doi:10.1016/j.jmb.2006.07.

032

J. Mol. Biol. (2006) 362, 567579

Folding Landscapes of the Alzheimer Amyloid-(12-28) Peptide

Andrij Baumketner and Joan-Emma Shea
Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA 93106, USA The energy landscape for folding of the 12-28 fragment of the Alzheimer amyloid (A) peptide is characterized using replica-exchange molecular dynamics simulations with an all-atom peptide model and explicit solvent. At physiological temperatures, the peptide exists mostly as a collapsed random coil, populating a small fraction (less than 10%) of hairpins with a -turn at position V18F19, with another 10% of hairpin-like conformations possessing a bend rather than a turn in the central VFFA positions. A small fraction of the populated states, 14%, adopt polyproline II (PPII) conformations. Folding of the structured hairpin states proceeds through the assembly of two locally stable segments, VFFAE and EDVGS. The interactions stabilizing these locally folded structural motifs are in conflict with those stabilizing the global fold of A12-28, a signature of underlying residual frustration in this peptide. At increased temperature, the population of both -strand and PPII conformations diminishes in favor of -turn and random-coil states. On the basis of the conformational preferences of A 12-28 monomers, two models for the molecular structure of amyloid fibrils formed by this peptide are proposed.
2006 Elsevier Ltd. All rights reserved. *Corresponding author

Keywords: protein folding; conformational space sampling; replica exchange molecular dynamics simulations; Alzheimer amyloid- peptide

Introduction
Alzheimer's disease belongs to a class of neurodegenerative disorders1 characterized by the presence of amyloid deposits in the brain.1,2 In the case of Alzheimer's disease, these deposits consist of aggregates 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 the monomeric species in solution remains poorly characterized. Determining the molecular structure of A monomers poses a serious experimental challenge, in large part because of the readiness of these peptides to aggregate. However, an understanding of the mechanisms of peptide self-assembly necessitates a structural characterization of the monomeric form of the peptide. Different monomeric conformations can give rise to different intermediates on (or off) pathway to aggregation,

Abbreviations used: PPII, polyproline II; SASA, solventaccessible surface area. E-mail address of the corresponding author: shea@chem.ucsb.edu

and hence to different aggregate end-products and fibril morphologies. Because investigations of the clinically relevant A peptides are prohibitive,5 model systems consisting of fragments of these peptides are emerging as viable tools for elucidating the general principles underlying protein aggregation. The 12-28 fragment, A12-28 (sequence VHHQKLVFFAEDVGSNK), the focus of this work, is a particularly attractive system, as it is toxic to the cell, forms amyloid fibrils and populates on-pathway aggregation intermediates (ADDLs, paranuclei, etc.) similar to those found for the full-length peptide.6 Recent work by Grslund and co-workers has begun to shed important new light on the nature of the conformations populated by the A12-28 peptide in aqueous solutions.79 Using a combination of spectroscopic probes, including circular dichroism and NMR, those authors showed that the peptide co-exists in a temperature-dependent equilibrium between polyproline II (PPII), -strand and randomcoil conformations. From a theoretical perspective, the nature of the transition from the -helical structure populated in apolar media to a -rich structure in water was probed by Tiana and coworkers through 100 ns simulations.1012

0022-2836/$ - see front matter 2006 Elsevier Ltd. All rights reserved.

568 In the present work, we use enhanced sampling simulation techniques to further probe the nature of the monomeric species populated by the A12-28 peptide over a range of temperatures.1318 The use of efficient sampling techniques is essential in order to obtain a correct statistical description of conformational space for peptides with energy landscapes dominated by high barriers and deep minima. Simulations are an invaluable complement to experiment, capable of providing a detailed atomistic picture of the folding process. In contrast to most experimental approaches that can probe only the highest populated states of a molecule, these simulations provide direct access to the entire conformational space sampled under a given set of conditions. This is especially critical for the study of the monomeric state of this A peptide, which appears to be mostly unstructured under normal physiological conditions, populating only a small fraction of ordered states. Although not detectable by ensemble-averaging experimental techniques such as NMR, structured conformational ensembles with low population may nonetheless play a major role in initiating protein aggregation. We model the A12-28 peptide using an all-atom force field,19 and an explicit water model,20 and perform our simulations using the replica-exchange molecular dynamics method.14,1618 Oursimulations reveal, in agreement with experiment, that the peptide exists predominantly in a collapsed, mostly unstructured state under physiological conditions. We also uncover the presence of structured hairpinlike conformations, co-existing with less structured states. While such states were not discussed in experimental studies, their presence is consistent with the experimental observations reported on this peptide, as well as with other theoretical investigations. Emerging from our studies is the interesting observation that the principle of minimal frustration is violated in the folding of these peptides. Frustration is present both in the hydrophobic forces and in the formation of local structured elements. The folding mechanism of these hairpins, their temperature-dependence, and their role in initiating aggregation into fibrils is discussed.

