Biophysical Properties of Membrane-Active Peptides Based on Micelle

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J. Phys. Chem. B 2010, 114, 13726–13735

Biophysical Properties of Membrane-Active Peptides Based on Micelle Modeling: A Case Study of Cell-Penetrating and Antimicrobial Peptides Qian Wang,† Gongyi Hong,‡,§ Glenn R. Johnson,| Ruth Pachter,‡ and Margaret S. Cheung*,† Department of Physics, UniVersity of Houston, Houston, Texas, United States; Air Force Research Laboratory, Materials & Manufacturing Directorate, Wright-Patterson Air Force Base, Dayton, Ohio, United States; General Dynamics Information Technology, Inc., Dayton, Ohio, United States; and ARFL/RX, Tyndall Air Force Base, Tyndall, Florida, United States ReceiVed: July 25, 2010; ReVised Manuscript ReceiVed: September 25, 2010

We investigated the molecular mechanisms of short peptides interacting with membrane-mimetic systems. Three short peptides were selected for this study: penetratin as a cell-penetrating peptide (CPP), and temporin A and KSL as antimicrobial peptides (AMP). We investigated the detailed interactions of the peptides with dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles, and the subsequent peptide insertion based on free energy calculations by using all-atomistic molecular dynamics simulations with the united atom force field and explicit solvent models. First, we found that the free energy barrier to insertion for the three peptides is dependent on the chemical composition of the micelles. Because of the favorable electrostatic interactions between the peptides and the headgroups of lipids, the insertion barrier into an SDS micelle is less than a DPC micelle. Second, the peptides’ secondary structures may play a key role in their binding and insertion ability, particularly for amphiphilic peptides such as penetratin and KSL. The secondary structures with a stronger ability to bind with and insert into micelles are the ones that account for a smaller surface area of hydrophobic core, thus offering a possible criterion for peptide design with specific functionalities. 1. Introduction During the past decade, a large number of short membraneactive peptides, typically composed of 12-45 amino acids, were shown to pass across cell membranes with different lytic activities. Cell-penetrating peptides (CPP)1 present low lytic activities whereas antimicrobial peptides (AMP)2 can rapidly kill microbes without exerting toxicity against the host. They share similar physicochemical properties and their modes of interaction depend on the target plasma membrane. However, little is known about a quantitative, physical picture that is related to the biological function of these short peptides (e.g., structure-activity relationship). In this work, we investigate the biophysical properties of three membrane-active peptides (i.e., a CPP penetratin3 and two AMPs temporin A3 and KSL4) by studying their structures and dynamics when interacting with two chemically distinct membrane-mimetic micelles (DPC and SDS) using all-atomistic molecular dynamics simulations. Penetratin belongs to a category of CPPs that have drawn much attention because their ability of internalization allows navigation into the nucleus of living cells without disruption of plasma membranes. It was derived from the third helix of the homeodomain of Antennapedia and its mechanism of translocation is energy-independent and receptor-independent,5 indicating that the uptake of this peptide does not involve endocytosis, although some have suggested a different mechanism that requires endocytosis;6 however, either does not rule out the possibility of the other. Nevertheless, it is worthwhile to investigate the relationship between the physicochemical properties and the structural characteristics of penetratin. Using * Corresponding author. E-mail: [email protected]. Tel.: 713-743-8358. † University of Houston. ‡ Wright-Patterson Air Force Base. § General Dynamics Information Technology, Inc. | Tyndall Air Force Base.

micelles as biomimetic modeling systems for biological membranes, a molecular understanding of penetratin interacting with lipid molecules without destroying the overall structure of a micelle will give useful insights into the development of pharmaceuticals for the purpose of noninvasive drug entry into a cell. The structural characteristics of penetratin in the presence of membrane-mimetic materials such as phospholipid vesicles and micelles were studied using a variety of spectroscopic methods7 such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance spectroscopy (NMR). These experiments suggested that penetratin shows high plasticity in its structure, which can be either R-helices, β-turns, or random coils, dependent on the phospholipids of selected membrane model systems. Although the roles of Arg and Lys of penetratin in the translocation were recently addressed from 13C, 31P, and 19F solid-state NMR,8 the overall structural characteristics of penetratin that account for its permeability across plasma membranes are still unclear. We also studied two AMPs, temporin A and KSL, and investigated their interaction with different membrane-mimetic models. First, temporin A is an antimicrobial peptide (AMP) from the temporin family derived from frog skins. Unlike the AMPs with bactericidal activities, the net charges in temporins are typically lower, their peptide lengths are shorter, and they can interact with both anionic and zwitterionic membrane models.9 Interestingly, temporin A and temporin B10 are also an antiparasitic AMP and can target and damage the plasma membrane of Leishmania protozoa which adopts different lipid compositions from bacteria. Second, KSL, a de novo designed decapeptide composed of a few types of amino acids, including charged residues, has shown a broad spectrum of antibacterial activities with little hemolytic actions.11 Experiments on its derivatives and analogues suggested that both the R-helical and

