Insights from Molecular Dynamics Simulations - ACS Publications

Aug 6, 2013 - Deparment of Physics, University of Cyprus, PO20537, CY1678, Nicosia, Cyprus. •S Supporting Information. ABSTRACT: Specific ion effect...
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Helix Formation by Alanine-Based Peptides in Pure Water and Electrolyte Solutions: Insights from Molecular Dynamics Simulations Filippos Ioannou,† Epameinondas Leontidis,*,† and Georgios Archontis*,‡ †

Department of Chemistry, and ‡Deparment of Physics, University of Cyprus, PO20537, CY1678, Nicosia, Cyprus S Supporting Information *

ABSTRACT: Specific ion effects on oligopeptide conformations in solution are attracting strong research attention, because of their impact on the protein-folding problem and on several important biological−biotechnological applications. In this work, we have addressed specific effects of electrolytes on the tendency of oligopeptides toward formation and propagation of helical segments. We have used replica-exchange molecular dynamics (REMD) simulations to study the conformations of two short hydrophobic peptides [Ace-(AAQAA)3-Nme (AQ), and Ace-A8-Nme (A8)] in pure water and in aqueous solutions of sodium chloride (NaCl) and sodium iodide (NaI) with concentrations of 1 and 3 M. The average helicities of the AQ peptide have been analyzed to yield Lifson−Roig (LR) parameters for helix nucleation and helix propagation. The salt dependence of these parameters suggests that electrolytes tend to stabilize the helical conformations of short peptides by enhancing the helix nucleation parameter. The helical conformations of longer oligopeptides are destabilized in the presence of salts, however, because the helix propagation parameters are reduced by electrolytes. On top of this general trend, we observe a significant specific salt effect in these simulations. The hydrophobic iodide ion in NaI solutions has a high affinity for the peptide backbone, which reflects itself in an enhanced helix nucleation and a reduced helix propagation parameter with respect to pure water or NaCl solutions. The present work thus explains the computational evidence that electrolytes tend to stabilize the compact conformations of short peptides and destabilize them for longer peptides, and it also sheds additional light on the specific salt effects on compact peptide conformations.

1. INTRODUCTION

useful models for the conformational preferences of the protein backbone in pure water and electrolyte solutions. Even though the addition of salt did not have a noticeable impact on the conformational properties of the dipeptide,33 it enhanced the fraction of tetrapeptide “α-helical” conformations and increased the formation probabilities of the α-helical hydrogen bond (i/i + 4) and other hydrogen bonds (i/i + 3, i/i + 2). The tetrapeptide result is seemingly at odds with simulations of the longer and more complex “neutral” EK peptide Ace-AEAAAKEAAAKA-Nme,21 which indicate a destabilization of helical conformations in the presence of high (2.5−3 M) NaI concentration. This destabilization was mainly attributed to an enhanced sodium affinity for the peptide carbonyl groups in the presence of iodide, due to the affinity of the latter for the hydrophobic Ala side chains. Other electrolytes (KF, KCl, NaCl, NaClO4, GdmCl) were also found to reduce the helicity of this peptide.34 Fedorov studied the conformational preferences of trialanine in sodium halide aqueous solutions and concluded that NaF tends to favor compact over extended conformations with respect to the situation in pure water, whereas NaCl, NaBr, and NaI in fact favor extended conformations.20,25 For peptides with a net

Proteins and peptides exist and function in living systems in a “sea” of electrolytes. A considerable amount of (largely experimental) work in the 1980s and 1990s investigated salt effects on peptide conformations in aqueous solutions.1−13 With the increase of computational power in recent years, a new interest in computer investigations of ion−peptide interactions in solution is now emerging. Numerous computational studies employ model oligopeptides and related compounds to assess specific ionic interactions with the peptide backbone and side chains, and their impact on secondary structure. Significant insights about this complex problem have started emerging from this work.14−38 We recently studied by MD simulations33 the conformational properties of two alanine-based peptides in pure water and in a range of aqueous NaCl and NaI solutions: the dipeptide AceAla-Nme and the tetrapeptide Ace-Ala3-Nme, with Ace and Nme denoting, respectively, the blocking groups CH3CO− and −NHCH3. The dipeptide contains one pair of backbone dihedral angles (φ,ψ) and two peptide groups and is a prototypical model of the protein backbone;26,39−42 the tetrapeptide has three (φ,ψ) pairs and four peptide groups and can form a full α-helix turn with an i/i + 4 α-helical hydrogen bond between the Ace CO group and the Nme NH group. Thus, despite their small sizes, both peptides constitute © 2013 American Chemical Society

