Changes to the Structure and Dynamics in Mutations of Aβ21–30

Article pubs.acs.org/JPCB

Changes to the Structure and Dynamics in Mutations of Aβ21−30 Caused by Ions in Solution Micholas Dean Smith and Luis Cruz* Department of Physics, Drexel University, 3141 Chestnut Street, Philadelphia 19104, Pennsylvania, United States S Supporting Information *

ABSTRACT: The structure and dynamics of the 21−30 fragment of the amyloid β-protein (Aβ21−30) and its Dutch [Glu22Gln], Arctic [Glu22Gly], and Iowa [Asp23Asn] isoforms are of considerable importance, as their folding may play an important role in the pathogenesis of sporadic and familial forms of Alzheimer’s disease and cerebral amyloid angiopathy. A full understanding of this pathologic folding in in vivo environments is still elusive. Here we examine the interactions and effects of two neurobiologically relevant salts (CaCl2 and KCl) on the structure and dynamics of Aβ21−30 decapeptide monomers containing the Dutch, Arctic, and Iowa charge-modifying point mutations using isobaric−isothermal (NPT) explicit water all-atom moleculardynamics simulations. Measurements of secondary structure populations, intrapeptide hydrogen bonding, salt bridging, secondary structure lifetimes, cation−residue contacts, water− peptide hydrogen bonding, and hydration-shell water residence times reveal a variety of ion and mutation-dependent modifications to the decapeptide’s structure and dynamics. In general, Ca2+ has the effect of increasing coil-state populations and lifetimes, modifying the behavior of the decapeptide’s hydration shell and diminishing intrapeptide hydrogen bonding, while K+ is found to diminish coil populations and lifetimes and, for the case of the Iowa mutant, dramatically increase the decapeptide’s propensity for β secondary structures. Mutation-dependent effects highlight the different roles of the Glu22 and Asp23 residues in either solvating or enhancing turn structures, respectively. Taken together, our results provide insights into the differential roles of different ionic species as well as specific effects on the Glu22 and Asp23 residues of Aβ21−30 mediated by ion− decapeptide interactions and the solvent, which could be important interaction mechanisms relevant to the peptide’s behavior in both in vitro and in vivo environments.

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al.15 with the discovery of a small protease-resistant decapeptide region in the full-length Aβ peptide, the 21−30 amino acid region. The protease-resistant nature of this region has suggested that this decapeptide region may act as a nucleation site for the pathological misfolding of the monomer15 and form a loop/bend16 that helps maintain a surrounding β-hairpin (between amino acids 17−21 and 30−36) with the capability of serving as a possible aggregation site.17,18 Additional study of the 21−30 region when isolated as a fragment, Aβ21−30, has also been demonstrated to be protease-resistant,15 a loop/bend former,19−22 and has the interesting feature of containing a number of important point mutations (at the 22nd and 23rd amino acids) associated with various familial forms of AD (FAD). 23−30 For these reasons, several computational19−22;31−40 and experimental12,15,30,33,41−44 studies have sought to characterize the various structures and dynamics of the wild-type (WT) Aβ21−30 and its point mutations, from which a wealth of information has been found. Some of the

INTRODUCTION Alzheimer’s disease (AD) is characterized by the deposition of amyloid plaques, neurofibrillary tangles, and eventual neuronal death. Initially, the amyloid cascade hypothesis (ACH)1,2 posited that Amyloid-β (Aβ) fibrils formed senile plaques that lead to the neuronal cell death associated with AD. Since the initial development of the ACH, new evidence has indicated that prefibril, low-weight oligomers of Aβ are potent neurotoxic agents3−11 and that they may be responsible for the loss of neuronal function and neuronal cell death associated with AD. This shift toward oligomeric neurotoxic agents introduces a plausible tactic for the development of therapeutics: by targeting dominant structures in the monomeric forms of Aβ that could contribute to oligomerization, it may be possible to prevent individual monomers from undergoing aggregation into the neurotoxic oligomeric forms of Aβ and thus potentially limit the deleterious effects of AD. This tactic, however, requires having a comprehensive understanding of the structure and dynamics of Aβ monomers, which is particularly difficult given that it is an intrinsically disordered protein.12−14 An important contribution toward revealing the structure and dynamics characteristics of Aβ was provided by Lazo et © 2013 American Chemical Society