10.1021/jp1069362  2010 American Chemical Society Published on Web 10/12/2010

Biophysical Properties of Membrane-Active Peptides β-turn structures of KSL can be the key characteristics that account for its antibacterial activities.12 The molecular mechanism of typical AMPs was extensively studied2,13 and a number of antimicrobial peptide membrane permeation mechanisms were proposed. However, whether these mechanisms may be applied to temporin A and KSL is still unknown. Nevertheless, it is of interest to explore the mechanism of their interactions with membrane-mimetic micelles that may account for their toxicity at an early stage of peptide-membrane interaction. Here, we used molecular dynamics (MD) simulations to study the interaction between these three peptides and membranemimetic micelles. MD provides an excellent approach to complement experimental findings of the structure-function relationship of a peptide in the presence of different lipid membrane models.14 All-atomistic MD simulations were performed on AMPs in lipid bilayers/micelles to probe their lytic activities.15 MD simulations were also used to investigate the translocation properties of cell-penetrating peptides.16 Although MD is a powerful tool, it is often limited by the large size of lipid bilayers causing it to require large-scale computing resources. In contrast, theoretical models such as those describing the association of cationic peptides with a lipid membrane17 can provide a valuable method in computing a comprehensive parameter phase space of lipid-peptide interaction. However, it often fails to deliver specific atomistic information that is crucial in the study of peptide-membrane interactions. In this regard, we adopted membrane-mimetic micelles as a minimalist’s framework to study some important aspects at an early and transient stage of the peptide binding and insertion process. DPC micelles are zwitterionic while SDS micelles are anionic. Despite its simplicity, studies based on these micelles have revealed key interactions in peptide-membrane systems.18 Moreover, detailed analysis of the roles of the secondary structure and amino acid type, as described in this work, could assist in peptide design for desired functionality. 2. Methods 2.1. Simulation Details. Molecular dynamics simulations were performed using the Gromacs 4.0.5 package.19 The GROMOS96 43a1 force field,20 an united atom force field, was implemented for the simulations. These simulations were performed in a 9 nm periodic box with explicit water molecules represented by the single-point charge (SPC) model. The typical number of water molecules was about 22 000 ( 1000. In addition, 0.02 M sodium chloride was added to match the conditions with the experiments on penetratin-membrane systems.21 Sodium ions as countercation were added to the SDS micelles such that the [Na+] reaches to 0.12 M in the systems with SDS micelle. Sodium ions play an important role in charge neutralization and remaining a reasonable ionic strength in the medium. The fourth order of the particle mesh Ewald (PME) method was used to calculate the long-range electrostatic interactions. The cutoff for the real space of Coulombic interactions and pair list was set to 0.9 nm and the cutoff for van der Waals interactions was set to 1.6 nm.22 The Nose´-Hoover method23 was used to maintain the temperature at 300 K and the Parrinello-Rahman method24 was used to maintain the pressure at 1.0 atm. Covalent bond length was constrained by the LINCS method.25 2.2. Preparation of Protein-Micelle Computer Models. Penetratin. The protein structure of penetratin (RQIKIWFQNRRMKWKK) was obtained from the protein data bank (PDB ID: 1OMQ).26 Its N-terminus was capped by acetylation and the C-terminus was capped by amidation to reflect the conditions

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Figure 1. Percentage of dominant secondary structures in the ensemble of a peptide: (A, B) penetratin, (C, D) temporin A, and (E, F) KSL.