Received: June 24, 2013 Revised: August 4, 2013 Published: August 6, 2013 9866

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Scheme 1. Peptide Ace-(AAQAA)3-NMe

same sequence (with a different C-terminal blocking group) [Ace-(AAQAA)3-NH2] and the closely related peptide Ace(AAQAA) 3 -Y-NH 2 were shown to adopt an α-helical conformation in NaCl solutions, with experimental stabilities (average helicity) that depend on the ionic strength.2 In addition, the same sequence (with −NHCH3 or an −NH2 Cterminal blocking group) has been employed in several computational studies of the helix/coil equilibrium in implicit and explicit solvent, but not in the presence of salts.15−17,31,32 We also study the alanine homopolymer Ace-Ala8-Nme (A8), which is a longer version of the di- and tetrapeptide studied earlier.33 We use sodium chloride (NaCl) and sodium iodide (NaI) as electrolytes. In our previous study of the alanine tetrapeptide, both salts tended to stabilize the formation of an α-helical turn.33 In general, NaCl is expected not to give strong specific salt effects, while iodide is a chaotropic anion and can be used to illustrate potential side-chain or backbone interactions of the more hydrophobic ions. A point that distinguishes the present work is our use of Lifson−Roig theory56 to obtain average helix nucleation and helix propagation parameters under various electrolyte conditions. Such calculations have been performed for peptides in pure water so far (ref 35 and references therein). By applying them to the study of salt effects, we hope to gain insights beyond those provided by pair correlation functions alone.

charge, on the other hand, salts appear to enhance the propensity for helical conformations, as was demonstrated by Asciutto et al.27 for the oligopeptide AAAAA(AAARA)3A and by Crevenna et al.34 for the oligopeptides (AK)6 and (AE)6. The combined experimental and computational work of refs 37 and 38 has illustrated that salt effects on peptide conformations can be quite complex, involving ion hydration, ion pairing, and specific ion−peptide group interactions. As hinted by the results discussed above, several factors could determine salt impact on helix stability. Force-field effects must always be assessed in similar investigations. Our previous simulations employed the all-atom CHARMM22 force field43 with the grid-based torsional (CMAP) correction,44,45 the TIP3P water model,46,47 and ion parameters from Joung and Cheatham.48 Dzubiella21,24,29,30,34 used the ff03 force field for the peptide,49 the TIP3P water model, and the Dang parameters for the ions.50 Asciutto et al.22,27 used the ffSB99 force field for the peptide,51 the TIP3P water model, and perchlorate ion parameters from Baaden et al.52 In his trialanine work, Fedorov used the OPLSAA-2001 force field for the peptide and ion parameters53 and the TIP5P-EW water model.54 Leaving aside the question of force field effects, there are two important open questions from the previous work regarding the structures of the peptides themselves: (a) Could salt effects on peptide helicity be different for short and long peptides? Differences for the relative stabilization of helical conformations in pure water, depending on the length of the alanine peptide, were already observed in the older simulations of Wang et al.14 Traditional statistical-mechanical models of the helix/coil transition55−57 make a distinction between helix initiation and elongation, based on the fact that these two processes are associated with different free energy changes (and corresponding entropic and enthalpic components). The addition of ions may affect these processes in a different manner, introducing a further complexity in the dependence of helix stability on the peptide length. To address this question, we simulate salt effects on alanine-based peptide models, whose behavior is mainly determined by interactions of the solution with the peptide backbone and simple side chains. (b) To what extent are side-chain charges important in the stabilization or destabilization of helices in the presence of salts? To address this question, it is necessary to simulate peptides with charged side chains.21,29,30,34 We mention some preliminary findings in the discussion section, but we address this more complex question in a forthcoming publication. To examine the impact of salts on the stabilization of helical sequences, we study here in detail the peptide Ace-(AAQAA)3Nme (AQ). This is an important model peptide, because the

2. METHODOLOGY Simulation System. The peptide Ace-(AAQAA)3-NMe (AQ) is shown in Scheme 1; Ace (CH3CO−) and NMe (−NHCH3) are respectively the N- and C- terminal blocking groups, A is alanine and Q is glutamine. We use sodium chloride (NaCl) and sodium iodide (NaI) as electrolytes, as stated before. To obtain a more complete picture of the helicity dependence on peptide length, we perform additional simulations of the alanine nonapeptide Ace-Ala8-Nme (A8) in pure water and a NaI solution. Force Field. We investigated the conformational properties of peptides AQ and A8 in a range of electrolyte concentrations. We represented the solution environment by a periodically replicated octahedral box of water molecules and a number of ions reproducing the desired salt concentration. Table 1 lists the total number of ions, water molecules, and water-box sizes for the various peptide simulations. Although the actual salt concentrations are listed in the table, for simplicity we will designate them as “1 M” or “3 M” concentrations in the ensuing discussion. We conducted all simulations with the AMBER 11 simulation package.58 The peptide atomic charges, van der 9867

