Radical Formation Initiates Solvent-Dependent Unfolding and β

We examined the effects of Cα-centered radical formation on the stability of a model helical peptide, N-Ac-KK(AL)10KK-NH2. Three, 100 ns molecular dy...
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Radical Formation Initiates Solvent-Dependent Unfolding and β‑sheet Formation in a Model Helical Peptide Michael C. Owen,*,† Birgit Strodel,†,‡ Imre G. Csizmadia,§,∥,⊥,○ and Béla Viskolcz∥,⊥,○ †

Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich, Germany Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany § Department of Chemistry, University of Toronto, Toronto, ON Canada M5S 3H6 ∥ Institute of Chemistry, Faculty of Material Science, University of Miskolc, Egyetemváros 1, H-3529 Miskolc, Hungary ⊥ Drug Discovery Research Center, 6720 Szeged, Hungary ‡

S Supporting Information *

ABSTRACT: We examined the effects of Cα-centered radical formation on the stability of a model helical peptide, N-AcKK(AL)10KK-NH2. Three, 100 ns molecular dynamics simulations using the OPLS-AA force field were carried out on each α-helical peptide in six distinct binary TIP4P water/ 2,2,2-trifluoroethanol (TFE) mixtures. The α-helicity was at a maximum in 20% TFE, which was inversely proportional to the number of H-bonds between water molecules and the peptide backbone. The radial distribution of TFE around the peptide backbone was highest in 20% TFE, which enhanced helix stability. The Cα-centered radical initiated the formation of a turn within 5 ns, which was a smaller kink at high TFE concentrations, and a loop at lower TFE concentrations. The highest helicity of the peptide radical was measured in 100% TFE. The formation of hydrogen bonds between the peptide backbone and water destabilized the helix, whereas the clustering of TFE molecules around the radical center stabilized the helix. Following radical termination, the once helical structure converted to a βsheet rich state in 100% water only, and this transition did not occur in the nonradical control peptide. This study gives evidence on how the formation of peptide radicals can initiate α-helical to β-sheet transitions under oxidative stress conditions.

1.0. INTRODUCTION Reactive oxygen species (ROS) is a consolidating term that describes compounds that form as a result of the incomplete reduction of oxygen.1 When cellular concentrations of ROS become elevated, the cell is considered to be in a state of oxidative stress, which is associated with a host of maladies, including, aging,2 diabetes,3 Alzheimer’s disease, Parkinson’s disease, and Creutzfeld−Jakob disease.4,5 In most cases, the causative factor in each of these diseases is related to the aggregation of misfolded proteins.6−9 Free radicals can abstract a hydrogen from the Cα atom of a peptide. The resulting peptide radicals have been detected by spin-trapping and EPR spectroscopy, however locating the radical center in an oxidized protein can be difficult.10 There are a vast number of experimental studies on the effects of radicals on proteins, however, a systematic experimental investigation is extremely difficult because of the multitude of and difficulty in differentiating the reaction products.11,12 Cα-centered radicals can be found in biological systems, and have been shown to be present at the active site of anaerobic enzymes and are stabilized by the capto-dative effect.13−17 Radicals that form on the outer surface of a protein can be repaired immediately by © XXXX American Chemical Society

antioxidants such as glutathione or ascorbate, however irreparable proteins are marked for degradation by proteases and may be toxic.18 The reactivity at the Cα is of particular interest due to the direct proximity to the amide bond and its direct influence on the ϕ and ψ angles of a protein, two parameters that define protein secondary structures. Studying the effect of radical formation on the peptide backbone using quantum chemical approaches has been done previously, which demonstrated an increased stability of the β conformation.14,19−23 This molecular dynamics (MD) study will examine the effect of protein oxidation on protein folding. Water is abundant in living systems; therefore, most studies employ pure water as the ideal solvent when representing a biologically relevant environment. However, water is highly compartmentalized within the cell, and the intracellular environment is crowded with organelles, proteins, and vesicle surfaces, all of which provide surfaces and nonaqueous media with which proteins and peptides can interact.24 As such, Received: January 7, 2016 Revised: April 7, 2016

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helicity of a hydrophobic peptide by minimizing the contribution from other secondary structural elements or factors such as aromatic interactions or salt bridge formation. This reductive system will serve as a model to which the effect of Hα abstraction on the structure of other helical peptides can be compared. We believe that the results obtained herein help to elucidate the effects of Hα abstraction on the folding of transmembrane and water-accessible peptides in oxidatively stressed conditions.