Alzheimer Amyloid-(12-28) Peptide

coils based on and angles. The results of our analysis at 280 K are shown in Figure 1 for all 17 residues of the peptide. A12-28 is seen to populate mostly random-coil states, that co-exist with PPII, strand and -turn structures. The highest population of PPII states (26%) is observed in the central and C-terminal regions of the peptide, for residues A21E22 and S26N27, respectively. Residues A21E22 were seen in the experiments reported by Grslund et al.9 to populate more PPII structure than any other residue in the central part of the sequence, while no experimental information is available for the secondary structure of the C-terminal part. The other residues of the peptide (with the exception of F19 and G25) exhibit non-vanishing amounts of PPII. A high propensity to form -strand is observed for residues F20V24 (populated 20% of the time) and H14-L17 (15% populated). These two -strand regions are connected by a -turn at V18F19, suggesting the presence of a -hairpin conformation. A non-vanishing propensity to turn formation is seen for the G25N27 and H14Q15 segments. No -turn structure was reported in the experiments reported by Grslund et al.,9 as this secondary structure element was not included in the analysis. Our observation of -turns, however, is consistent with other simulations for A12-28,1012 as well as experiments for a number of other A peptides.2225 A detailed comparison with experimental findings on this peptide is given in a later section. Nature of structured states: -hairpin formation In order to further characterize the structured conformational states of A12-28, we performed a clustering analysis of all conformations visited by the peptide at 280 K. Our analysis (described in Methods) reveals two main families of structures,

Results
Conformations populated by the A12-28 peptide We first turn to an analysis of the conformations populated during the replica exchange molecular dynamics simulations at low temperatures. Secondary structure analysis: co-existence of PPII, random coil, -strand and -hairpin conformations The secondary structure of the conformations sampled in our simulations was assigned using the PROSS protocol. 21 This protocol distinguishes between -helices, -strands, -turns and random
Figure 1. Residue-specific secondary structure of A12-28 at 280 K. The analysis is carried out according to the PROSS protocol,21 which distinguishes five different types of secondary structure: -helix, PPII, -strand, turn and random coil. The population of -helices is vanishing. All other secondary structure types are distributed non-uniformly along the sequence.

Alzheimer Amyloid-(12-28) Peptide

569 state on the folding map. The second most populated ensemble corresponds to conformations belonging to C2. Clusters C1 and C2 have a population of approximately 10%. Two other clusters, noted C3 and C4 in Figure 3 have populations below 5%. Due to the low population of these clusters, we focus the analysis presented here on the structured clusters C1 and C2. Residual frustration in folding Frustration in hydrophobic forces The possible contribution of hydrophobic forces to the stability of the folded states is investigated by considering the solvent-accessible surface area (SASA) of the peptide as a measure of the strength of the hydrophobic forces.26 Hydrophobic forces are known to drive the folding of globular proteins,27 and may play a role in the folding of A12-28. The mean SASA, averaged over all recorded conformations at 280 K, is plotted as a function of the first two principal components PC1 and PC2 in Figure 3(b). All clusters identified have low SASA values, with cluster C1 displaying the lowest value. The low SASA regions correlate well with the highly populated regions in the free energy map shown in Figure 3(a), implying an important role for the hydrophobic effect in

which we refer to as cluster 1 (C1) and cluster 2 (C2), that were sampled more frequently than other conformations. The most representative conformations 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 extending over residues F20D23 and H14L17. The Cterminal D23K28 segment is mostly unstructured. Cluster C1 is stabilized by a number of hydrogen bonds and salt-bridges, including a hydrogen bond between L17 and F20 (defining the turn region) and salt-bridges D23N terminus, D23K28 and K16 E22. Conformations of cluster C2 show a bend at residues L17F19, with strands formed by residues F20D23 and H13K16. Hydrogen bonds stabilizing these structures include interactions H14E22, H13E22, V18K16, F20V18 as well as D23G25, and S26N terminus. A salt-bridge K16E22 is present, as was the case for the C1 structures. In order to probe the relative population of the conformational states sampled by the peptide, we generated a free energy surface for A12-28 as a function of the first two principal components at 280 K. (Figure 3(a)). The centroid of cluster C1 was chosen as the reference state for building the principal components, and is the lowest free energy