in the experimental preparation.27 Penetratin was energyminimized with the steepest descent method and then equilibrated at 500 K for 20 ns to produce randomized structures as initial conditions. In order to investigate the role of the peptide’s secondary structure in interacting with the micelles, we selected a penetratin structure with the most helical content (PR), and a peptide structure with the most beta content (Pβ) as initial configurations (Figure 1). The percentages of each dominant secondary conformations in Figure 1 were computed by the program STRIDE28 from the ensemble. Temporin A and KSL. There are no solution structures available for temporin A and KSL; hence we produced the coordinates of the temporin A (FLPLIGRVLSGIL-NH2) and KSL (KKVVFKVKFK-NH2) peptides from their sequences by using the xLEaP program in the AMBER9 package.29 Temporin A and KSL were capped by amidation to reflect the conditions in the experimental preparation.4,10 The same steps of energy minimization as well as the conditions to produce randomized initial structures described above were applied on temporin A and KSL. The structural criteria of the selection of the initial conditions, TR and Tβ, of temporin A are the same as penetratin (Figure 1). However, for KSL, there is no dominant helical structure; we chose a dominant turn shape (KT) and a dominant β-strand structure (Kβ) (Figure 1). Micelle Models. Two micelle models were investigated. The DPC micelle model with a radius of 2.5 nm, consisting of 65 DPC lipid molecules, was downloaded from http://moose. bio.ucalgary.ca. The SDS micelle model with a radius of 2.35 nm, consisting of 60 SDS lipid molecules, was obtained from the work of MacKerell.30 The aggregation number 60 is close to the experimental value of 63.31 The topological files of the micelles were produced by PRODRG.32 DPC and SDS molecules are represented in Figure S1 in the Supporting Information. 2.3. Umbrella Sampling. The umbrella sampling (US) method33 was used to compute the free energy of peptide insertion as a function of the distance between the center of

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mass of a peptide (e.g., penetratin) and the center of mass of a micelle (Dcom) at 300 K. The initial configurations for US were prepared by pulling the peptides from the outside to the inside of a micelle using the initial conditions provided in Figure 1 for the two micelles, DPC (D) and SDS (S). The pulling rate was controlled at 0.001 nm/ps. US was performed along the distance between the center of mass of a micelle and a peptide from Dcom ) 2.5 nm to Dcom ) 0.5 nm with a bin size of 0.1 nm (i.e., 21 windows). For each window, a 20 ns molecular dynamics with harmonic constraints was performed. The harmonic potential U(Dcom) was imposed between the center of mass of a peptide and the center of mass of each micelle at a distance of Dcom with a force constant of 1000 kJ/mol/nm2. The free energy as a function of Dcom was obtained by reweighing the density of states.34 The error values on the free energy calculations were estimated by the bootstrap method.35 2.4. Kinetic Trajectories of a Peptide Binding to Micelles. Different initial configurations (i.e., PR, Pβ, TR, Tβ, KT, and Kβ) were randomly distributed around the proximity of a micelle. The minimum distances between peptides and micelles were in the range of 0.3-0.6 nm. Once each configuration was positioned for initial binding, we also chose another two configurations as initial conditions by rotating this peptide to another 90° and 180° against the peptides’ longitudinal axis. A peptide’s longitudinal axis of each figuration is defined by the two CR atoms in the following residues: PR, residue 1 and residue 16; Pβ, residue 1 and residue 10; TR, residue 1 and residue 13; Tβ, residue 1 and residue 7; KT, residue 1 and residue 10; Kβ, residue 1 and residue 5. A 1 ns equilibration at T ) 300 K was performed, followed by a 20 ns simulation at a constant pressure of 1 atm and temperature 300 K. Each simulation under different peptide orientations was repeated four times using different random seed numbers; therefore, a total of 12 trajectories starting from the same initial conformations were produced for the calculation of temporal averages. A shell with a radius of 3.5 nm located at the center of mass of a micelle is defined as a collision zone. For any trajectory, whenever any part of a peptide enters the collision zone the data was collected for binding analysis. 3. Results 3.1. Kinetic Analysis of Penetratin’s Binding to Micelles. Dependence on the secondary structures and the type of micelles for the mechanism of penetratin binding to micelles was investigated by all-atomistic molecular dynamics simulations with explicit water models. Several trajectories of 20 ns simulations, which differ by the initial positions and secondary structures of penetratin, were collected for the kinetic analysis of penetratin’s binding to a DPC micelle as shown in Figure 2A, and a SDS micelle as shown in Figure 2B. There are two interesting features from the kinetic analysis that underpin the importance of penetratin’s secondary structure for micelle binding. First, the overall temporal decay of Dcom for an R-helical dominant structure (PR) is much faster than a β-strand in the presence of a DPC micelle (Figure 2A). We fitted the curve in Figure 2A with a single exponential (Table 1) and showed that the binding rate for PR was 5.45 × 10-3 ps-1, about 2-fold greater than Pβ (2.91 × 10-3 ps-1). Interestingly, the same feature that favors an R-helical dominant structure for binding was observed for SDS micelles in Figure 2B. The binding rate of PR was fitted to 3.67 × 10-3 ps-1 in Table 1 and it is ∼3-fold greater than Pβ (1.33 × 10-3 ps-1). Second, both parts A and B of Figure 2 demonstrate that after 10 ns the distance between the center of mass of a peptide and