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316.2, 321.3, 326.5, 331.8, 337.2, 342.7, 348.3, 354.1, 360.1, 366.1, 372.4, 378.0, 385.3, 392.0, 398.9, 406.0, 413.2, 420.7, and 428.4 K. The simulation length in this case was also 30 ns per replica. Secondary Structure Calculations and Conformational Analysis. Definition of Helical States. The computation of the peptide helicity was based on the Lifson−Roig (LR) model.56 In the LR definition, a residue i is considered α-helical if and only if its backbone torsional angles (φi,ψi), and the pairs (φi−1,ψi−1) and (φi+1,ψi+1) of the adjacent residues fall in the αhelical region of the Ramachandran map (φ = −65 ± 35°, ψ = −37 ± 30°). A helical segment must contain at least two successive residues in helical conformation; a single helical residue between residues in a coil (nonhelical) state is not counted as a helical segment. Calculation of Lifson−Roig (LR) Parameters. The LR model characterizes the helix−coil transition in terms of a helix nucleation parameter (v) and a helix propagation parameter (w).56 Residues in a coil state are assigned a weight of 1. Residues in a helical segment are assigned weights v and w when located, respectively, at the two boundaries or the interior; values v ≪ w reflect a free-energy penalty associated with the start of a helix. The total statistical weight of a given conformation is the product of weights of the individual residues. The partition function for a blocked peptide with n amino acid residues can be written in a compact matrix form as18,56

Table 1. Composition of Simulated Solutions solution 0M 1.1 M 3.2 M 1.1 M 3.0 M

NaCl NaCl NaI NaI

0M 3.0 M NaI

no. of waters 2623 2524 2491 2557 2556 1282 1259

no. of cations Peptide AQ 0 54 160 54 160 Peptide A8 0 80

no. of anions

box length (Å)

0 54 160 54 160

47.32 46.92 47.38 47.60 49.04

0 80

37.27 38.78

Waals, and stereochemical parameters were taken from the ff03* force field.23 Ion parameters, optimized for highconcentration simulations, were taken from the work of Joung and Cheatham.46 We employed these parameters in our previous work with the shorter alanine peptides.33 Water molecules were represented by the TIP3P model,47,48 which is consistent with the peptide and ion parameters used in this work. Simulation Protocol. Starting from a helical conformation, the peptide was first minimized, heated up gradually from 0 to 300 K in six 30 ps steps (every 50 K), and then subjected to a 50 ns production simulation at 300 K with the Hawkins/ Crammer/Truhlar GB model.59 From this simulation, we extracted the lowest-energy structure and solvated it in a truncated octahedron simulation cell with the appropriate number of TIP3P water molecules and ions (Table 1). Prior to the simulation, we alleviated bad contacts by energy minimization. Next, we heated the system from 0 to 300 K by a 20 ps MD run at constant-volume conditions with weak harmonic restraints on the peptide, switched off the restraints, and conducted an 1 ns run at 300 K under constant pressure of 1 atm, with a pressure relaxation time of 2 ps. Using the average cell size of this constant-pressure run, we conducted 0.5 ns runs at constant volume conditions, with temperatures in the range T = 280−520 K (in 20 K intervals). We computed the mean potential energies of these systems and used the method of ref 60 to calculate optimal temperatures for replica-exchange runs, targeting 15−20% exchange probability between neighboring replicas. We finally employed 48 replicas with temperatures 293.2, 296.3, 299.6, 302.9, 306.2, 309.6, 313.0, 316.5, 320.0, 323.6, 327.2, 330.9, 334.7, 338.5, 342.4, 346.3, 350.3, 354.3, 358.5, 362.7, 366.9, 371.3, 375.7, 380.2, 384.8, 389.4, 394.2, 399.0, 403.9, 408.9, 414.0, 419.2, 424.6, 430.0, 435.5, 441.2, 447.0, 452.9, 458.9, 465.1, 471.4, 477.9, 484.6, 491.4, 498.4, 505.6, 513.0, and 520.6 K. The temperature was controlled by Langevin dynamics with a collision frequency of 1.0 ps−1. In order to prevent possible trans−cis conformational changes at the highest temperatures of the replica simulations, we applied chirality restraints on the peptide bonds. Long-range electrostatic interactions were calculated by the PME method61 with a 0.99 Å grid spacing and a 9 Å cutoff. The simulations were performed at constant-volume conditions with a 2 fs time step, and had a total length of 1.44 μs (30 ns per replica). Exchanges between neighboring replicas were attempted every 1 ps, and the obtained exchange probabilities were 16−18%. All replicas performed random walks in the temperature space, spanning several times the entire range of temperatures. For the simulations of the A8 peptide we employed 24 replicas with temperatures 292.5, 297.0, 301.7, 306.4, 311.3,

⎛0⎞ Z = (0 0 1)M ⎜ 1 ⎟⎟ ⎝1 ⎠ n⎜

(1)

where the weight matrix M is defined as ⎛w v 0⎞ ⎜ ⎟ M = ⎜0 0 1 ⎟ ⎝v v 1 ⎠

(2)