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

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Alzheimer Amyloid-(12-28) Peptide

A12-28 are not ideally smooth. Further evidence that the hydrophobic interactions are not well optimized for the most populated states observed in our simulations can be seen from an analysis of the specific hydrophobic interactions in the structured conformations of the peptide. Specifically, hydrophobic residues V12 and V24 form a contact in the conformations belonging to cluster C1, thereby reducing the exposure of hydrophobic surface to the solvent. In contrast, in conformations of the equally populated cluster C2, both residues V12 and V24 fully expose their hydrophobic sidechains to water. The presence of residual frustration in the hydrophobic interactions in A12-28 was observed in earlier simulations.10 Our results are at odds with the principle of minimal frustration,2830 in that we observe a discrepancy between the states favored by the hydrophobic forces and those actually populated in the simulations. The residual sequence frustration observed here may be a hallmark of amyloidogenic peptides that sets these systems apart from more foldable sequences.31 We note that it is possible that current force fields overestimate the degree of frustration that proteins really possess. Frustration in local structured elements: implications for the folding mechanism The clustering and secondary structure analysis presented in the preceding section revealed that the amount of structured states populated by A12-28 is low. We probe here whether shorter segments within this peptide can exhibit a greater extent of structuring than the peptide as a whole, and whether they may play a role in folding the hairpins. We divided the peptide into segments of length N = 414 residues and the resulting conformations were clustered based on mutual RMSD among C atoms. The result of this analysis revealed two segments, each of five residues, with enhanced propensity for structuring: the C-terminal E22-S26 (EDVGS) segment and the V18-E22 (VFFAE) segment located within the central hydrophobic cluster. Representative structures of the most highly populated conformations of both segments are shown in Figure 4(a). Both the VFFAE and EDVGS segments adopt a bend structure as their most highly populated state. Interestingly, the EDVGS bend is identical with the structure adopted by this sequence in the context of the A21-30 peptide.32 The A21-30 shows a bend located at residues 2428, stabilized partly by Coulombic interactions between residues 22 and 28. This peptide is resistant to proteolysis and is

Figure 3. (a) Free energy map (in units of kBT) of the A12-28 peptide at 280 K projected onto the first two principal components PC1 and PC2. The centroid of cluster C1 was used as the reference state to compute the principal components. (b) Mean solvent-accessible surface area (in nm2), averaged over all recorded conformations at 280 K. The data are shown as a function of the first two principal components PC1 and PC2. The location of the four most populated clusters is shown by contour lines.

folding. Interestingly, however, not all conformations with a small exposed surface area are highly populated. For instance, the conformations with small surface area located at approximately (PC1 = 1.1; PC2 = 0.5) delineate the free energy barrier linking the conformations of cluster C3 with the rest of the states visited and are, as a result, not highly populated. The fact that conformations with low SASA values exist that are not populated significantly, implies that the folding landscapes of

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 fragments were seen in our simulations to be most structured. Unfolding of clusters C1 and C2 is seen readily at the higher temperature.

Alzheimer Amyloid-(12-28) Peptide

571

Figure 4 (legend on previous page)