Wang et al. the center of mass of a micelle (Dcom) begins to plateau. However, these profiles plateau at a different value of Dcom, where Dcom is much shorter for PR than for Pβ in the presence of DPC micelles. Dcom of PR remains at ∼1.9 nm and Dcom for Pβ remains at ∼2.1 nm. 3.2. Penetratin’s Structural Analysis upon Binding to Micelles. In section 3.1 we identified the importance of secondary structures as initial conditions for penetratin’s binding to micelles. We evaluated the participation of each residue in the binding process of penetratin by comparing the averaged minimal distance (Dmin) between a residue and any lipid molecule in a micelle at the last 2 ns of the simulation. If the Dmin of a residue is less than 0.35 nm, which is approximately the size of a water molecule, we considered this residue as a binding residue that makes essential contact with a micelle. For PR in the presence of a DPC micelle, charged residues (i.e., Arg1, Arg11, Lys15) and hydrophobic residues (i.e., Trp6, Phe7, Trp14) were binding residues (Figure 3A), indicating that both the electrostatic and hydrophobic interactions were important to binding. However, for Pβ, only charged residues (Arg1, Arg10, and Arg 11) were most important to binding in the presence of a DPC micelle (Figure 3B). In the presence of a SDS micelle, for PR, charged residues (i.e., Lys13, Lys15 and Lys16) and hydrophobic residues (i.e., Trp14) are important to binding (Figure 3C). For Pβ, charged residues (i.e., Lys13, Lys15, and Lys16) are important to binding as shown in Figure 3D. Interestingly, there are a significant number of binding residues, both PR and Pβ, located at the C-terminus of penetratin, indicating a tilted structure necessary for binding a SDS micelle. Particularly for PR, there is a relatively good agreement between our results and a prior NMR study in which the position of the C-terminus of a helical penetratin is positioned deeper inside the micelle than its N-terminus.7b In contrast to SDS, when in the presence of the DPC micelle, the distances between the residues at the N-terminus and the surface of a DPC micelle are nearly the same as the distances between the residues at the C-terminus and the surfaces of a DPC micelle, indicating that penetratin approaches a DPC micelle without a tilted preference. 3.3. Mechanism of Penetratin’s Binding to DPC Micelles. Because differences between PR and Pβ in penetratin’s binding mechanism are most evident when interacting with a DPC micelle as seen from analysis in prior sections, we took this system as an example to investigate the driving forces of PR and Pβ for binding. Temporally averaged electrostatic energy and van der Waals (VDW) potential energy as a function of time are plotted in Figure 4, A and B, respectively. The profile of VDW potential energy for PR decays faster than that of Pβ, while there is little difference in the electrostatic interactions for both PR and Pβ, indicating that the LJ interaction is responsible for a greater binding rate of PR than Pβ as observed in Figure 2 and Table 1. Following the discussion in section 3.2 on the analysis of binding residues, this driving VDW potential energy for PR is attributed to the interactions between its hydrophobic binding residues (Trp6, Phe7, and Trp14) and a zwitterionic DPC micelle, while there are no hydrophobic residue as binding residues for Pβ. The role of these three hydrophobic residues in PR’s and Pβ’s binding to a DPC micelle is illustrated in a typical 20-ns trajectory in Figure 5. For PR, at t ) 1 ns, Trp6 (yellow) makes contact to a DPC micelle. At t ) 10 ns, Trp6 and Arg10 (red) insert into the micelle, while Trp6, Phe7 (orange) and Trp14 (orange) form a hydrophobic core. The hydrophobic surface area of this hydrophobic core involving Trp6, Phe6 and Trp 14 is

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Figure 2. Temporally averaged distance (Dcom) between the center of mass of a peptide and the center of mass of a micelle (DPC or SDS) as a function of time: (A) penetratin with a DPC micelle, (B) penetratin with a SDS micelle, (C) temporin A with a DPC micelle, (D) temporin A with an SDS micelle, (E) KSL with a DPC micelle, and (F) KSL with an SDS micelle. Error values shown are the standard error of the mean.