Received: August 27, 2013 Revised: November 6, 2013 Published: November 8, 2013 14907

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simulations with mutations at positions 22 (Arctic/Dutch) and 23 (Iowa) of Aβ21−30 within aqueous ionic solutions of CaCl2 and KCl salts at various concentrations. These mutants were chosen to further evaluate the previously proposed31 salt− decapeptide interaction mechanisms and to reveal possible saltdependent modifications to the structure and dynamics of these pathologically relevant variants. By measuring secondary structure populations, structure lifetimes, intrapeptide and water−peptide HBs, hydration-shell residence times, and residue−ion contacts, a plethora of modifications to the decapeptide’s structure and secondary structure lifetimes are found to be salt- and mutation-specific. From these measures, this work demonstrates the importance of the Asp23 residue in the stability of the hydrophobic turn between residues 24−27 and additional details concerning the mechanisms behind the CaCl2 specific effects found by Smith et al.31

results from these studies are: the formation of salt-bridges between amino acids 22/23 and 28, dominant hydrogen bonding (HB) between the 23rd and 26/27th residues, and a bend/turn-motif from the 24th through the 28th amino acids attributed to intrapeptide hydrophobic and electrostatic interactions. In addition to these results, Chen et al.22 demonstrated that the conformational space of WT Aβ21−30 could be divided into three distinct families. Meanwhile, the Dutch and Arctic mutants (charge-neutralizing point mutations at position 22) have been shown to exhibit a reduced formation of salt-bridges between amino acids at positions 22 and 28 and reduced stability in the bend/turn-motif between amino acids at positions 24 and 28 compared with the WT form while still preserving much of the WT form’s HB network.33,36,44 The Iowa mutant (charge-neutralizing point mutation at position 23) has been shown to exhibit significantly different behavior and may sample a different conformational space compared with the WT and the Dutch and Arctic mutations.33,34,36,44 Minority conformations of the Aβ21−30 monomer, such as metastable β-turns and β-hairpins, have also been shown to be important in the WT15,31,35,42 decapeptide as well as in Dutch, Arctic, and Iowa mutations.32 The interest in such metastable structures stems from the possibility of enhancing or stalling the peptide’s folding and thus its aggregation by manipulating the behavior of these metastable conformational states.32,45,46 Although the abundance of these structures is small (≤12% for WT, Arctic, and Dutch, and ∼32% for Iowa), lifetimes of the βhairpins are found to be on the order of hundreds of nanoseconds; WT and Arctic forms are found to have lifetimes of ∼100 ns, while the Dutch and Iowa mutants have β-hairpin lifetimes equal to or larger than 500 ns, and as a group these measures may have a correlation to the pathogenesis and severity of AD.32 Recently, computational work by Smith et al.31 has addressed the question of whether neurobiologically important salts have an effect on the structure and dynamics of the WT Aβ21−30. Using molecular dynamics (MD) simulations of the WT Aβ21−30 decapeptide under various aqueous group I/IIAChloride salt environments, this study showed substantial differences in the populations and lifetimes of unstructured conformations and the bend/turn-motif when the decapeptide was exposed to 0.1 to 0.5 M salt concentrations. It was observed that increasing concentrations of CaCl2 (and to some extent MgCl2) increased the populations and lifetimes of unstructured random-coil structures, with an accompanying decrease in intrapeptide HBs. Increasing concentrations of KCl did not significantly alter the distribution of secondary structure populations but increased intrapeptide HBs and lifetimes of turn structures. Another group IA salt tested, NaCl, did not show any effects compared with any of the other salt-rich environments and bulk water conditions tested. The mechanisms behind the previously reported phenomena were attributed to a combination of residue-cation contacts and ion-induced changes to the hydration shell of the decapeptide. However, these mechanisms were found to only adequately explain the increases in coil structures and lifetimes and decreases in HBs by the group IIA salts, while the effects observed under KCl concentrations could not be attributed to the tested contact-based or hydration-shell modification mechanisms. To further evaluate the validity of the previously attributed mechanisms and investigate the responses of the familial mutations under salt conditions, this work uses all-atom MD