The average fractional helical content (⟨nH⟩) and the average number of helical segments (⟨nS⟩) are linked to the LR helix nucleation (v) and propagation (w) parameters by the following equations: ⟨nH⟩ =

1 w dZ , n − 2 Z dw

⟨nS⟩ =

v12 dZ Z dv12

(3)

In eq 3, v12 is the element in the first row and second column of matrix M. The MD simulations can be used to evaluate the expectation values ⟨nH⟩ and ⟨nS⟩. Then, eqs 1−3 can be used to derive v and w values consistent with these quantities through an appropriate fitting procedure. Recent studies23,35 point out that LR (and Zimm−Bragg) parameters from simulation and experiment are difficult to compare. Even so, a comparison of these quantities for the same peptide at different temperatures or salt conditions can provide a valuable additional analysis tool. The LR parameters of eqs 1−3 are linked to the Zimm− Bragg (ZB) helix parameters55 by the following relations:57 s=

w , 1+v

σ=

v2 (1 + v)4

(4)

The temperature dependence of the Zimm−Bragg parameter s and the following equation18,23 9868

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Figure 1. Temperature dependence of the average helicity for peptide AQ.

nucleation and propagation steps, thus introducing a dependence of helical stability on the peptide length. 3.2. Impact of Salt and Temperature on the LR Propagation and Nucleation Parameters. We examine in more quantitative detail the effects of salt and temperature on helicity by analyzing the helix propagation (w) and nucleation (v) Lifson−Roig parameters56 of peptide AQ. The obtained values correspond to averages over the AQ sequence. Figure 2 displays the temperature dependence of the LR parameters for the various simulated solution. The strong similarity between the propagation (upper plot of Figure 2) and helicity curves (Figure 1) indicates that the helicity is

−kB ln s(T ) = ΔH(T0) − T ΔS(T0) + ΔCp(T − T0) − T ΔCp ln

T T0

(5)

provide a thermodynamic model for helix elongation. Equation 5 assumes a temperature-independent heat capacity change ΔCp.

3. RESULTS In what follows, we describe in detail the results for the longer peptide Ace-(AAQAA)3-Nme (AQ) used in this work. When examining the helicity dependence on peptide length, we also present results for Ace-Ala8-Nme (A8). 3.1. Impact of Salt on Helicity. Figure 1 displays the temperature dependence of the average AQ helicity in the various solutions considered in the present work. In pure water the helicity is 18.7% at 299.6 K, in excellent agreement with the result of Best and Hummer23 for the related peptide Ace-(AAQAA)3-NH2 with the same force field (18.8% at 303 K). The helicity decreases with temperature, reflecting the gradual stabilization of unstructured conformations. As in other work,23,62 this reduction is more gradual than the experimental observations, which suggest a loss of helical populations above ∼350 K. Best and Hummer attributed this discrepancy to force-field inaccuracies, noting that the computed enthalpic and entropic contributions to helix propagation were ∼50% of the corresponding experimental values.23 The helicity changes with salt type and concentration. This dependence is more apparent at lower temperatures (below ∼380 K) and higher concentration. Interestingly, the 3 M results show clearly that the two salts have opposite impact: NaCl weakly promotes and NaI clearly disfavors the formation of helices. An analogous strong destabilization of helical conformations by NaI, and a mild stabilization by NaCl, were reported in a simulation study of the peptide Ace-(AE)6Nme.24 Our earlier simulations with the alanine tetrapeptide Ace-Ala3-Nme,33 as well as results with the A8 peptide (see below), demonstrate a stabilization of helices by NaI. As shown in the next section, salts have an opposite impact on the helix

Figure 2. Temperature dependence of the LR propagation (upper plot) and nucleation (lower plot) parameters for peptide AQ. 9869

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Figure 3. Profile of the main-chain CO group probability (%) to participate in internal α-helical bonds. The last three CO groups of the C-terminal end are omitted, as they cannot participate in α-helical hydrogen bonds.