TABLE 1: Fitted Single Exponential of Temporally Averaged Distance between the Center of Mass of Penetratin and the Center of Mass of a Micelle (Dcom) as a Function of Time (t) in Figure 2 Using Dcom ) A0 exp(-A1t + A2) A0 (nm) A1 (×10-3 ps-1) A2 (nm)

PDR

PDβ

PSR

PSβ

TDR

TDβ

TSR

TSβ

KDT

KDβ

KST

KSβ

0.98 5.45 1.91

1.04 2.91 2.14

1.25 3.67 1.82

1.02 1.33 1.74

0.88 4.62 1.87

1.02 4.37 1.84

1.90 4.54 1.52

1.31 4.13 1.60

1.15 5.00 1.93

0.81 2.28 2.02

1.17 2.23 1.62

1.06 3.50 1.77

1.74 nm2. At t ) 20 ns, the hydrophobic core residues Trp6, Phe7 and Trp14 as well as Arg10 form a complex and insert themselves under the surface of a DPC micelle. As a result, the orientation of lipid molecules of a DPC micelle is interrupted and tiled away from a radial direction. In contrast to the hydrophobic residues in PR that are able to pack themselves closely into a core, and allow favorable cation-π interactions between Trp6 and Arg10 as suggested in an experimental study,36 the same hydrophobic residues in Pβ are unlikely to

make the same impact to binding when they are separated in a rather extended configuration. The hydrophobic surface area of Pβ involving Trp6, Phe7, and Trp14 is 2.15 nm2, increased by 23.6% compared to that of PR. 3.4. Temporin A Binding to DPC and SDS Micelles. Temporally averaged Dcom between temporin A and the micelles is shown in Figure 2C (DPC micelle) and 2D (SDS micelle). For TR and Tβ, differences in the profiles of Dcom regarding binding to SDS and DPC micelles are small. After being fitted

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Figure 3. Average minimal distance (Dmin) between each residue of a penetratin and a micelle in the last 2 ns of the kinetic binding simulations: (A) PR and DPC micelle, (B) Pβ and DPC micelle, (C) PR and SDS micelle, and (D) Pβ and SDS micelle.

with a single exponential (Table 1), the binding rates of TR (4.62 × 10-3 ps-1) and Tβ (4.37 × 10-3 ps-1) are almost the same in the presence of a DPC micelle. In the presence of a SDS micelle, the binding rate for TR was 4.54 × 10-3 ps-1 and for Tβ, 4.13 × 10-3 ps-1. Dcom levels out at 1.9 nm for the DPC micelle and 1.6 nm for the SDS micelle. The minimal distance (Dmin) between each residue of temporin A and the micelles was investigated in Figure S2 (see Supporting Information). In the presence of a DPC micelle, the binding residues of TR are hydrophobic: Phe1, Leu4, Leu9, indicating that the major driving force is the hydrophobic interaction. The same three residues were characterized for Tβ in binding with a DPC micelle. In the presence of a SDS micelle, the hydrophobic residues Phe1 and Gly11 were shown to be essential in binding for both TR and Tβ. These results demonstrate the importance of hydrophobic interaction over electrostatic interactions for temporin A in binding DPC and SDS micelles because all the binding residues are hydrophobic. We further investigated the electrostatic interactions and the LJ potential energy between temporin A and a DPC micelle as a function of time in Figure 4, C and D for TR and Tβ, respectively. There is little difference between TR and Tβ in both parts C and D of Figure 4. In addition, in the presence of a SDS micelles (Figure S3 in the Supporting Information), their differences are insignificant. In contrast to penetratin, the secondary structure of temporin A makes little impact to binding with either DPC or SDS micelles. This may be attributed to the chemical composition of temporin A, which is mostly composed of hydrophobic residues. Therefore, as long as there is a strong hydrophobic interaction between temporin A and the micelles, the selection of secondary structures matters little to binding.