572 believed to nucleate folding of the full-length, A40/42 peptide.33 The most highly populated VFFAE conformations (18%) are present in both clusters C1 and C2, while those of EDVGS (13%) are present in C2 (as well as in some other less highly populated A12-28 clusters), but not in cluster C1. In fact, the EDVGS bend appears to be incompatible with the structures belonging to C1. Indeed, the bend requires the folding of the V24 N27 backbone onto the side-chain of D23, which would disrupt the C1 -hairpins, due to steric clashes between C-terminal and N-terminal atoms. Energetically, the disruption of -hairpins is very unfavorable, as it entails breaking some of the inter-strand hydrogen bonds, in particular the bond between the carboxy oxygen atoms of D23 and the amino group of the N terminus. Thus, it appears that the interactions that stabilize local structured elements in A12-28 are in discord with those that are needed to stabilize global folds assembled from these local structures, another manifestation of the residual frustration present in A12-28. It is interesting to note that foldable proteins can satisfy the principle of minimal frustration, even though their fragments may possess thermodynamically stable traps. Amyloidogenic 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 the C1 and C2 structures. The free energy map for folding of the A12-28 peptide as a function of C RMS deviation over VFFAE and EDVGS residues from the most stable conformations adopted by these segments is shown in Figure 4(a). The folding of C1 conformations follows a primary folding pathway that does not involve the formation of the EDVGS motif. A second, less-populated pathway leads to C1 conformations indirectly, by passing through C2 states. Two major pathways exist for the folding of C2 conformations as well. In the first, the EDVGS motif is formed first and then the central loop is assembled. Alternatively, a restructuring of C1 conformations leads to C2 states through the breaking of the central -turn accompanied by the formation of the EDVGS motif. It is clear from the non-diagonal shape of Figure 4(a) that the EDVGS and VFFAE motifs do not fold concurrently. Effects of temperature on the structure of A12-28 Temperature-dependence of the secondary structure The total amounts of secondary structure as a function 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, (consistent with CD experiments,7,9 and the fact that PPII structures are stabilized by energetic rather than entropic terms34), while the content of -turn and

Alzheimer Amyloid-(12-28) Peptide Table 1. Secondary structure (SS) content of A12-28 as a function of temperature
SS content (%) T (K) 280 297 315 333 354 376 PPII 13.7(2) 13.3(2) 13.0(2) 13.1(2) 13.0(2) 12.3(2) -Strand 10.9(4) 9.2(2) 8.7(3) 8.6(2) 7.1(2) 6.3(2) -Turn 9.2(4) 10.4(2) 10.8(2) 10.9(2) 11.5(2) 11.8(2) Coil 66.4(4) 67.2(3) 67.5(4) 67.3(3) 68.2(3) 69.4(3)

Estimated errors in the first digit after the decimal point are shown in parentheses.

random coil increases. The change in secondary structure content per residue upon raising the temperature is presented in Figure 5(a). It is seen that the general trend for PPII and -strand structure in most residues is to decrease with temperature. In particular, the -strands centered around segments H14K16 and F20V24 lose about 10% in population as the temperature is raised from 280 K to 376 K. Similarly, the PPII population of segment S15N16 and residues H13 and Q15 drops by about 5% following this change in temperature. The highest increase in -turn structure is observed for residues F19F20. Although the change in secondary structure with temperature is small, it is statistically significant, as illustrated in the plots of secondary structure as a function of temperature in Figure 5(b). Nature of the hairpin conformations at high temperatures We now probe more specifically the effect of temperature on the conformations belonging to C1 and C2. At elevated temperatures, structured conformations of both clusters C1 and C2 break up, gradually taking on more flexible states. As the temperature is raised, the population of these clusters drops dramatically from about 10% at 280 K to less than 1% at 376 K. The molecular size of A12-28 is not affected strongly by temperature. The radius of gyration computed over C atoms remains at 0.7-0.8 nm at both high and low temperature. The high-temperature ensembles are not structurally homogeneous and cannot be clustered efficiently. Hence, to gain further insight into the nature of the high-temperature structures of A12-28, we examine the temperature behavior of the local structural motifs VFFAE and EDVGS. The free energy surface as a function of the C RMS deviation over fragment VFFAE (denoted RMSD1), and the C RMS deviation over fragment EDVGS (denoted RMSD2), at 376 K is shown in Figure 4(b). The VFFAE motif does not survive heat-denaturation, its population dropping from 18% at 280 K to 6% at 376 K. In contrast, the conformations of the EDVGS segment are almost unaffected by temperature. Their relative population of 13% at 280 K remains at the same level at

Alzheimer Amyloid-(12-28) Peptide

573

Figure 5. Change in secondary structure content (%) of A12-28 (a) for each residue, as the temperature is raised from 280 K to 376 K and (b) as a function of temperature.