studies of protein and peptide structures in nonaqueous solvents can also provide biologically relevant results. Alcohol-based cosolvents, such as 2,2,2-trifluoroethanol (TFE), have been used for decades to stabilize secondary structures in peptides, and these solvents display excellent solvent properties.25,26 The physical properties of TFE which mediate its secondary structure stabilizing effects are not fully understood, however two possible mechanisms have been discussed. The first is the direct binding of TFE to the peptide,27 while the second is the weakening of the hydrogen bonds between water and the CO and NH groups of the peptide bond.28 The TFE effect also depends on the primary structure of the peptide or protein under investigation and on the TFE concentration.29 Moreover, TFE has been shown to enhance the internal hydrogen bonding of the peptide chain, to reduce the hydrophobic effect, and to preferentially solvate certain side chain groups of amino acid residues.30 Solutions containing TFE have a preference for stabilizing helical conformations by enhancing existing helical propensities, as opposed to the induction of random α-helices.27,30,31 Other secondary structure elements such as turns, β-hairpins, β-sheets, and hydrophobic clusters can also be stabilized by TFE, however this has a higher dependence on the primary structure of the peptide.32−36 This study will also examine the role of TFE in mediating helicity in the oxidized and nonoxidized peptides. To this end, the Lys-flanked α-helical peptide, Ac-K2(AL)10K2-NH2 (AL10) and its respective peptide radical (AL10(R)) were used as model peptides to determine the effect of hydrogen abstraction on the stability of a peptide helix. Their respective primary structures are shown in Figure 1. The

2.0. METHODS 2.1. Molecular Dynamics Simulations. The molecular dynamics simulations were carried out with the GROMACS 4.5.5 program package,38−40 the OPLS-AA force field41 and the recently developed parameters for the Cα-centered radical of the alanyl residue for use with the OPLS-AA force field using periodic boundary conditions.42 TIP4P water molecules43 and the standard TFE parameters for the OPLS-AA force field were used.44 Long-range electrostatic interactions and nonbonded interactions were treated by the twin-range method, and were effective over a range of 0.9−1.4 nm cutoff, for which the particle-mesh Ewald method was employed.45 The initial structure of the AL10 and AL10(R) (with an ALR radical at position 11) peptides in the α-helical conformation were created with the tleap module of the AmberTools 1.5 program package, and was the same for all simulation runs.46 The experimental value for the isothermal compressibility of 2,2,2trifluoroethanol was used,47 and the compressibility of the TFE/water mixtures were scaled according to their relative concentration, as was done previously.48 The peptides were solvated with pre-equilibrated binary solvents in each of the respective TFE/water concentrations. Four chloride ions were added to neutralize the charge of each system. The electrostatically neutral systems containing the AL10 peptide or AL10(R) peptide were solvated in binary solvent systems containing increasing concentrations of TFE (0%, 20%, 40%, 60%, 80%, and 100%) with H2O as a cosolvent. Each system was energy-minimized by the steepest descent method until the maximum force on each atom was less than 1000.0 kJ/ mol/nm. The temperature of the systems was coupled to a modified Berendsen thermostat49 at 310 K to mimic physiological conditions. Simulations under NVT conditions of the positionally restrained peptide were performed for 100 ps followed by NPT simulations at a pressure of 1 bar by using the Parrinello−Rahman method.50 The bonds between atoms in each system were constrained by the LINCS method.51 The dynamics of the peptides were then simulated three times, each for 100 ns, using a 2 fs time step under NPT conditions from the same α-helical starting structure yet using different starting velocities. The coordinates of the simulations were saved every 2 ps and the trajectories were subsequently analyzed. The starting conditions of the AL10 and AL10(R) simulations in each of the solvent systems are summarized in Table 1. 2.2. Trajectory Analysis. The three runs of each peptide in each of the solvent systems were concatenated before they were analyzed. The trajectories were subjected to cluster analysis using the algorithm of Daura et al., with a backbone RMSD of 1.0 Å.52 The middle structure of the three largest clusters from each trajectory was selected as the representative structure for the AL10 and AL10(R) peptide in each of the solvent systems. The occurrence of α-helix, 310-helix, β-bend, β-turn, and random coil secondary structure elements during the trajectory was determined using the define secondary structure of

Figure 1. A schematic representation of the two Lys-capped hydrophobic peptides. Peptide A (AL10) contains a closed-shell Ala11 residue, whereas peptide B (AL10(R)) contains a Cα-centered alanyl radical (ALR11).

name is derived from the 10-fold repetition of the Ala and Leu residues in the peptide’s primary structure. This peptide belongs to a family of transmembrane peptides that are designed to maintain an α-helix in the hydrophobic environment of the lipid core, while the dilysine caps anchor the peptide to the surface of the lipid bilayer.37 Binary solvent systems containing TFE and water have been used to reproduce the hydrophilic/hydrophobic character of lipid bilayers.29 The effect of hydrogen abstraction on the helicity of the peptide will be examined in MD simulations in solutions of increasing TFE concentrations (0%, 20%, 40%, 60%, 80%, 100%) with H2O as a cosolvent. This will enable the effects of Hα abstraction from Cα on the stability in helices to be studied both in water and under the influence of TFE. To the best of our knowledge, this is the first MD study on the structural consequences of Hα abstraction from an amino acid residue to be determined in different solvent systems. The repetitive AlaLeu motif provides an easily transferable model to study the B