3.5. KSL Binding to DPC and SDS Micelles. Temporally averaged Dcom between KSL and micelles is shown in Figure 2, part E (DPC micelles) and part F (SDS micelles). When fitted by a single exponential (Table 1), in the presence of a DPC micelle, the binding rate for KT binding is 5.00 × 10-3 ps-1, which is approximately 2-fold greater than that for Kβ (2.28 × 10-3 ps-1), indicating that the overall temporal decay of KT is much faster than Kβ. Indeed, when only the LJ potential energy is plotted as a function of time for KT, its profile decays faster than that of Kβ (Figure 4F), while the profiles of electrostatic interactions in Figure 4E remain the same for both KT and Kβ. In contrast, in the presence of a SDS micelle, both the profiles of electrostatic energy and LJ potential energy remain indistinguishable (Figure S5 in the Supporting Information), indicating that the secondary structure of KSL plays a lesser role in binding to SDS micelles. We further investigated the binding residues of KSL when interacting with a DPC micelle in Figure S4, A and B, and with a SDS micelle in Figure S4, C and D, in the Supporting Information. The binding residues of KT are charged (i.e., Lys6, Lys8, Lys10) and hydrophobic (i.e., Val3, Phe5, Phe9), indicating that both electrostatic and hydrophobic interactions are important for KT to bind to DPC micelles (Figure S4A). For Kβ in Figure S4B, the binding residues are charged (i.e., Lys1 and Lys6) and hydrophobic (i.e., Val3 and Phe5). Phe9, however, is not a binding residue for Kβ, indicating that the hydrophobic interaction between Kβ and a DPC micelle is weaker than for KT. In the presence of a SDS micelle, for KT, the charged residues (i.e., Lys1, Lys6, Lys9) and the hydrophobic residues (i.e., Val3, Phe5, Phe9) are binding residues (as shown in Figure S4C), whereas for Kβ, the charged binding

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Figure 4. Interaction between a peptide with distinct secondary structures as initial conditions and a DPC micelle as a function of time: (A) penetratin, electrostatic energy, (B) penetratin, van der Waals potential energy, (C) temporin A, electrostatic energy, (D) temporin A, van der Waals potential energy, (E) KSL, electrostatic energy, and (F) KSL, van der Waals potential energy.

residues are Lys1, Lys2 and Lys6, and the hydrophobic residues are Val3 and Phe5, as shown in Figure S4D in the Supporting Information. A typical 20 ns trajectory of KSL using two distinct secondary structures as initial conditions to bind a DPC micelle is illustrated in Figure 6. The two hydrophobic residues Phe5 and Phe9 are colored in yellow and orange, respectively, for visual guidance. At t ) 1 ns, the two residues in KT collapse and form a hydrophobic core attributed to an increase in the hydrophobic interactions of KT. The hydrophobic surface area involving Phe5 and Phe9 is 1.17 nm2. At t ) 10 ns, Phe5 in KT makes contact to the lipid molecules, interrupting the alignment of lipid molecules. At t ) 20 ns, the hydrophobic core is inserted under the surface of a DPC micelle. In contrast, these two hydrophobic residues stay separated apart in Kβ and the structure of Kβ remains at the surface of a DPC micelle. The hydrophobic surface area of the two residues of Kβ is 1.53 nm2 and it increases by 30.8% when compared to that of KT. 3.6. Free Energy of Peptide Insertion into Micelles. The free energy profiles as a function of the distance between the center of mass of the peptide and the center of mass of a micelle (Dcom) are shown in Figure 7. The free energy differences (∆Gb) between the free energy minimum, often positioned near the micelle surface from Dcom ) 1.5 nm to Dcom ) 2.1 nm, and the free energy near the center of mass of the micelle (Dcom ) 0.7),

can give a reasonable estimate on the free-energy cost of peptide insertion inside a micelle. Despite the selection of peptide and secondary structures, ∆Gb ranges from 150 to 200 kJ/mol for a DPC micelle and from 50 to 125 kJ/mol for a SDS micelle. Such an insurmountably large free energy barrier makes a spontaneous peptide insertion nearly impossible on a nanosecond time scale. It is unlikely that the system can reach the thermodynamic equilibrium positions within several tens of nanoseconds whereas in the kinetic studies the simulation time stops when the trajectories reach plateau. As a result, the position of free energy minima of peptide insertion is slightly off with respect to the position when the trajectories reach plateau in the kinetic study. Interestingly, for each peptide, ∆Gb in a SDS micelle (a negatively charged micelle) is about 75 kJ/mol less than that in a DPC micelle (a zwitterionic micelle) (Figure 7). In order to investigate the key interactions that account for such differences in the free energy barrier, we measured the electrostatic energy (averaged over several trajectories as described in the Methods section) between each peptide and the solvated water molecules at the surface and at the center of a micelle under different micelle conditions as provided in Table 2. In the presence of a DPC micelle, the difference in the electrostatic energy between C-S ) is about the center position and the surface position (∆EDPC 60-90 kJ/mol greater than that in the presence of a SDS micelle

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Figure 5. Illustration of penetratin binding to a DPC micelle. The residue in yellow is Trp6, the residues in orange are Phe7 and Trp14, and the residue in red is Arg10.