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METHODS This study examines and compares three, net-charge zero, point mutations and the WT form of the 10 amino acid (Aβ21−30) decapeptide under KCl and CaCl2 aqueous environments. The primary structure of the decapeptide used is Ala21-X22-Y23Val24-Gly25-Ser26-Asn27-Lys28-Gly29-Ala30, where the point mutations are defined as: X = Gly with Y = Asp (Arctic, E22G), X = Gln with Y = Asp (Dutch, E22Q), and X = Glu with Y = Asn (Iowa, D23N). The decapeptide is modeled using all-atom MD simulations in explicit solvent with the TIP3P47,48 water model, OPLS-AA49,50 force field, and the NPT ensemble. The simulations were performed using the GROMACS 4.0.751−53 package. Temperature and pressure coupling for the performed simulations were obtained through the use of the Berendsen thermo/barostats,51,54 with the simulation temperature and pressure set to 283 K and 1 atm, respectively. The choice of this particular value for the temperature was made to correspond with previous experiments15 and to compare with previous computational work.19,31,32 Aqueous salt conditions were created by first constructing a cubic periodic box with a gap of 0.7 nm between the initial conformation of peptide (and its associated atoms) and the periodic wall and then solvating the simulation box with TIP3P waters. The initial conformation of the peptide used in this construction was an extended randomcoil structure. Because of slight variations in the size of each mutant type, the sides of the box are between 40 and 44 Å in size. To obtain the salt concentrations of 0.1, 0.3, and 0.5 M CaCl2 (and KCl), we introduced dissociated salt ions to the simulation system by replacing a predetermined number of water molecules at random locations. We note that even the smallest ionic concentration used here (0.1 M) is between 45 and 65 times greater55 than typical in vivo extracellular environments. Our use of this and the higher concentrations, however, is justified in two ways: (1) biologically, the environment surrounding the Aβ after its cleavage from the larger amyloid precursor protein (APP) close to the membrane is higher than the average in vivo concentration, and (2) computationally, a higher concentration of salt ions increases the rate of peptide−ion interactions, allowing us to adequately sample these interactions in a reasonable amount of computational time, but as shown below, without increasing the number of peptide−ion contacts per simulation frame. The number of replaced water molecules as well as additional details regarding the simulation system are available as Supporting Information (see SI Table 1). 14908

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second solvation shell of the individual cations and any atom on each amino acid residue. The solvation shells were defined as the location of the second peak in the empirical radial distribution functions of water molecules centered about a dissolved salt ion. The values for K+ and Ca2+ ions used were 0.033 and 0.0306 nm, respectively.31 Water−Peptide Interactions. Calculations of the mean residence lifetimes of water molecules within the decapeptide’s hydration-shell were obtained following the same procedure described in previous work.31 In brief, a summary of this method is as follows: (i) identify individual water molecules within 5 Å of the decapeptide at each time step, (ii) monitor the lifetimes of individual water molecules existing within the hydration-shell boundary, and (iii) compute the average of these lifetimes. In addition to hydration-shell lifetimes, water− decapeptide HBs were also monitored. (See the Hydrogen Bonds section.)