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The Journal of Physical Chemistry B Table 1. Isothermal Compressibility (bar−1) and the Number of Water and TFE Molecules in Each of the Simulations

these extensions were identical to those described in Section 2.1.

number of solvent molecules solvent of system

isothermal compressibility/bar

100% H20, 0% TFE

4.50 × 10−5

80% H20, 20% TFE

5.90 × 10−5

60% H20, 40% TFE

7.30 × 10−5

40% H20, 60% TFE

8.70 × 10−5

20% H20, 80% TFE

1.01 × 10−4

0% H20, 100% TFE

1.15 × 10−4

‑1

water

TFE

11366 11367 5746 5746 3164 3164 2075 2075 702 702 0 0

0 0 1261 1261 1773 1773 1961 1960 2347 2349 2516 2516

3.0. RESULTS AND DISCUSSION 3.1. Conformational Sampling. The AL10 and AL10(R) peptides were chosen as model peptides because of their high content of Ala and Leu residues, both of which have high helical propensities in water.54 The α-helix was chosen as the starting structure for each system, however the configurational sampling was enhanced with the employment of three runs for each system, each with distinct starting velocities. Moreover, we employed six solvent systems, each containing an incremental increase of 20% TFE, to determine the effect of different environments on the helical structure. Despite the abundance of water in the cell, protein structures are present in all areas of the organism, from aqueous media to hydrophobic, solvent-free environments. Therefore, it is important to include solvents that vary in water content, to not only more effectively represent the different environments in which proteins exist, but also to further our understanding of the influence of the solvent on the stability of secondary structures. This study focuses on the water−helix interactions, but also includes the effect of radical formation in the different environments. A total simulation time of 7.2 μs was sampled in this study. The trajectories indicate that radicalization affected the helical conformation within the first 5 ns of the trajectory, which is well-within the sampling time of the radical initiation phase. Moreover, the similarity between the observation made during the first 100 ns to that recorded during the subsequent 50 ns indicates that the simulation time is sufficient to study the effects of solvent and radical formation on the secondary structure stability. The solvent and structural effects during the peptide radical phase, as well as the termination phase will be discussed in the subsequent sections. 3.2. Cluster Analysis. The results of the cluster analysis and the solvent-accessible surface area of each peptide are summarized in Table 2. The largest cluster identified in each simulation contained between 3% and 11% of the total number of structures, which indicates that the peptide was flexible under

proteins (DSSP) algorithm.44 The ratio of these structural elements compared to the total number of frames in each trajectory was plotted for each residue in the peptide, whereas the structure of each peptide in each solvent is shown explicitly. The solvent-accessible surface area was computed using the method of Eisenhaber et al. as implemented in GROMACS.53 The interactions between the solvent molecules of each solvent system and the peptide backbone were computed during the simulations. The frequency of the number of the following interactions were plotted: the hydrogen bonds (H-bonds) between water and the peptide backbone, H-bonds between TFE and the peptide backbone, the number of TFE molecules within 3.5 Å of the peptide backbone, and the number of intrapeptide H-bonds between the i and i + 4 residues. Changes to the position of the backbone atoms from the initial peptide structure during the simulations were monitored by computing the root-mean-square deviation (RMSD) and the radius of gyration (Rgyr) of the peptide backbone with the GROMACS program package. The population of the sampled structures was plotted on the RMSD-Rgyr surface. One of the three runs of each system was extended a further 50 ns, to see if the results of the subsequent 50 ns deviated from those of the first 100 ns of each system. A convergence test on the helicity of the system was also done, in which the peptide helicity during the first 30 ns, the first 60 ns, and the first 120 ns and during the entire 150 ns was compared, to determine if a significant drift in this parameter can be observed as the interval time increased. The data suggested that some systems took longer to equilibrate than other did; however, we infer that the marginal drift does not dispute the overall conclusions of the study, which showed a good agreement with experimental findings. The DSSP and clustering analyses of these trajectories, and the results of the convergence test are presented in the Supporting Information. The dynamics of the AL10 and AL10(R) peptides in 100% water, 40% water, and 100% TFE solvent systems were selected for further analysis. The AL10 simulation in each of the three solvent systems was extended a subsequent 350 ns to increase the sampling time to 500 ns. However, before the 350 ns extension, the ALR radical parameters of the respective AL10(R) peptide were converted to the Ala residue. This was done to test the ability of the AL10(R) peptide to recover its helical conformation after radical reparation takes place. The simulation conditions of

Table 2. Number of Clusters in Each Simulation Resulting from an RMSD Cutoff of 1.0 Å and the Proportion of Structures in the Most Populated Clustera

solvent of system 100% H20, 0% TFE 80% H20, 20% TFE 60% H20, 40% TFE 40% H20, 60% TFE 20% H20, 80% TFE 0% H20, 100% TFE

peptide

number of clusters

proportion of structures in most populated cluster/%

solventaccessible surface area of structure/Å2

AL10 AL10(R) AL10 AL10(R) AL10 AL10(R) AL10 AL10(R) AL10 AL10(R) AL10 AL10(R)

4921 3359 1910 6980 2790 3452 3576 4675 2750 3205 1349 1870

3.7 10.4 17.2 3.2 11.8 6.7 7.4 2.9 9.7 3.1 19.0 8.0

2283 2144 2473 2212 2362 2421 2336 2538 2411 2474 2394 2429

a

The solvent-accessible surface area of the central structure of each cluster is also shown.

