured.

A second model that is compatible with the resultsof the present simulations is shown Figure 8(b). Thismodel envisions that fibril nucleation occurs fromparallel -sheet complexes, built by adding con-formations of cluster C2 on top of each other.Hydrogen bonds stabilizing the complex are formed

between each pair of consecutive monomers and runparallel with the fiber axis. Several -sheet con-structs may be needed to form a protofilament. Thefibril model shown in Figure 8(b) is closely related tothe models proposed recently for full-length A40/42 fibrils.4850 Although our simulations of the

monomeric species do not allow us to favor one ofthe proposed protofilament models over the other,they seem to rule out fully extended conformationsas building blocks of A12-28 protofilaments.Extended conformations may play such a role forsmaller fragments such as A16-22,5157 however,such conformations were scarcely populated in oursimulations and seem unlikely candidates to giverise to a fibril nucleus. Ongoing research in ourgroup aims at probing the stability of the twoprotofilament morphologies proposed here.

Methods

We model A12-28 peptide, amino acid sequenceVHHQKLVFFAEDVGSNK, using the all-atom OPLS forcefield.19 The peptide was solvated in a cubic box filledwith 7059 TIP3P water molecules.20 The length of thesimulation box, 60.4 , was determined in short constantpressure simulations at 280 K, which were equilibrated ata physiological external pressure of 1 atm (=101,325 Pa).We considered the protonation states of all titratableresidues to be appropriate for neutral pH, whichrendered the total charge of the simulated system zero.

The simulations were carried out using GROMACSsoftware.58,59 Covalent bonds of the water moleculeswere held constant by the SETTLE algorithm.60 The bondsinvolving hydrogen atoms of the peptide were constrainedaccording to the LINCS protocol.61 This allowed arelatively long simulation time-step of 2 fs to be employed.Non-bonded Lennard-Jones interactions were tapered,starting at 7 and extending to 8 cut-off. Neighborlists for the non-bonded interactions were updated everyten simulation steps. Electrostatic interactions wereincluded, using the reaction field approach.62 The tem-perature was controlled by the Nose-Hoover algorithm63

with a 0.05 ps time constant. In total, 60 replicas of theoriginal system were considered, at temperatures expo-nentially spaced between 280 K and 600 K. Exchanges ofreplicas at adjacent temperatures were attempted every500 simulation steps. The same time-interval was used toperiodicallysave atomic coordinates. The simulationswerestarted from a random extended conformation, the same

for all replicas, and equilibrated for about 4 ns. Subsequentequilibrium sampling runs were performed over 28 ns.

To classify all sampled conformations into groups ofsimilar/dissimilar states, we performed a clusteringanalysis. The analysis was conducted according to the

gromos protocol,64

using RMS deviations over C

atomsof residues 216 as a measure of structural similarity. Wefound that the two terminal residues V12 and K28 are toomobile to be clustered efficiently. In our analysis,conformations with a mutual RMS< 2 were consideredas belonging to the same cluster.

Acknowledgements

The support of the NSF Career Award #0133504,the A. P. Sloan Foundation, the David and LucilePackard foundation and the Institute for Collabora-tive Biotechnologies through grant DAAD19-03-D-0004 from the U.S. Army Research Office is grate-fully acknowledged. Computational resources of theCalifornia NanoSystems Institute (CNSI) obtainedthrough an NSF grant (CHE-0321368) are alsoacknowledged. Some of the simulations wereperformed using the NSF TERAGRID facilities,through the allocation grant # MCA05S027.

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Edited by F. E. Cohen

(Received 19 April 2006; received in revised form 12 June 2006; accepted 17 July 2006)Available online 21 July 2006


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Baumketner 2006 - Folding Landscapes Abeta 12-28