parallel alignment of four peptide groups, which takes place during the initiation of an α-helical structure (formation of the first α-helical bond), is disfavored electrostatically due to repulsive interactions among the peptide group dipole moments; screening of electrostatic interactions by salt should facilitate this process, in line with the increase of parameter v. This stabilization of α-helical conformations by salt was predicted theoretically by Kirkwood,63 on the basis of interactions between the electrolyte ions and the helix macrodipole. This prediction was verified experimentally by Baldwin and co-workers for the peptide Ace-(AAQAA)3YNH2.2 Inspection of Figures 1 and 2 shows that the net average helicity depends on the interplay between nucleation and propagation parameters. The destabilization of helical conformations in 3 M NaI (relative to 0 M) arises mainly from the significant reduction in the propagation parameter, despite the concomitant increase of the nucleation parameter (at lower temperatures). In the presence of 3 M NaCl and 1 M NaI, both parameters increase (with respect to pure water), in accordance with the overall stabilization of helical conformations (Figure 1); this is especially apparent for 3 M NaCl in the entire temperature range. Finally, in the presence of 1 M NaCl the propagation parameter remains unaltered, yielding a similar helicity as in pure water. The increase in the nucleation parameter (Figure 2) suggests that salts promote helix initiation. This seems to be a general effect, and could be attributed, as mentioned before, to the screening of electrostatic interactions, which facilitates the alignment of peptide-bond dipoles during helix initiation. At least for low temperatures (with the exception of the 3 M NaI system), this effect appears to depend little on salt concentration, in agreement with the recent observation of Dzubiella that beyond 0.5 M salt the screening is essentially complete.34,64,65 The decrease in the propagation parameter (Figure 2) suggests that salts tend to hinder helix elongation. This behavior seems to be dependent on salt type and concentration (and the peptide sequence). For our model system, it is especially prominent in the 3 M NaI solution. In the LR formalism, the propagation parameter is related to the free-energy change due to the addition of a helical hydrogen bond in a long, pre-existing helix.56 On the other hand, the

dominated by the propagation parameter for peptides of this length. The value of w in pure water is 1.08 at 299.6 K, in very good agreement with the computational estimate of Best and Hummer (1.10) for the same peptide.23 Experimental estimates for related peptides are somewhat larger. For example, Scholtz et al. obtained an average value of 1.44 at 273 K for the sequence Ace-Y(AEAAKA)n-FNH2, with n = 2−6;3 Rohl and Baldwin obtained a value of 1.58 for peptide Ace-(AAKAA)n-YNH2 at 278 K (n = 1−10);5 Chakrabartty et al. obtained a value of 1.61 for alanine residues in model peptides at 273 K and 1 M NaCl.7 Best and Hummer23 as well as Vitalis and Caflisch35 have noted recently that LR (and ZB) values obtained from simulations and experiment are based on different assumptions and are hence not directly comparable to each other. The addition of NaI at high concentration (3 M) reduces noticeably the propagation parameter in the entire range of simulated temperatures. At smaller NaI concentration (1 M), or in the presence of NaCl, the effect on this parameter is much weaker. We present an interpretation of this behavior of w later, using radial distribution functions as measures of affinities of the various solution components (water, salt, and peptide). The temperature dependence of the nucleation parameter is displayed in the lower plot of Figure 2. The value in pure water at 299.6 K is 0.214, in good agreement with the values 0.18− 0.21 obtained by Best and Hummer for the same peptide.23 As in other computational applications of the LR model, the v values are an order of magnitude larger than the experimental estimates of 0.03−0.05 at 0 °C,9,11 due to the sensitivity of v on the chosen dependence of the circular dichroism signal on helix length12,23 and limitations in the LR model.35 The results in Figure 2 imply that temperature and salt type have markedly different impact on the nucleation and propagation parameters. The propagation parameter decreases monotonically with temperature. The nucleation parameter increases up to ∼350−380 K (except in 3 M NaI, where it maintains an approximately constant large value), and decreases slowly afterward. A similar temperature dependence of these parameters was found in the simulations of Ace-(AAQAA)3NH2 in pure water, reported in ref 23. The nucleation parameter increases in the presence of salts. This increase can be understood as follows: The approach and 9870

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Figure 4. Na−O rdf between sodium and the oxygen atoms of the peptide main-chain CO groups, for the NaCl (a) and NaI (b) solutions.

Figure 5. Upper panels: Rdf between the anion and main-chain amino-group hydrogen, for the NaCl (a) and NaI (b) solutions. Lower panels: Rdf between anion and Ala side-chain Cβ atoms for the NaCl (c) and NaI (d) solutions.

hinder or promote the formation of internal α-helical hydrogen bonds in relatively short model peptides, such as the one of the present study. The net salt result on helix stability is a combination of the impact on helix initiation and propagation. A potential explanation of the salt impact on the propagation parameter w can be offered by analysis of the affinities among the various solution components. In fact, a strong affinity of sodium for the main-chain CO groups has been invoked to explain the NaI-induced destabilization of helical conformations for peptide Ace-(AE)6-Nme24 and also its effect on Nmethylacetamide and N-isopropylacrylamide conformations in solution.28,66 This affinity was based on the analysis of the corresponding salt−peptide radial distribution functions (rdf). However, even though sodium also manifests a propensity to interact with CO in the presence of chloride (see Figure 4), the addition of NaCl has a stabilizing effect on the helical

average helicity (Figure 1) is related to the total number of hydrogen bonds. Even though most of these bonds are “internal” (formed between backbone groups in the helix interior), their stability depends on both propagation and nucleation parameters, due to the small average size of the formed helices (e.g., ⟨nH⟩ = 6.2 residues at 299.6 K). Figure 3 displays the probability of participation of the various mainchain CO groups in internal α-helical bonds. We observe a uniform suppression of hydrogen bonds in the presence of 3 M NaI (in line with the large reduction in the propagation parameter w), and an increase in the presence of NaCl (due to the combined effects of the nucleation and propagation parameters), except in the central peptide region (CO groups of residues 5−6). An analogous NaCl-induced stabilization of helical conformations has also been observed in simulations of other model peptides.24 In conclusion, the addition of salt may 9871