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Figure 2. Central structure from the largest cluster for the concatenated trajectories of AL10 (A) and AL10(R) (B) in each of the solvent systems, as computed using the clustering of Daura et al.52 The Ala residues are colored in red, the Leu residues in pink, and the Lys residues in cyan. The water molecules within 3.5 Å of the peptide backbone are also shown.

the loop region when the concentration of TFE is 40% or greater. Concurrently, the loop disappears, and is replaced by a slight kink in the helix. DSSP analysis indicates that the kink occurs at the position of the Ala radical, which adopts a turn conformation at this residue in an otherwise helical peptide. These representative structures elegantly illustrate the ability of the H-bonds of the water molecules to usurp the H-bonds within the peptide when present in 80% water or higher. This effectively eliminates the intrapeptide H-bonds that stabilize secondary structures in peptides and replaces them with Hbonds with water that decrease the helicity of the peptide. Small angle X-ray measurements of binary mixtures of TFE/water and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP)/water indicate strong clustering of the fluorinated solvents, particularly in the 25−35% v/v range.56 This clustering stabilizes H-bonds within the peptide and increases helicity. 3.3. DSSP Analysis of Secondary Structure. The fraction of each secondary structure in each residue are shown in Figure 3A. The α-helix was by far the most abundant secondary structure element in the AL10 peptide when TFE was present. In solvents containing greater than 20% TFE, residues 9 through 16 displayed a greater than 90% α-helicity. This value decreased as the residue number approached the N-terminus and C-terminus, and was zero at residues one and 24, respectively. The N terminus had a higher prevalence for forming α-helices in 20% TFE than at the other concentrations, whereas the C terminus had a higher prevalence of helicity in 100% TFE than in the other solvents. Assuming that TFE interferes with the ability of water to compete with the peptide backbone for hydrogen bonds (which will be discussed in more detail in subsequent sections), the concentration-dependent TFE 100% water contained 20−50% less α-helix content then when TFE was present. Moreover, a β-bend centered around residues 6 and 7 formed in this solvent. The effect of TFE on the helicity of the AL10(R) peptide was similar to that of the AL10 peptide, as shown in Figure 3B. 100% TFE elicited a

the respective simulation conditions. Figure 2 displays each structure and the water molecules that are within 3.5 Å of the peptide backbone. The AL10 peptide in 100% water contains a mixed helix/turn structure, with the helix present in residues 8 to 24. The N-terminal residues contain a bend and a turn, which enables the peptide to reduce its solvent accessible surface area by folding these residues to form a region that is absent of water molecules. All 24 residues of AL10 are helical when in solutions that contain both water and TFE. A total of 12 water molecules were within 3.5 Å of the peptide backbone in 20% TFE, which dropped to two or less (zero water molecules in 40% TFE, 2 molecules in 60% TFE, and 1 in 80% TFE) when the TFE concentration increased further. As shown in Figure 2, the water molecules form a spiral around the peptide helix, which can be attributed to the alternating Ala and Leu residues that comprise the primary structure. The smaller Ala residues enable the water molecules to come within the 3.5 Å threshold, whereas the larger Leu residue shields the peptide from the water molecules. This shielding reduces the ability of the water molecules to form H-bonds with the peptide backbone. This effect could explain the higher helical propensity of the larger hydrophobic residues such as Val, Leu, and Ile, despite the greater loss of entropy associated with the formation of helices comprised of these residues.55 The ability of TFE to cluster around specific residues is directly related to the ability of fluorinated alcohols to induce secondary structure conformational changes in a sequence-dependent manner.29 The representative structures of the AL10(R) are drastically different from those of AL10 (Figure 2). In 100% water the AL10(R) peptide contains a loop in the center of the peptide, whereas the residues near the N and C-terminus retained their helicity. Water molecules are uniformly distributed around AL10(R) in this solvent. The length of the loop region shrinks in 80% water, with the flanking helical segments forming a pocket that is free of water molecules. Water does not solvate D

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Figure 3. Time-averaged secondary structure assignment of each residue of the AL10 (A) and AL10(R) peptide (B) during the first 100 ns of simulation using the DSSP method.