Figure 6. Illustration of KSL binding to DPC micelle. The residue in yellow is Phe5 and the residue in orange is Phe9. C-S (∆ESDS ), which is on the same order as the difference in an overall free energy barrier as mentioned above (75 kJ/mol). Thus, our results indicate that the electrostatic interaction from the surface charges of a SDS micelle can greatly reduce ∆Gb by narrowing the difference in the peptide-water electrostatic energy between the surface and the center of a SDS micelle. Next, the secondary structure of a peptide may also affect the free energy of insertion, particularly on the position of free

energy minima. For penetratin in Figure 7A, the free energy minimum for PR insertion (Dcom < 1.8 nm) is closer to the center of a micelle than for Pβ insertion (Dcom > 2 nm). In addition, ∆Gb for PR is about 25 kJ/mol less than Pβ. These results are in agreement with the above binding simulations when Dcom between PR and a micelle is less than that between Pβ and a micelle after a 20 ns simulation. For temporin A, however, there is no clear trend in how the secondary structure affects the

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J. Phys. Chem. B, Vol. 114, No. 43, 2010 13733 closer to the center of a micelle than for Kβ insertion (Dcom > 2 nm) (Figure 7C). In addition, ∆Gb for KT is about 25 kJ/mol less than Kβ. These results are in accord with the earlier binding results, for which Dcom of KT is less than that of Kβ at the final stage in a 20 ns simulation.

Figure 7. Free energy of peptide insertion as a function of the distance (Dcom) between the center of mass of a peptide and the center of mass of micelles (DPC or SDS): (A) penetratin, (B) temporin A, and (C) KSL. The error value, estimated by the bootstrap method, of each data point is less than 0.5 kJ/mol and too small to be shown in the figures.

insertion of temporin A as illustrated in Figure 7B. For KSL, the free energy minimum for KT insertion (Dcom < 1.8 nm) is

4. Discussion 4.1. Secondary Structure Affects the Amphiphilic Peptides’ Binding and Insertion Process. Our investigation demonstrates that the selection of peptides’ secondary structures may play a key role in its ability of binding and insertion (not toxicity), particularly for amphiphilic peptides such as penetratin and KSL. For example, PR binds faster and inserts deeper into DPC and SDS micelles than Pβ. The form of the secondary structures determines residue interactions in a peptide, which dictates its binding and insertion mechanism. In penetratin, formation of Trp6 and Arg10 through π-cation interactions is enabled in an R-helical PR rather than in an extended strand of Pβ. As a result, stronger π-cation interactions may impact the alignment of lipid molecules of a micelle. In addition, the hydrophobic core of PR involving Trp6, Phe7, and Trp14 enhances the hydrophobic interactions between penetratin and the micelle. Therefore, the free energy minimum of PR insertion is found to be under the micelle surface, while Pβ is found at the surface. Our simulation results emphasize the importance of Arg and Trp, consistent with prior experimental studies on the effect of Arg and Trp interactions on lipids, where the two types of amino acids form a complex36 and remain at the surface of the lipid molecules through hydrogen bond formation with lipid head groups.37 Although the types of amino acids important to binding were characterized, the experimental finding on the form of secondary structures pertinent to binding and insertion is still unclear.7a,c-e,26,38 In some experiments using CD, a helix-β transformation of penetratin was found to be necessary,7a while based on another NMR experiment a β-turn was important to penetratin’s insertion process.7e Our results on penetratin based on micelles may not appropriately address the importance of peptide-lipid ratios, nor can micelles adequately represent lipids; nevertheless, our simulations provide a thorough investigation of the biophysical properties of peptides when interacting with membrane-mimetic materials. 4.2. Chemical Composition of a Micelle Affects the Amphiphilic Peptides’ Binding and Insertion. Our results show that the electrostatic interaction between an amphiphilic peptide (rich in Arg and Lys) and a micelle facilitates not only the kinetics of binding but also its insertion into micelles. Both in the CPP and in the AMP families, the positively charged residues Arg and Lys were found to be important when interacting with lipid systems.8,39 In our study, we examined the potential interactions and provided details on how the charged residues bind better in negatively charged micelles. We