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Figure 6. Upper panels: Rdf between anion and water hydrogen for the NaCl (a) and NaI (b) solutions. Lower panels: Rdf between sodium and anion for the NaCl (c) and NaI (d) solutions.

a minimal perturbation of the peptide hydration by the electrolytes even at quite high concentrations. Figure 4 displays rdf curves of the sodium−peptide carbonyl oxygen for the various solutions. The peaks are enhanced in the presence of iodide (at 3 M), as also observed in our earlier alanine dipeptide and tetrapeptide simulations33 and other studies.66 The enhancement has been related to the strong affinity of iodide for the hydrophobic alanine side-chain groups.24,28 The rdf curves of the anion (Cl− or I−)−amide hydrogen atom pairs are displayed in Figures 5a,b. The contact peaks are much smaller than unity, showing that both anions are excluded from the vicinity of the amide group; the more pronounced I− NH contact peak reflects the stronger tendency of iodide to approach the peptide. This tendency is enhanced with concentration. Figure 5c,d displays the rdf function of the anion−alanine side-chain methyl carbon pairs. As for the alanine dipeptide and tetrapeptide,33 Cl− actually avoids the methyl groups, and an increase in NaCl concentration does not enhance the weak interaction between Cl− and the side-chain carbon; I−, on the contrary, has a stronger interaction with the methyl carbons, which increases with NaI concentration. These findings are in agreement with the behavior of the anions toward the hydrophobic groups observed in other works,21,24,28,66 and are consistent with the stronger iodide affinity for the NH group (Figure 5b) and the stronger sodium affinity for the CO group in the NaI solutions (Figure 4b). Figure 6 plots the anion−water and anion−cation rdf curves. Chloride demonstrates a much stronger affinity for water

conformations of the present peptide. Furthermore, the addition of NaI increased the population of helical conformations of the alanine tetrapeptide.33 This complex behavior implies that sodium−peptide interactions alone cannot entirely account for the conformational preferences of these peptides, but the overall stability is determined by the competition among salt−peptide, salt−water, and peptide− peptide interactions. It should also be noted that recent spectroscopic experiments with electrolyte solutions of butyramide suggest that the interactions between metal cations and the carbonyl oxygen of amides are weak.68 It is thus possible that ion−peptide interactions are overestimated by current force fields. Taking these facts into consideration, we will base our arguments on the relative height of contact peaks for salt−peptide, salt−water, and peptide−peptide rdf. All rdf curves are obtained from the T = 299.6 K trajectory of the REMD simulations. An alternative measure is the preferential solvation based on differences of Kirkwood−Buff integrals of the rdf peaks, which provides the relative preference of one species over another for interaction with a particular group.66,69,70 Figure S1 in the Supporting Information section displays water−peptide group rdf curves. The addition of ions causes a very slight decrease of these curves, reflecting a small reduction in the hydration of the peptide due to competition between ions and water for the peptide groups. The decrease in the first peak is more noticeable in the water hydrogen−carbonyl oxygen curves (Figure S1a,b), and less so in the water oxygen− amide hydrogen curves (Figure S1c,d), presumably due to the stronger sodium affinity for the carbonyl group, relative to the anion affinity for the amide group. Overall, these results suggest 9872

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general, we expect that salt effects on helicities cease to depend on peptide length beyond a certain size, for which the helix elongation step dominates, as shown in ref 34 with simulations of peptides with 10−15 monomers. This is reflected in Figure 7 by the decline in the slope of the curves in pure water and salt at larger peptide lengths. To further check the prediction of Figure 7, we computed the average helicity of the alanine peptide Ace-Ala8-NMe (A8) in pure water and a 3 M NaI solution as a function of temperature from replica-exchange simulations with the same force-field as for the Ace-(AAQAA)3-Nme peptide. The calculated helicity of A8 is shown in Figure S2 in the Supporting Information as a function of temperature. Indeed, the addition of 3 M NaI salt causes a helicity increase in Ace-A8Nme. The actual helicities and the crossover value n = 6 predicted in Figure 7 do not agree with the results of Figure S2. Presumably, this is due to the pure alanine content of the A8 peptide; in contrast, Figure S2 uses average v and w values from the 15-residue peptide, which has an 80% alanine and 20% glutamine composition. 3.4. Impact of Salt on Thermodynamics of Helix Formation. Using the Zimm−Bragg propagation parameter s and eqs 4 and 5, we can compute a thermodynamic model for the elongation of a helix in a sequence with the characteristics of the Ace-(AAQAA)3-Nme peptide. Setting T0 to the lowest value of the REMD runs (293.2 K), and assuming a temperature-independent heat capacity ΔCp, we obtained values reported in Table 2.