whereas an increase in random coil was also shown. The helicity in the subsequent 50 ns also showed a similar distribution of secondary structures in both the AL10 and AL10(R) peptide residues (Figure S2), yet the unfolding continued for AL10 in 100% water and AL10(R) in solvents containing more than 80% TFE. In AL10, the α-helicity was highest in the central region of the peptide and when TFE was present. In the absence of TFE the N-terminal region had a lower helical content than the C-terminal region had. The βbend and β-turn structures were prevalent in 100% water, particularly at the N-terminus, whereas the β-bend structure in 40% TFE was slightly higher than what was shown during the first 100 ns. As was shown during the first 100 ns, the α-helical content of AL10(R) was reduced at the radical center and

higher helicity at the C-terminus; however, 80% TFE, rather than the 20% TFE shown in AL10, elicited the highest helicity at the N-terminus. The Cα-centered radical (ALR) at position 11 disrupted the α-helicity of this residue in a solventdependent manner, which was lowest in 100% water. The significantly reduced α-helicity at the radical center (Figure 3B) was converted to a β-bend in 100% water, which was present in 30%−50% of the structures when TFE was present. The effect could preferentially modulate the stability of helices at either termini. The AL10 peptide in β-turn content increased up to 20% as the TFE concentration increased, whereas the random coil content was highest (35−40%) when 20−60% TFE was present. The loss of α-helicity shown in other residues was primarily coupled to an increase in 310-helix and β-turn, E

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Figure 4. RMSD-Rgyr density plots of the AL10 peptide (A) and the AL10(R) peptide (B) in each of the solvent systems. A representative structure from the trajectory corresponding to the RMSD and Rgyr values of the labeled maxima are also shown.

neighboring residues. The α-helix was only present in the residues 1−10 when the TFE concentration was at 80% or higher. The random coil dominated at the N-terminus and Cterminus, particularly when the TFE concentration was less than 60%. In summary, the α-helix is stable in AL10 in the presence of TFE and increases with TFE concentration, while radicalization of AL10 leads to an increase in other secondary structures. The ϕ and ψ angles of the Ala11 residue in AL10 remained close to −47°, −57°, corresponding to those of an α-helix. The ϕ and ψ angles of the ALR11 residue largely remained close to 0°, −60°, however the ϕ and ψ angles of 0°, 150° (0−40% TFE) and 0°, 0° (60−100% TFE) were also observed (data not shown).

3.4. RMSD-Rgyr Population Surfaces. The RMSD-Rgyr population surfaces shown in Figure 4 enable the compactness of the peptide to be measured in relation to the backbone deviation from the starting helical structure and provide information on the distribution of the structures that occur during the trajectory. Representative structures that correspond to each maxima are also shown in each surface, which are in good agreement with the representative structures depicted in the cluster analysis shown in Figure 2. The AL10 peptide in 100% water has the most varied structure, as demonstrated by the large area occupied in the RMSD-Rgyr space. The DSSP plots in Figure 3 indicate that the N- or C-terminus had a large tendency to unfold, which should account for most of the F

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The Journal of Physical Chemistry B RMSD from the reference structure. The decreased Rgyr values compared to the starting structure shown by these structures is due to the turn formation that enables the terminal residues to interact with the central region of the peptide helix, as shown by the representative structure of the largest cluster in this solvent system. The AL10 peptide remained closest to its starting structure in 20% TFE, covering less than half of the plot area as the AL10 peptide did in 100% water. This structure also showed the greatest tendency to adopt an extended conformation in 100% water and in 60% TFE, as demonstrated by the Rgyr values that approach 16 Å. The decreasing Rgyr values for solvents with TFE indicate that the structures became more compact as they deviate from the starting structure. However, since the DSSP results show the persistence of the α-helical structure when TFE is present, the increase in RMSD can likely be attributed to structural changes near the N-terminus or C-terminus, with residues Ala5Leu20 retaining the α-helical conformation. In 100% water, the most populated geometry of the AL10(R) peptide had an RMSD of 10 Å from the starting structure and was more compact than that of the helix, with an Rgyr of approximately 7 Å. The remaining structures in water were generally less compact and deviated less from the starting helix. Increasing the TFE content to 20% increased the density of the respective plot in the areas occupied by the AL10 peptide, however, the most populated AL10(R) structure was more compact than the starting helix. The solvent that contained 20% TFE caused the AL10(R) peptide to adopt the largest variety of structures. As the TFE concentration increased further, the distribution of the RMSD and Rgyr values of AL10(R) became closer to the more helical structures of the AL10 peptide, as also shown in the DSSP plots in Figure 3. However, the most populated regions of the AL10(R) density plots in these solvents had RMSD values of approximately 4 Å. This discrepancy can be attributed to the presence of the “kink” in the center of the helical region. Quantum chemical calculations have previously shown that the Cα-centered peptide radicals prefer planar conformations. The high Rgyr values of AL10(R) in 80% water indicates that a binary solvent unfolds the AL10(R) peptide more effectively than homogeneous solvents of water or TFE do. This is also demonstrated by the highest prevalence for the random coil according to the respective DSSP plot in Figure 3, and the high number of clusters found using cluster analysis (Table 2). Moreover, it is interesting to note that 20% TFE elicited the highest α-helicity for AL10, while it promoted the highest degree of unfolding in AL10(R). 3.5. Interactions between Solvent Molecules and the Peptide Backbone. The number of TFE molecules within 3.5 Å of the peptide backbone is taken as a measure of the degree of interaction between the TFE molecules and the respective peptide backbone. These results are shown in Figure 5A, wherein each curve of the AL10 peptide shows a near symmetric distribution. The curves indicate a distribution of interactions, however in the following sections only the maximum number of each interaction will be mentioned for brevity. In 20% TFE, only one or two TFE molecules had a 0.17 probability of being within 3.5 Å of the AL10 backbone. The number of interacting TFE molecules increased as the TFE % increased, with five TFE molecules within the cutoff distance when the TFE concentration was 80% or 100%. The maxima decreased in moment of TFE (2.03 D) to that of water (1.85 D) demonstrates that both molecules can interact magnitude