Simulation trajectories were generated using a three-step process: (i) energy minimization of the initial spatial coordinates of the decapeptide, waters, and ions (through steepest descent), (ii) position restraint relaxation, and (iii) generation of production runs. Energy minimization was achieved using 0.1 ps of steepest decent steps with a time step of 1 fs and a maximum of 1000 steps. Position restraint relaxation, using SETTLE56 and LINCS,57 was performed on the decapeptide by generating short (20 ps) simulations with a time step of 2 fs. Finally, production runs were generated using a 4 fs time step (shown in prior work31 to be a sufficient time step for this decapeptide), with positions and energies recorded at 4 ps intervals for 400 ns to match prior work.31 For each mutant (Arctic, Dutch, Iowa), 10 trajectories of 400 ns at each salt concentration (0, 0.1, 0.3, 0.5 M CaCl2 and KCl) were generated. For comparison purposes, 10 WT 400 ns trajectories at each tested salt concentration (0 to 0.5 M KCl and 0 to 0.5 M CaCl2) were used from prior work,15,31 which followed the same simulation methodology used here. The trajectory simulation time lengths of 400 ns were found to provide sufficient sampling of the available phase space by calculating the convergence of the coverage of the (ϕ/ψ) peptide backbone angles58 and verifying that at the conclusion of the simulations this convergence did not substantially increase with simulation time (SI Figure 1). The total simulation time from all forms of Aβ21−30 and concentration combinations used in this work amounts to 112 μs. Secondary Structure Classification and Lifetimes. Secondary structures of the decapeptide were classified as belonging to one of four categories per frame (coil, turn, β, or helix) using a STRIDE-based59,60 classification scheme defined in Smith et al.31 with the measurements implemented through the VMD software package61 for each frame of a given trajectory. The time series obtained from these measurements were then used to calculate the average distribution of secondary structure populations for each mutant and salt concentration combination and lifetimes for the coil, turn, and β structures. To obtain the lifetimes of the coil, turn, and β structures, we broke the secondary structure time series generated for each trajectory into four different binary series, one for each structural category (coil, turn, β, and helix) and an autocorrelation function for each binary series using a maximum lag (the maximum number of frames skipped in the computation of frame to frame correlations) equal to onequarter of the total length of the trajectory was then computed. The autocorrelation functions for each binary series from each trajectory were then truncated at the first region of both positive slope and autocorrelation value below 0.1 or the first zero crossing, whichever occurred first. After these truncations, fitting to an exponential function, exp(−t/τ), was performed for each truncated autocorrelation function, and the corresponding secondary structure lifetime was taken to be the time-constant (τ) multiplied by the frame saving rate (4 ps). Hydrogen Bonds. Intrapeptide and water−peptide HBs were measured using a distance cutoff of 3 Å and a bonding angle of 20° between hydrogen donor−acceptor pairs. Intrapeptide HBs were measured using VMD, while water− peptide HBs were measured using the ‘g_hbonds’ utility provided by GROMACS. Additionally, the number of specific HBs between Glu22/Asp23 and Lys28 were computed using VMD and using the same cutoffs listed above. Cation−Residue Contacts. Contacts between individual residues and cation atoms were defined as overlaps between the

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RESULTS Measurements of the average secondary structure populations, number of intrapeptide HBs and oppositely charged residue− residue HBs, and secondary structure lifetimes for the Arctic, Dutch, Iowa mutants, as well as the WT are presented to show differences, trends, and general effects of the decapeptide with increasing KCl and CaCl2 concentrations. In addition, cation− decapeptide contacts, hydration-shell residence times, and water−decapeptide HB measurements are presented to explore possible interaction mechanisms between the salt environments and the decapeptide. Secondary Structure Populations. In Figure 1, normalized histograms of secondary structure populations are presented for each mutant and WT at each tested concentration of CaCl2 (top) and KCl (bottom). Salient results in this Figure are that the Arctic mutant and WT under increasing CaCl2 concentrations have increasing populations of coil structures and decreasing populations of turn structures. Additionally, at high concentrations (0.5 M) of CaCl2, the Dutch, Iowa, and WT have decreased β structures. Increasing concentrations of KCl provided negligible changes to the Arctic mutant, minor decreases in turn structures, and minor increases in β structures for the Dutch mutant, and significant increases in β structures at high concentrations (0.5M) for the Iowa mutant. Coil secondary structures decreased with increasing concentrations of KCl for all mutants, while they remained unaffected in the WT. Of additional interest in this Figure are the secondary structure tendencies for each mutant under bulk (pure) water conditions (0 M). Under these bulk conditions, we note that when comparing the mutants and WT the largest deviation in secondary structure is found for the Iowa mutant with a pronounced β-structure population. This result is comparable to similarly high β-structure measurements made by Cruz et al.32 for the Iowa mutant. In contrast with the turn, β, and coil structures, helix structures appear with negligible propensity, in agreement with previous work.19−21,31,40 A recent study by Lin et al.,62 however, finds substantial helical content in the decapeptide region of the full-length peptide. A possible explanation is that Lin’s work uses the Amber99SB force-field, which may favor helical structures, as shown by Nguyen et al.63 Intrapeptide HBs. Figure 2 shows the mean number of intrapeptide HBs per frame. From this Figure, it is clear that under bulk conditions the number of HBs per frame is lowest in the Iowa mutant. Also, increasing concentrations of CaCl2 14909