(Figure 6a) or sodium (Figure 6c), than for the peptide (Figure 5a,c). Because of the strong intrapeptide interactions that accompany the formation of helices, helical conformations promote ion−water and ion−ion interactions, at the expense of ion−peptide interactions. In the case of NaCl solutions, the stronger anion affinity for water and its accompanying cation seems to stabilize helical conformations. The much weaker iodide affinity for water and sodium (Figure 6b,d) is in line with the lack of a similar enhancement of helical conformations in the NaI solutions. The stronger sodium−chloride affinity might also account for the decrease of the sodium−CO rdf first peak with NaCl concentration (Figure 4a). The increase of the sodium−CO rdf with NaI concentration (Figure 4b) might be due to the combined effect of the weaker iodide−sodium affinity and the stronger iodide−nonpolar side chain affinity. In conclusion, the overall salt effect on the peptide conformations is determined by a complex competition between the various solution components. 3.3. Dependence of Helix Stability on Peptide Length. In the LR formalism, the average number of helical hydrogen bonds depends on the nucleation and propagation parameters and on the peptide length (see eq 1). Using the average nucleation and propagation parameters from the T = 299.6 K simulations in pure water (w = 1.08, v = 0.21) and in 3 M NaI (w = 0.91, v = 0.35) and eq 1, we computed a theoretical helicity as a function of peptide length (n). This estimate ignores the actual dependence of w and v on the peptide length,23,67 and is a qualitative indicator of the helicity fraction versus length for a peptide with the average characteristics of the simulated sequence Ace-(AAQAA)3-Nme. The resulting graph (Figure 7) shows that the addition of 3 M NaI stabilizes helical conformations for “short” peptides

Table 2. Enthalpy, Entropy and Heat-Capacity Change Associated with the Conversion of a Residue from Coil to Helical State at the End of a Long α-Helix with the Average Characteristics of the Peptide Ace-(AAQAA)3-Nmea salt−water water ΔH0 (kcal/mol) ΔS0 (cal/mol/K) ΔCp (cal/mol/K)

−0.70 −2.57 −1.04

1 M NaCl 1 M NaI 3 M NaCl 3 M NaI +0.05 +0.12 +0.16

+0.25 +0.80 −2.86

+0.26 +0.83 −1.61

+0.57 +1.34 −3.18

a

The various quantities are obtained from the temperature dependence of the ZB parameter s and eq 5, assuming a temperatureindependent heat capacity ΔCp and setting T0 = 293.2 K (the lowest temperature of the replica simulations).

The various thermodynamic quantities are evaluated at 293.2 K. In pure water, helix elongation is enthalpically favored (ΔH = −0.70 kcal/mol), and entropically opposed (ΔS = −2.57 cal/ mol/K), as expected. Best and Hummer obtained a similar enthalpy value at 300 K (−0.61 kcal/mol) and a less negative value for the entropy (−1.81 cal/mol/K) in their MD study.23 Experimental measurements by circular dichroism71 and calorimetry71,72 place the enthalpy change in the range −0.9 to −1.3 kcal/mol for model peptides. The parameters evaluated above are based on the temperature dependence of the LR parameter s in the simulations. Parameter s (equivalently, w) mirrors the peptide helicity (Figures 1 and 2), and therefore has a weaker dependence on temperature in our simulations relative to experiment (see discussion following Figure 1). This systematic error is likely to be present in pure water and salt solutions, and to partly cancel in the differences (salt − water). Table 2 shows that helix elongation becomes slightly less favored enthalpically and slightly less opposed entropically in the presence of salt; the

Figure 7. Dependence of helicity on peptide length. The helicity is computed from the LR parameters for the AQ peptide at 299.6 K, and eq 1.

(due to the increase in the nucleation parameter), whereas it destabilizes helical conformations for “long” peptides (due to the decrease in the propagation parameter). For a 15-residue peptide, the helicities are respectively ∼20% and ∼12% in 0 and 3 M NaI, in agreement with the simulation estimate. With the LR parameters from the AQ simulations, the crossover between helix destabilization and stabilization occurs at a length of ∼6− 7 residues. This result is in agreement with the stabilization of the helical turn in the alanine tetrapeptide in the presence of 3 M NaI.33 It is also compatible with the results of Dzubiella, who found destabilization of the helical conformations of longer “neutral” oligopeptides in the presence of salts.21,24,34 In 9873