Figure 5. Peptide-solvent and intrapeptide interactions for AL10 and AL10(R) in each of the solvent systems. The number of TFE molecules within 3.5 Å of the peptide backbone (A). The frequency of the number of H-bonds between TFE and the peptide backbone (B), the number of H-bonds between water and the peptide backbone (C), and the number of H-bonds between residues i and residue i + 4 (D) are shown.

and their distributions became broader as the TFE% increased, indicating a greater variation in the number of TFE molecules that were within 3.5 Å of the AL10 backbone. This increase is due to the greater number of TFE molecules present in the system. The number of TFE molecules that comprised Hbonds with the backbone of AL10 followed a similar trend. The number of H-bonds increased from four to nine as the TFE% increased from 20% to 100%. With a lower dielectric constant (εr) than water εr(TFE) = 26.1, εr(H2O) = 80.4, the accumulation of TFE molecules around the peptide can enhance the intramolecular interactions which would stabilize secondary structures.57,58 Despite the differences in dielectric constant the similarity of the dipole with polar side chains of amino acids as well as the amide bond of the peptide backbone. The difference in molecular size could affect the clustering pattern of TFE in water, and the rationale behind the maximal helicity of the peptide occurs at 20% TFE in water. Experiments have shown that TFE can surround a peptide to create a local G

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Figure 6. Radial distribution function of the solvent with respect to the Cα of residue Ala11 in AL10 and ALR11 in AL10(R).

TFE demonstrates that the H-bonds between the peptide and water are favored over those between the peptide and TFE, a finding that is supported by experiments, and is likely due to the greater number of hydrogen bond donors in water, the smaller molecular size, and stronger polarity of H2O compared to TFE.28,50 It seems that the effect of the larger dipole of TFE is offset by its comparatively larger size. The reduced number of α-helical H-bonds in AL10(R) demonstrates the loss of helicity in the peptide radical. Three H-bonds between i, i + 4 residues were found in the AL10(R) peptide in 100% water, which is less than in the case of AL10. In the other solvent systems the number of interactions between the solvent molecules and the peptide depends on the solvent-accessible surface area (data shown in Table 2). Surprisingly, an increase to 100% TFE did not increase the number of H-bonds between TFE and the backbone of AL10(R), as was shown in the case of AL10 in Figure 5B, but instead decreased it. This suggests that the interaction between TFE and water are driven by the hydrophobic interactions instead of hydrogen bonding. This is in agreement with a study by Fiorini et al.,29 who used NMR to demonstrate that the concentration of TFE around a tetrapeptide is higher than it is in the bulk media, whereas the concentration of ethanol is the same as that in the bulk media. This also suggests that the interaction between TFE and peptide is driven by the fluoro groups of TFE, rather then the alcohol. Surprisingly, there were very few hydrogen bonds between water and AL10(R) in 100% water, as well, whereas the opposite was shown for AL10 in 100% water, which was the system with the most hydrogen bonds with water. The only difference between AL10 and AL10(R) in 100% water is the shape of the peptide, the latter of which formed radical-induced loop. Unfortunately. we can only speculate that the decreased solvent accessible surface area, as well as perhaps the peptide curvature made hydrogen bonding