376 K. This difference in temperature behavior may be related to the different nature of forces stabilizing the VFFAE and EDVGS segments. The hairpin topology is retained at high temperatures The nature of the conformations populated at high temperatures are further explored through free energy maps (Figure 6) at 280 K and 376 K plotted as a function of two parameters D1 and D2 quantifying the degree of structuring present in the central -hairpin. The parameter D1 denotes the distance between C atoms of L17 and F20, and characterizes the degree of formation of the L17F20 (LVFF) -turn. The parameter D2 denotes the distance between the C atoms of A21 and K16, and is a measure of the extent to which structure is preserved further away from the turn. At 280 K four major minima in the D1D2 free energy map are observed. 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 C2 conformations). The third minimum (III) is characterized by both large values of D1 and D2 and denotes all the conformations that do not form L17F20 and K16A21 contacts. The fourth minimum (IV) denotes conformations in which the LVFF turn is present but the entire hairpin is not well developed. The different conformations that can possibly be classified according to two distances, D1 and D2, are shown schematically in Figure 6(a), next to the free energy minimum with which they correspond. Increasing temperature results in a strong depopulation of states II and enhanced population of the fully unfolded states (III). More conformations are seen to occupy the halfopen hairpin states (IV) at high temperature, while the total number of conformations with both L17F20 and K16A21 contacts present does not change much with temperature. Taken together, these results

574

Alzheimer Amyloid-(12-28) Peptide

Figure 6. Free energy map (in units of kBT) 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 structural completeness of the LVFF turn while D2indicates how much hairpin-like structure is maintained further away from the turn. The drawings show four different conformations, (I), (II), (III) and (IV), that can be populated by the central KLVFFA segment of A12-28.

indicate that the hairpin-like structures populated at low temperatures do not disappear from the conformational ensembles completely. Rather, they transform into a looser or swollen conformations that still maintain the topology of a hairpin. This is particularly true for the central -hairpin LVFF whose population is not affected by temperature. The presence of hairpin-like conformations populated at high temperature is consistent with the conclusions illustrated by Figure 5 for the temperature-dependence of the secondary structure. Comparison with experiment Grslund et al. have analyzed residue-specific changes of secondary structure content in A12-28 using 1H NMR.9 Dihedral angles evaluated from J-coupling measurements were used to distinguish between different secondary elements, leading to the conclusion that residues V18F20 and V24 populate substantial amounts of -strand structure at 280 K. In our simulations, on the other hand, we find that residues V24 and F20 adopt predominantly -strand conformations at 280 K, while residues V18 and F19 populate mostly -turns and that, as a result, hairpin structures are seen at low temperatures. The apparent discrepancy in the experimental and computational results (i.e. the presence or the absence of hairpins) can be reconciled as follows. The -turn motif, present in our simulation (and missing from the analysis presented by Grslund et al.8) is characterized by a wide range of angles, from 140 to 50,35 and thus 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 of different structural elements. Theoretical angles for residues V18F20 and V24, calculated from our

simulation data are compared to the experimental values shown in Figure 7. In general, we observe a good agreement between theoretical and experimental results. In particular, the experimental and theoretical values coincide within statistical errors for residues V18 and F19 while for residues F20 and V24 our simulations appear to slightly overestimate the -strand content. Whether the structure populates a strand or a -turn may appear to be merely a matter of semantics, we believe that our finding of a -turn in the monomeric state of A1228 may have important implications for the aggregation of this peptide into fibrils. Indeed, it has been noted recently that the amount of -turn content present in other A peptides is positively correlated with the ability of these peptides to aggregate.36 In Discussion and Conclusions, we propose a fibril model based on the hairpin structures seen in our simulations. The differences in temperature-dependence seen experimentally and computationally can also be attributed to insensitivity of the angle overlap as a probe of secondary structure. Experimentally, residues V18F20 and V24 (determined to be strands at low temperature) undergo a structural transition into random-coil states at high temperature. In our simulations, we find that at high temperatures, only residues V18 and V24 undergo a transition to random-coil states, while residue F19 actually becomes more structured, depopulating coil states in favor of PPII, -strand and -turn conformations. For F20, the population of randomcoil structures remains constant, with -strand conformations 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 -turn structure observed in our simulations for F19F20, produces temperature changes in angles

Alzheimer Amyloid-(12-28) Peptide

575

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

consistent with a structural shift toward disordered random-coil states.