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addition of NaCl, because the engagement of peptide groups in intrapeptide interactions facilitates the formation of anion− cation and salt−water interactions. In running work, we examine the impact of solvent-exposed salt bridges on the α-helix/coil equilibrium of neutral peptides with anionic and cationic side chains. The effect on helix stability due to ion interactions with charged side chains has been investigated in a series of papers by Dzubiella.21,29,30,34 Preliminary analysis of our results suggests that salts affect the stability of helical conformations not only through interactions with the backbone but also by their ability to screen potentially stabilizing electrostatic interactions among suitably positioned charged side chains. The results of these simulations will be presented in a forthcoming publication. The present work shows that the impact of salt on the conformational stability of peptides in aqueous solutions depends on several factors that need to be considered separately. Ongoing developments in additive32,74 and polarizable36 protein and ion force fields, and increasing computer power, suggest that simulations will offer very dependable results on this problem in the near future. The successful coupling of simulations to experimental measurements of peptide conformations is already emerging and providing exciting opportunities in this area.27,28,34,37,38

enthalpic contribution prevails slightly, yielding a net destabilization of helix elongation by salt. These trends are more apparent in the 3 M NaI solution, in line with the reduced helicity in the presence of this salt (Figure 1). The smaller enthalpic stabilization of helix elongation by 3 M NaI (relative to pure water) could be due to the competition between peptide−peptide and ion/water−peptide interactions, as discussed above. The obtained heat capacity values lie within the estimated range of values for helix formation, −8 to +8 cal/mol/K.73 Differential scanning calorimetry measurements have reported a value of −7.6 cal/mol/K.72 Best and Hummer estimated a negative heat capacity (≈−3.6 cal/mol/K).

4. CONCLUSIONS AND PERSPECTIVE In this work, we have examined the conformational stability of the model peptide Ace-(AAQAA)3-Nme (AQ) in pure water and various electrolyte solutions. Our simulations show a destabilization of α-helical structures in high-concentration NaI solutions, and a stabilization in high-concentration NaCl. Analysis of the corresponding LR parameters shows a general enhancement of the helix-nucleation step by salts. This observation is in line with the previously observed increase in helicity of the alanine tetrapeptide by both NaCl and NaI.33 It may be understood on the basis of electrostatic screening by salt, which is expected to reduce the free-energy penalty for peptide group ordering during helix initiation. The same LR analysis suggests that salts hinder the propagation of helices in a salt-specific manner: the LR propagation parameter of AQ is significantly reduced (relative to pure water) in the presence of 3 M NaI, and slightly reduced in all other salt solutions studied here. An important conclusion is that the net salt effect on helix stability can be best understood by considering separately its impact on the nucleation and propagation steps. In shorter peptides, where the nucleation step has a much larger impact, facilitation of the nucleation step may lead to a net stabilization of helical conformations, as shown explicitly by our Ala tetrapeptide33 and nonapeptide simulations (this work) in 3 M NaI. The same salt environment can destabilize helical conformations in longer peptides, as shown here for AQ. Furthermore, in a peptide of given length, the addition of different salts stabilizes or destabilizes helical conformations, depending on their effects on nucleation and propagation. This is manifested here by the 3 M NaCl and 3 M NaI simulations of AQ. We expect that salt effects on helicities cease to depend on peptide length beyond a certain size for which the helix elongation step dominates, as observed in recent simulations of peptides with 10−15 monomers.34 The destabilization of helical conformations of longer peptides by NaI (relative to NaCl) can be understood in terms of the differences in ion sizes. The larger iodide size renders it less polar (more hydrophobic). As a result, it has a smaller affinity for sodium, and a larger affinity for the peptide nonpolar side chains and main-chain peptide groups, relative to chloride. Overall, ion−peptide nonpolar and polar contacts are enhanced in the NaI solution (relative to the NaCl solution), at the expense of intrapeptide polar interactions, and the formation of helical structures. The helix stabilization by NaCl (relative to pure water) may be understood in terms of the small chloride size. Chloride has a large affinity for sodium and water, and small affinity for the peptide. Helical conformations may be enhanced by the

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 contains water−peptide rdfs for the AQ peptide. Figure S2 contains average helicity vs temperature plots for the A8 peptide in pure water and in 3 M NaI solutions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.A.); [email protected] (E.L.). Tel.: +357 22892767. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.A. and P.I. thank the Cyprus Research Promotion Foundation, the government of Cyprus, and the European Union Structural Funds for Cyprus for financial support (project RPF/PENEK/ENISX/0308/24). G.A. and E.L. thank the A. G. Leventis Foundation for partial financial support of this work. Simulations were carried out in the Biophysics clusters at the University of Cyprus supported partially by a grant from the A. G. Leventis Foundation, and at an IBM cluster of the Cyprus Institute, financed by the Cyprus Research Promotion Foundation grant INFRASTRUCTURE/ STRATEGIC/0308/31, “Cy-Tera: A Multi- Teraflop/s computing facility for Science and Technology in Cyprus”, that is cofunded by the European Regional Development fund for Cyprus.

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