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keep intrapeptide HBs at the bulk value or in some cases slightly increased. Figure 3 shows the average number of HBs per frame between the charged residues Glu22/Asp23 and Lys28 (salt-

Figure 3. Mean HBs per frame between Glu22/Asp23 and Lys28. The dashed horizontal line represents the mean value calculated under pure water conditions. Error bars are standard errors of the mean.

bridges). The data show that bulk water values of salt-bridges per frame are lowest in the Dutch mutant. The data also show that CaCl2, in general, brings down the number of salt-bridges, but when compared with the respective bulk water values, only the Arctic and (to a weaker extent) WT forms of the decapeptide are reduced, and that the amount of this reduction is minimal (∼0.4 HBs/frame) as opposed to the significantly larger value (1 HB/frame) from Figure 2. In addition, the data shows that KCl has a negligible influence on salt-bridges. Secondary Structure Lifetimes. To gauge changes to the dynamics of the decapeptide’s mutants under the bulk, KCl, and CaCl2 rich environments, we computed the mean lifetimes of the turn, coil, and β secondary structures (Figure 4). Focusing first on turn structures, turn lifetimes (top row) under the bulk water conditions are found to be lower in the Iowa mutant when compared with the other mutants and the WT. This would seem to suggest that Asp23 is essential to have the longest turn lifetimes. From the mean values of the turn lifetimes, one can construct the ranking in bulk (although not significant): Arctic > Dutch > WT ≫ Iowa. When looking at the effects of salt, the data indicate that both KCl and CaCl2 have the effect of reducing the turn lifetimes in the mutants relative to their respective bulk values. Whereas these data are consistent with previous findings for CaCl2 in the WT form,31 KCl has the reverse effect. Turning to the measured coil lifetimes in bulk (middle row − Figure 4), the data suggest the ranking of coil lifetimes in the bulk to be Iowa > Dutch ≫ Arctic ≈ WT, which is a reversal of the turn lifetime ranking with the Iowa mutant now on top. In relation to the salts, CaCl2 seems to not change coil lifetimes in the mutants relative to their respective bulk lifetimes in contrast with the WT, where coil lifetimes dramatically increased; while, increasing concentrations of KCl brought all coil lifetimes of the mutants and the WT to approximately the same absolute small value. When compared relative to their respective bulk coil lifetimes, however, the biggest reduction is in the Iowa mutant, followed by Dutch, Arctic, and WT, where there was no significant difference between bulk and KCl coil lifetimes. This comparative decrease also follows the ranking given above.

Figure 1. Histograms of secondary structure for: (a) Top, CaCl2; (b) Bottom, KCl. Error bars are standard error of the mean.

Figure 2. Mean intrapeptide HBs per frame. The dashed horizontal line represents the mean value calculated under pure water conditions. Error bars are standard errors of the mean.

decrease intrapeptide HBs for all mutants and the WT, with approximately the same absolute number of HBs at high concentration. In contrast, KCl concentrations are found to 14910

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Figure 4. Mean secondary structure lifetimes. Each graph in a particular column corresponds to lifetimes of turns, coils, and β structures. Error bars are standard errors of the mean. The dashed horizontal lines represent the mean values calculated under pure water conditions.*Note: Because of the small number of β events that prevented good sampling (see Smith et al.31), the presented β lifetimes for the WT form of the decapeptide correspond to the average of both sporadically formed and preformed β structures, while all other forms of the decapeptide show lifetimes only for sporadically formed β structures.