Discussion and Conclusions

It is well established that conformations of the Alzheimer amyloid peptides, when incorporated into amyloid fibrils, are rich in -structural motifs such as -strands or -hairpins.4 However the nature of the monomeric forms of these peptides in solution is poorly characterized at an atomically detailed level. Most spectroscopic probes point to a predominantly random-coil conformation in aqueous solutions at a low concentration of peptide and normal temperature and pressure,3740 and the results of our simulations largely support this view. Our simulations indicate that the A12-28 fragment populates a structurally diverse ensemble of conformations, co-existing with one another in a dynamic equilibrium. The conformations populated under physiological temperatures are mostly collapsed coils, with radius of gyration at least half that of a fully extended polypeptide chain. The molecular size of the populated states is largely unaffected by temperature, a result that is in-line with the finding reported by Grslund et al., who concluded that the hydrodynamic radius of A12-28 increases by less than 4% as the temperature is raised from 0 C to 25 C.8 Approximately 20% of the conformations sampled by the peptide at 280 K are seen to populate structures with a hairpin topology. Half of these possess a turn located at L17F20 and constitute true hairpins, while the other half possess a bend in this region and correspond to hairpin-like loops. The

conformations belonging to these two classes are very similar in terms of their size, as evaluated by radius of gyration and end-to-end distance. Experimentally, the monomeric conformations of A12-28 in aqueous solution were characterized by Grslund et al. using NMR and CD spectroscopy.79 Focusing primarily on the secondary structure of the peptide, these authors found that A12-28 monomers exist in equilibrium between -strand, PPII and randomcoil conformations. At increased temperature, the total amounts of -strand and PPII structure were seen to decrease in favor of random-coil states.9 Our simulations, on the other hand, report the presence of a hairpin at low temperatures, with increased turn structuring of the F19 residue. Our results are consistent with those reported by Grslund et al.: the difference in secondary structure assignment results from differences in resolution of the secondary structural probes used. (The angles used in the experimental assignment do not distinguish between -strands and -turns.) Our observation of -strands and -turns is in agreement with previous studies on various A peptides.22,23,41,24,25 In particular, -hairpins were seen in a number of earlier simulations on A12-28.1012 A transition from an -helical state of A12-28, the native state of this peptide in apolar medium, to a -hairpin was followed in 100 ns constant-temperature molecular dynamics simulations by Daidone et al.,10 and by Simona et al.11 The most frequently sampled conformation in the work by Daidone et al. was a -hairpin characterized by a type II turn located at residues F19F20A21E22.10 In addition to the hairpin, a compact bend intermediate was found, stabilized by K16E22 and K16D23 saltbridges and close-packing of the 1721 (LVFFA)

576

Alzheimer Amyloid-(12-28) Peptide

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 selfassembly. Several -sheets make up a protofilament. In model (b), loop-like A12-28 monomers (from cluster C2) line up on top of each other to form two parallel -sheets. Several of these -sheet complexes self-associate, possibly through the exposed hydrophobic residues, to form a protofilament. In both models, the inter-strand hydrogen bonds run parallel with the fiber axis.

cluster of hydrophobic residues. While our results agree with the previous simulations,1012 in that hairpins constitute a significantly populated conformational ensemble, we observe some discrepancy regarding details of the structural nature of A12-28. In particular, the -turn seen in the present work is located at positions L17V18F19F20, which represents a two residue shift along the sequence toward the N terminus with respect to the -turn of the earlier simulations.1012 We note that the -hairpins sampled in these simulations were visited in our simulations (with a C RMS2 from the most representative state) but they were not populated significantly. In addition, the temperature-dependence of the hairpins seen here differs from that seen by the same authors, who reported a non-monotonic tem-perature-dependence.12 In their simulations, the hairpin population, negligible at 273 K, rose to a maximum value of 90% at 315 K, before decreasing to levels of 60% as the temperature was raised further. In contrast to these observations, our simulations indicate clearly that the population of both hairpins (C1) and hairpin-like (C2) structures in A12-28 decreases in a monotonous manner with temperature, from 20% at 280 K, to less than 1% at 376 K, without any increase in population at an intermediate temperature. The observed differences between our results and those reported by Tiana et al.1012 are likely due to differences in simulation methodologies (NVT ensemble simulations1012 and the replica exchange simulations performed here) as well as to differences in force fields. Different force fields have different propensities for the formation of secondary structure elements,42 a factor that could very well account for the discrepancies between Tiana's results and ours. Finally, our simulations may provide new insights into the internal organization of A12-28 fibrils, whose molecular structure is unknown.6
G. Tiana, personal communication.