TFE concentration that is higher than that in the bulk media, which can cause the helical stabilizing effect of TFE to have a maximum value.29 This phenomenon is true in the case of TFE, but not ethanol, for all residue types, and has been demonstrated experimentally to occur between 25% and 40% TFE in the case of a tetrapeptide.29 The results in Figure 5 show that 20% TFE was able to stabilize helicity by promoting the formation of intrapeptide hydrogen bonds. The broad distribution of TFE molecules within 3.5 Å of the AL10(R) backbone when the TFE concentration was 60% or less (Figure 5A) can be due to the large conformational changes shown in the corresponding Rgyr-RMSD figure. The cluster analysis, DSSP analysis, and Ramachandran maps all indicate a greater helicity at TFE concentrations of 80% or higher, wherein the distribution of TFE molecules around AL10(R) approach those of the AL10 peptide. This agreement shows the positive effect of TFE on helical stability, however, the effect is maximal at 20% TFE for AL10 and at 100% in AL10(R). The number of H-bonds between water molecules and the peptide backbone is plotted in Figure 5C, whereas the number of i, i + 4 intramolecular H-bonds are plotted in Figure 5D. The highest number of H-bonds between water and the peptide backbone of AL10 occurred in 100% water, as supported by the lower amount of α-helicity shown in the DSSP analysis. This solvent system also stabilized the fewest number of i, i + 4 H-bonds, which also indicates that the AL10 peptide had the lowest helicity in 100% water. The highest number of α-helical H-bonds was found in 80% water, wherein 12 such bonds formed, whereas 80% water also stabilized the fewest number of H-bonds between the AL10 backbone and both water and TFE. This correlation also demonstrates that TFE stabilizes the helicity of the AL10 peptide by promoting the formation of H-bonds within the peptide. The high number of α-helical H-bonds in solutions that contain more than 20% H

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Figure 7. DSSP analysis of the AL10 and AL10(R) peptides in 100% water, 40% water and 100% TFE, demonstrating the formation of the β-sheet conformation following radical termination. Cluster analysis also shows the most prevalent structures during the first 175 ns and last 175 ns of the post-termination trajectories.

3.6. Peptide Radical Termination. The extension of the AL10 simulations in 100% water, 40% water and 100% TFE from 150 to 500 ns showed secondary structures that were similar to what was shown during the initial 150 ns. The helix collapsed into bend and turn structures in 100% water, while the helicity of AL10 increased as the TFE concentration increased. The first and last 175 ns were clustered separately, largely due to the changes that occurred in the AL10(R) peptide after this duration. The clustering of AL10 in 100% water shows that the helix largely disappears during the second half of the trajectory, however, the helical instability was already observed during the first 175 ns. In the case of AL10(R), the final frame from the 150 ns simulation was used as the starting structure each of the three 350 ns simulations, however the peptide was converted to its closed-shell by replacing the ALR parameters for the Ala parameters. It is still referred to as AL10(R) in Figure 7 to facilitate comparison to AL10, which did not originate from an AL10(R) configuration. The DSSP analysis of the AL10(R) peptide in 100% water depicted in Figure 7 shows a predominantly β-bend and β-turn structure during the first half of the trajectory, which later, and rather unexpectedly converts to a β-sheet-rich structure during the second half of the trajectory. The sheets primarily affect residues at the Nterminus and residues ten through 14, which includes the “former” radical center at residue 11 and are separated by a βhairpin. This change is facilitated by the different conformations of ALR11 compared to that of the Ala11 residue. The inclusion of 60% TFE enables the C-terminal region to remain helical

with water less favorable in the case of AL10(R) in 100% water. As shown in Figure 6D, the correlation between the number of intramolecular i, i + 4 H-bonds and the other interactions is strong for a linear and helical peptide such as AL10, but less so for AL10(R), which contains a bend. A plot of the radical distribution functions of TFE and water around the Cα of Ala11 and ALR11 is shown in Figure 6. The distribution of water was lowest when the TFE concentration was 20%, and in this solvent system the maximum number of αhelical H-bonds was observed for AL10. This indicates that the solvation of the peptide backbone by water molecules disrupts the helical structure. The radial distribution functions around one atom is less dependent on the solvent-accessible surface area than the total number of hydrogen bonds between the solvent and the peptide is, therefore the relationship between water solvation and α-helicity is presented more distinctly. In the case of AL10, the amplitude of the distribution of water from the Ala11 Cα correlates to the decreased α-helicity observed in the solvents containing more water: the more water near the protein backbone, the lower the α-helicity. The trend is the opposite in the case of TFE: the higher the distribution of TFE around the backbone, the higher the helicity. The AL10(R) peptide shows a similar effect. In both peptides, the distribution function of TFE is highest when the TFE concentration is 20%, the concentration at which TFE is closest to the peptide backbone. However, due to the preference of ALR11 to be in planar conformations (Figure 5), the presence of TFE cannot prevent the loss of helicity. I