TABLE 2: Electrostatic Potential Energy Interaction (E) between a Peptide in Distinct Secondary Structures (shown in Figure 1) and Its Solvated Water Molecules in the Presence of DPC (or SDS) Micellesa PR Pβ TR Tβ KT Kβ

C EDPC

S EDPC

C-S ∆EDPC

C ESDS

S ESDS

C-S ∆ESDS

-871 ( 6 -873 ( 11 -284 ( 4 -264 ( 10 -757 ( 9 -760 ( 4

-1107 ( 6 -1092 ( 5 -437 ( 4 -437 ( 5 -984 ( 8 -999 ( 8

236 ( 12 219 ( 16 -153 ( 8 -173 ( 15 227 ( 17 239 ( 12

-908 ( 5 -927 ( 9 -392 ( 8 -388 ( 3 -757 ( 8 -772 ( 6

-1067 ( 6 -1086 ( 8 -472 ( 10 -471 ( 8 -900 ( 3 -920 ( 5

159 ( 11 159 ( 17 -80 ( 18 -103 ( 11 143 ( 11 148 ( 11

a A superscript “S” represents the location of a peptide at the surface of a micelle and a superscript “C” represents the location of a peptide at the center of a micelle. The difference in the electrostatic potential energy interaction (∆E) is also presented. The unit is in kJ/mol. Error bars are included.

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found that, when a target micelle is neutrally charged (e.g., DPC), it is unlikely for peptides to be found beneath the micelle surface where the hydrophobic tails of lipids are present, due to a stronger electrostatic attraction between a peptide and solvent molecules than van de Waals interactions between a peptide and the hydrophobic lipid tails. However, when a target micelle is negatively charged such as a SDS micelle, the hydrophilic phosphate headgroups of a micelle provide stabilizing interactions with a peptide that attracts peptide insertion. As a result, the free energy barrier ∆Gb instead reduces in the presence of SDS micelles. 5. Conclusion We have employed all-atom molecular dynamics simulation to investigate the influence of the secondary structure of peptides (penetratin, temporin A, and KSL) and the chemical properties of target micelles on the binding and insertion process. Our results demonstrate that for penetratin an R-helical structure better facilitates the binding and insertion process than a β-strand structure in the presence of both DPC and SDS micelles. From comparison of the free energy of peptide insertion for both types of secondary structures, we suggest that the activity of penetratin for insertion is likely to have a small hydrophobic surface area, which may benefit from less disruption of lipid orientation around this hydrophobic core. For temporin A, there is no secondary structure preference in both the binding and insertion processes. Because it is composed of a high percentage of hydrophobic residues, it appears to be irrelevant for temporin A to adopt either of the secondary structures to maximize amphiphilicity. For KSL, a turn shape, which enhances its hydrophobic interaction to a micelle, is more favorable than the β-strand structure. In addition to the selection of secondary structures, the chemical composition of micelles influences the free energy minima of peptide insertion, mainly driven by interactions between a peptide and the hydrophilic head groups. For an amphiphilic peptide with a given set of hydrophobic amino acids, when interacting with membrane or membranemimetic structures, it is likely to adopt a secondary structure with a small hydrophobic surface area. We will further employ bioinformatics studies on membrane-active AMPs and CPPs to compare with existing experimental results. Acknowledgment. M.S.C. thanks the support partly from the Texas Center for Superconductivity at the University of Houston (TcSUH), UH Grants to Enhance and Advance Research (GEAR), and the Materials & Manufacturing Directorate, Air Force Research Laboratory. G.H. gratefully acknowledges support from the Defense Threat Reduction Agency (DTRA). Computations were partly supported by the Texas Learning and Computation Center (TLC2), Texas Advanced Computing Center (TACC), and 2010 IBM Shared University Research (SUR) Award on IBM’s Power7 high performance cluster (BlueBioU) to Rice University as part of IBM’s Smarter Planet Initiatives in Life Science/Healthcare and in collaboration with the Texas Medical Center partners, with additional contributions from IBM, CISCO, Qlogic, and Adaptive Computing. Q.W. thanks Dr. Alexander Mackerell for sharing the SDS coordinate files and Dr. Hugh Nymeyer for the discussion on the interaction cutoffs in Gromacs. M.S.C. thanks Dr. Huey W. Huang for bringing penetratin to her attention. Supporting Information Available: Figure S1: The representation of DPC and SDS molecule. Figure S2: The average minimal distance between temporinA and a micelle in kinetic

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