The structural transition from random-coil to sheet forms the key element in many recent theories of protein aggregation.4345 Growth of a fibril from a pre-formed seed must involve the conversion of the unstructured coiled states populated by monomeric A peptides into conformations rich in -structure, as demonstrated by CD and NMR experiments.7,8,3740 What experiments fail to tell us, however, is how these structured seeds, which are required for fibrillization to proceed, are nucleated. The importance of the nucleation event for the fibrillization process can be appreciated from recent work by Tycko et al.,46 who showed that A40 peptides can grow into fibrils of differing morphologies, depending on the structure of the particular seed that happened to be present in the solution first. While fibril nucleation that involves a major structural reorganization of its constituent monomeric units is certainly a viable scenario for fibrillization, it is plausible also that the formation of the fibrillar seeds occurs from pre-formed monomers. In this latter scenario, fibrillar seeds are nucleated from the structured states, rather than unstructured coils, as this entails a less drastic conformational entropy loss associated with the oligomerization. On the basis of the observation of two classes of the most structured monomeric species sampled in the present simulations, we propose two models for the molecular structure of A12-28 protofilaments. The schematics of these models are shown in Figure 8. In the first model (Figure 8(a)), the hairpin states of cluster C1 are assembled side-toside to form anti-parallel -sheets. Several of these -sheets are required to form a protofilament. The assembly of protofilaments into fibrils may occur due to the favorable hydrophobic interactions among residues lined up at one side of the sheet. This model bears close resemblance to the model of A11-25 fibrils proposed by Fraser et al.47 Structurally, the two models share the -hairpin motif folded around the V18F19 -turn, but differ

Alzheimer Amyloid-(12-28) Peptide

577
for all replicas, and equilibrated for about 4 ns. Subsequent equilibrium sampling runs were performed over 28 ns. To classify all sampled conformations into groups of similar/dissimilar states, we performed a clustering analysis. The analysis was conducted according to the gromos protocol,64 using RMS deviations over C atoms of residues 216 as a measure of structural similarity. We found that the two terminal residues V12 and K28 are too mobile to be clustered efficiently. In our analysis, conformations with a mutual RMS < 2 were considered as belonging to the same cluster.

in the conformational preferences of their Cterminal segments. While the model described by Fraser et al. assumes that the entire A11-25 peptide adopts a -hairpin conformation,47 in the model proposed here, the five C-terminal residues VGSNK are unstructured. A second model that is compatible with the results of the present simulations is shown Figure 8(b). This model envisions that fibril nucleation occurs from parallel -sheet complexes, built by adding conformations of cluster C2 on top of each other. Hydrogen bonds stabilizing the complex are formed between each pair of consecutive monomers and run parallel with the fiber axis. Several -sheet constructs may be needed to form a protofilament. The fibril model shown in Figure 8(b) is closely related to the models proposed recently for full-length A40/ 42 fibrils.4850 Although our simulations of the monomeric species do not allow us to favor one of the proposed protofilament models over the other, they seem to rule out fully extended conformations as building blocks of A12-28 protofilaments. Extended conformations may play such a role for smaller fragments such as A16-22,5157 however, such conformations were scarcely populated in our simulations and seem unlikely candidates to give rise to a fibril nucleus. Ongoing research in our group aims at probing the stability of the two protofilament morphologies proposed here.

Acknowledgements
The support of the NSF Career Award #0133504, the A. P. Sloan Foundation, the David and Lucile Packard foundation and the Institute for Collaborative Biotechnologies through grant DAAD19-03-D0004 from the U.S. Army Research Office is gratefully acknowledged. Computational resources of the California NanoSystems Institute (CNSI) obtained through an NSF grant (CHE-0321368) are also acknowledged. Some of the simulations were performed using the NSF TERAGRID facilities, through the allocation grant # MCA05S027.

References
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Methods
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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