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The Journal of Physical Chemistry B during the first half of the trajectory, with a mixture of helix and β-turn persisting during the second half. A turn also forms in the latter half of the trajectory at residue 11, which is stabilized by β-bridge and β-sheet on either side of Ala11. As expected, in 100% TFE the N-terminal and C-terminal regions retain their helicity, however the kink that was observed during the initial 100 ns simulation persisted, despite the replacement of the ALR parameters with those of Ala. Our results suggest that following radical termination, even helices containing residues of the highest helical propensity can convert to a β-sheet following radical attack. To the best of our knowledge, this is the first time this has been demonstrated in silico, however this has been shown in experiments. For example, Fouriertransform infrared spectroscopy has been used to monitor the formation of β-sheet-rich structures in the hydroperoxide-rich region of the cortex, which did not form in the hydroperoxidefree region of the hippocampus.59 Moreover, internal reflection infrared spectroscopy was used to demonstrate that the interaction of Amyloid-β42, an amyloidogenic peptide central to the pathology of Alzheimer’s disease, with oxidatively damaged lipid monolayers initiated protein misfolding and aggregation, whereas undamaged lipids had no such effect.60 We feel that the results of our all-atom MD simulations provide an atomistic view of how oxidative stress can facilitate the peptide misfolding. Along the pathway to protein aggregation, it has been proposed that slight conformational changes result in the formation of a misfolded intermediate, which is unstable in the aqueous environment due to unfavorable interactions between the protein and the solvent.61,62 Our results indicate that the initiation of peptide backbone radicals by the abstraction of hydrogen followed by free radical termination, can initiate such a conformational change to produce the β-sheet-rich state so often observed in amyloidogenic peptides. Free radical oxidation can speed this process, and cause amyloidogenesis in structures that may otherwise remain in their folded state.

deviated from the helical starting structure were generally more compact in higher water concentrations due to the hydrophobic effect. The AL10(R) peptide formed a turn that centered on residue 11 within the first 5 ns of the simulation. The turn was stabilized by solvating water molecules and formed a loop at higher water concentrations. The clustering of water molecules around the radical center was proportional to the helical content, which illustrates how the solvation of the peptide backbone by H2O destabilizes peptide α-helices. The helical content of AL10(R) was proportional to the TFE content and was highest in 100% TFE. During the post-termination phase the ALR is converted back to Ala and the AL10(R) simulation is continued from the previous simulation, and the trajectories in 100% water, 40% water and 100% TFE were analyzed. The systems containing TFE showed a concentration dependent relationship between TFE and helicity and the peptide structure in these systems remained helical. However, in 100% water the peptide converted to a β-sheet-rich state, despite the high helical propensity of the Ala and Leu residues that comprise the central region of this peptide. For AL10, used as a control, the helix reemained fully formed in 100% TFE and 40% water, and most importantly, no β-sheet was formed in 100% water. However, a future study with a longer sampling of the post-termination phase could increase the statistical significance of this result. These results indicate that radical formation on a peptide backbone can induce large structural changes when exposed to water molecules, and this effect is enhanced by interactions between water and the peptide backbone. Radical termination caused peptide to complete the conformational transition and formed the β-sheet conformation observed in amyloidogenic peptides that undergo a similar transition in oxidative stress conditions. Further studies will elucidate the role of oxidative stress on the structure of amyloidogenic peptides, in systems that are closely related to relevant pathological conditions.

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4.0. CONCLUSIONS Hydrogen abstraction by free radicals is an event which occurs in the presence of free radicals such as OH under oxidative stress conditions. We studied the effect of this process on the stability of a model helical peptide in different binary solvents to delineate the peptide-solvent interactions that dictate helical stability and to include environments in which the water molecules may not have access to the peptide or protein. The Lys-flanked peptide, Ac-K2(AL)10K2-NH2 (AL10), was studied in solvents in which the TFE concentration increased in 20% increments from 0% to 100% TFE in water. The structure of AL10 in these solvents was compared to that of its oxidized analogue AL10(R), which was formed by the incorporation of parameters for the Cα-centered alanyl radical (ALR) in place of Ala11. The trajectories following both the radical initiation and radical termination phases were analyzed. During the first 100 ns after radical initiation, the helicity of AL10 was enhanced when TFE was added to the solvent, and was at a maximum at 20% TFE with only a slight decrease in helicity observed when the TFE content increased further. In 20% TFE, the TFE molecules clustered around AL10, a tendency which is more pronounced in 20% TFE than in the other TFE/water mixtures, and preventing H2O from forming H-bonds with the peptide backbone. The increased helical stability was shown in the results of the DSSP analysis and the increase in intrapeptide hydrogen bonds. The structures that

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b00174. An additional 50 ns simulation on the first run of the AL10 and AL10(R) peptide under the same conditions as described in section 2.1, in addition to a convergence test (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(M.C.O.) E-mail: [email protected]. Telephone: +492461-61-9486. Notes

The authors declare no competing financial interest. ○ Drug Discovery Research Center: www.drugcent.eu.

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ACKNOWLEDGMENTS We thank László Müller and Máté Labádi for the administration of the computer systems used for this work. This project was supported by the project “New functional material and their biological and environmental answers” (Project ID: TAMOP-4.2.2.A-11/1/KONV-2012-0047) and the German Research Foundation (DFG). M.C.O. also thanks the Helmholtz Postdoc Program for financial support. J

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