For the lifetimes of the β structures (bottom row − Figure 4), the bulk results suggest lifetimes following the ranking WT ≈ Iowa ≫ Arctic ≈ Dutch. Of note is that in contrast with previous work,32 the mean values of β-structure lifetimes in bulk are seemingly lower than previously reported. The difference between this work and previous work is that here measurements of lifetimes of all types of β structures are performed together (not individually as in Cruz et al.32), which brings down the resulting value of the lifetimes, and our classification of β structures includes several conformations that have very short lifetimes. When looking at the effect of salts, the data show that for mutants, in general, β-structure lifetimes are at the level of their respective bulk values or above, in contrast with previous work,31 where β structure lifetimes were shown to be largely unaffected by KCl and CaCl2. In relative terms to the bulk conditions, β structure lifetimes are increased with increasing CaCl2 concentrations in the Arctic and Dutch mutants and increased with increasing KCl concentrations in the Dutch and Iowa mutants. Interaction Mechanisms. In seeking to elucidate the mechanisms behind the observed changes, two measures are utilized: (a) ion−residue contacts and (b) water−peptide interactions. The first aims to measure effects in terms of substitution of water by ions, interruption of intrapeptide HB, and screening of salt-bridges. The second aims to measure modifications to the HB behavior and dynamics of the water in the vicinity of the decapeptide (decapeptide hydration-shell water molecules). Ion−Residue Contacts. Ion−residue contacts are measured as the mean number of contacts per frame per residue and are presented in Figure 5. At first glance, this Figure shows that the contacts found under CaCl2 environments are predominately located at the charged residues (locations 22/23), and the charged C-termi (Ala30) of the decapeptide, while for KCl the contacts seem to be uniformly distributed throughout the decapeptide. More importantly, however, the data in this Figure show that whenever there is a contact, the Ca2+ has a

Figure 5. Mean cation−residue contacts between the peptide and (a) Ca2+ and (b) K+. Error bars are standard error of the mean.

significantly higher propensity than K+ to make the ion-residue contact regardless of mutation, with Ca2+ having a maximum number of contacts per frame of ∼0.9, while K+ had a significantly smaller value of ∼0.04. Water−Peptide Interactions. To investigate changes to the dynamics of the hydration-shell water under the salt environments, we provide in Figure 6 the mean residence (lifetimes) of hydration-shell water molecules as a function of salt concentration. Data from the bulk water environments indicate 14911

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Figure 6. Mean hydration shell water residence times. The dashed horizontal lines correspond to the mean values calculated under pure water conditions. Error bars are standard errors of the mean.

that the lifetime of the hydration-shell water is approximately the same across the mutations and the WT; however, as a function of salt, it is clear that the dynamics of the hydrationshell are insensitive to KCl concentrations, while CaCl2 significantly increases the lifetime of waters near the hydration-shell, with the WT having the largest increase, while the increases in the Arctic, Dutch, and Iowa mutants are lower and of roughly the same magnitude. These results suggest that the hydration-shell water under CaCl2 environments becomes less mobile close to the peptide. On examining the mean number of water−peptide HBs per frame, shown in Figure 7, the data show that for bulk the WT

Figure 8. Hydration-shell water-residence times versus mean number of water−peptide HBs. Error bars correspond to standard errors of the mean.

a linear trend (see SI Figure 2). Thus, the hydration-shell water residence time and the number of intrapeptide HBs are similar to the one for water−peptide HBs (see SI Figure 3).

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DISCUSSION This work investigated, using all-atom explicit solvent MD simulations, the interactions of KCl and CaCl2 ionic salts with the Dutch, Iowa, and Arctic point mutations of the Aβ21−30 decapeptide. From the measures of secondary structure populations, intrapeptide and water−peptide HBs, hydrationshell behavior, cation−residue contacts, and secondary structure lifetimes it was found that dynamic and structural properties of the Aβ21−30 decapeptide could be altered as a function of concentration of KCl and CaCl2 salts. First, however, without any salts (at 0 M), it was found that turn lifetimes and the number of intrapeptide HBs were greater in the Arctic and Dutch mutants and the WT than in the Iowa mutant. This trend in these two properties indicates that an intact Asp23 could be a main driver behind intramolecular HBs that form turn structures, which corresponds to a similar observation from NMR data from Grant et al,44 simulations from Cô té et al.,64 and combined computational and experimental work by Rosenman et al.18 It was also found that the WT and Iowa mutant had greater numbers of water− peptide HBs than the Dutch and Arctic mutants, which suggests that an intact Glu22 could mediate water−peptide interactions and thus could be critical to the solvation of the decapeptide. These two properties are complementary because, for example, in the Arctic mutant there is the least number of water−peptide HBs, but it has the longest turn lifetimes. Upon having both Glu22 and Asp23 intact, as in the WT, the effects on the number of water−peptide HBs dominate (having the largest 0 M number of water−peptide HBs), as opposed to a comparatively (relative to Arctic and Dutch mutants) small turn lifetime. This Glu22 versus Asp23 behavior is also present in the secondary structure population measurements, where for the Arctic and Dutch mutants (no Glu22) turn structures still dominate (consistent with the larger turn lifetimes and increased intrapeptide HBs), but for the Iowa mutant (no Asp23) values for turn populations substantially decrease (consistent with the smallest turn lifetimes and decreased intrapeptide HBs). In conclusion, an intact Asp23 (Arctic and

Figure 7. Mean water−decapeptide HBs per frame. The dashed horizontal lines correspond to the mean values calculated under pure water conditions. Error bars are standard errors of the mean.

and the Iowa mutant have a higher number of water−peptide HBs than the Arctic and Dutch mutants, suggesting that Glu22 is important for these interactions. As a function of salt, the data show that KCl has a negligible effect on water−peptide HB interactions, while CaCl2 greatly reduces HBs between the water and peptide in the mutants and the WT. When these two sets of data are combined for CaCl2, as shown in Figure 8, the data reiterate that: (i) mutations have, in general, a lower hydration-shell water residence time than the WT, (ii) mutations also have a lessened number of water− decapeptide HBs, and (iii) increasing the concentration of Ca2+ increases the hydration-shell water residence time while decreasing the water−decapeptide HBs. Because Ca2+ has the same diminishing effect with concentration on intrapeptide HBs as with the water−decapeptide HBs, when plotting the number of HBs as a function of water−peptide HBs we observe 14912

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intrapeptide HBs. However, these mechanisms have differing levels of effectiveness on the structural lifetimes and populations of each mutation. Coil populations only increase modestly in the Arctic and Dutch mutants and seem to stay the same in the Iowa mutant. Coil lifetimes also show upward trends in the Arctic and Dutch mutants but much less than in the WT, while in the Iowa mutant they do not increase. These muted effects in the Arctic and the Dutch mutants could be attributed to the fact that there is one less charged residue in these mutations and thus one less contact center for the Ca2+, as shown in the mean cation−residue contact data. One less contact may also mean that there are fewer Ca2+−water interactions that pin down the hydration-shell water and thus result in a reduced hydration-shell water residence time for the mutations, as shown in Figure 8 (and SI Figures 2 and 3). The lack of effects in the turn and coil lifetimes in the Iowa mutant despite the obvious influence of the Ca2+ on the hydration-shell water residence time and the decreased number of water− decapeptide HBs may be a consequence of the increased role of the formation of β structures that dominate the structural dynamics, as is shown by the highest β-structure lifetimes and their pronounced populations in the Iowa mutant. In contrast with the CaCl2, the mechanisms behind the effects found in the KCl are more elusive. The KCl salt does not have a pronounced effect on the properties of the WT except in the turn lifetime.31 For the mutations, however, KCl is found to reduce coil populations and coil and turn lifetimes while enhancing β-structure populations in the Iowa mutant at high concentrations. These effects are happening without any evidence of cation−decapeptide contacts nor changes to the water−peptide interactions from the bulk environment. In fact, one can rule out electrostatic interactions between the K+ and the peptide because the results shown in Figure 5 clearly illustrate that for the most part the K+ does not discriminate between the charged and noncharged amino acids. It is possible that indirect contact mechanisms are at work, and in particular crowding could be playing a role because the excluded volume by the K+ (plus its solvation water shell) in the neighborhood of the decapeptide (locally) can be as high as 20% at high concentration (SI Table 2, SI Figure 4). For the case of the Ca2+, the volume exclusion is much higher (twice as much per concentration) than in K+, so we cannot rule out that there is also a crowding effect in the Ca2+, but the contact mechanism is so strong in the Ca2+ that it could be possible that any crowding effect takes a secondary role. In summary, this work has shown that the addition of CaCl2 and KCl salts to the environment surrounding the Aβ21−30 WT and three-point mutations can substantially modify the structure and dynamics of this decapeptide. There are mutation specific as well as general effects due to the salts. While in bulk environments decapeptides with an intact Asp23 have enhanced turn structures, adding Ca2+ counters this effect by enhancing coil lifetimes at the expense of turn lifetimes. For the case of an intact Glu22 (Iowa mutant), bulk environments exhibit a reduced turn structure with considerable β structure, and adding K+ further enhances the β-structure lifetimes at high concentration. These results highlight the importance of the Asp23 in the formation of turns and the specificity of the Ca2+ in reversing this trend by targeting the Asp23. On the other hand, the high number of water−peptide HBs in the decapeptides with intact Glu22 may indicate that Glu22 is important to the solvation of the decapeptide. Additionally, the other effects due to the Ca2+ in the WT and mutations are

Dutch mutants and the WT) will ensure that turn structures will have enhanced stability (demonstrated by longer turn lifetimes) and intrapeptide HBs, while an intact Glu22 but no Asp23 (Iowa mutant) is not sufficient to preserve the turn structures, consistent with previous results on Asp23 and its relation to turn stability.18,44,64 When adding salts, it was observed that there were profound changes in many, but not all, of the measured quantities relative to their bulk environments. For example, increasing concentration of salts only moderately modified secondary structure populations for the turn, coil, and β secondary structures but did not change the population rankings for a given mutation. Also, salt bridges were mostly unchanged from their bulk values. When considering each salt separately, KCl was found to be particularly weak in its effects on the Aβ21−30 properties. Specifically, intrapeptide HBs, cation−decapeptide contacts, mean hydration shell water residence times, and mean water− peptide HBs did not exhibit significant changes from their bulk values at any of the concentration values of KCl tested here. However, KCl did have significant effects by increasing βstructure populations in the Iowa mutant (at 0.5M) and on coil structures by: (i) keeping equal or reducing the amount of secondary structure population as a function of concentration and (ii) reducing all coil lifetimes of all mutations and the WT to approximately the same reduced value. These two results clearly indicate a relationship between lifetimes and structure populations. Interestingly, KCl also lowered the turn lifetimes for the mutations while increasing them for the WT. This lowering and increasing do not have a corresponding change in the secondary structure populations as the error bars overlap, but because of this, the data cannot exclude the existence of such a correlation between turn lifetimes and populations. As already reported for the WT,31 CaCl2 displayed a much more pronounced effect on almost all quantities. In particular, in the WT and all tested mutations, CaCl2 environments: (i) kept equal or increased coil populations for all forms of Aβ, (ii) decreased intrapeptide HBs in absolute numbers and relative to bulk water conditions, (iii) decreased water−peptide HBs, (iv) and resulted in long residence times for the hydration-shell water molecules. CaCl2 also had mutation-specific effects as a function of concentration by showing decreasing trends in the turn lifetimes and by displaying increasing trends in the coil lifetimes on decapeptides with an intact Asp23 (Arctic and Dutch mutants and the WT). Again, these results on the lifetimes may have a corresponding behavior in the secondary structure populations of these decapeptides, but overlapping error bars prevent a clear correlation. The data suggest that the mechanisms behind the many changes due to the CaCl2 are similar to those previously reported for the CaCl2 in the WT.31 In that work, the proposed mechanism was that the cation−residue contacts slowed the dynamics of the hydration-shell water molecules through electrostatic interactions that “pinned” the Ca2+ to the charged residues and subsequently interacted with the hydration-shell water. This slowing down in turn hindered the water from being expelled from the inside of the decapeptide, thus precluding the formation of turns and enhancing coil structures. Here all of the same elements are present: a high number of direct cation−decapeptide contacts that increase residence times of the hydration-shell water and decrease the number of water−peptide HBs (the increased cation−water contacts would decrease the number of water−peptide HBs) for all mutations and the WT, with a corresponding low number of 14913

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found to be a result of direct contacts between this cation and the decapeptide, while the effects of the KCl may originate from volume exclusion due to crowding. The results presented here could be of importance in understanding the misfolding, and early aggregation of the full-length Aβ as salts such as the ones tested here is ubiquitous in neurobiological settings in in vivo environments. A full understanding of these phenomena may be of help in advancing prevention and treatment of Alzheimer’s disease.

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ASSOCIATED CONTENT

S Supporting Information *

Additional simulation details, supplementary measurements of hydrogen-bond hydration-shell correlations, and volume exclusion measures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 215-895-2739. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Computational time was partially provided by XSEDE computational resources grant TG-MCB110142. REFERENCES

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