Surface Activity of Amphiphilic Helical β-Peptides from Molecular

Jan 27, 2009 - Behavior of a Peptide During a Langmuir–Blodgett Compression Isotherm: A Molecular Dynamics Simulation Study. Benedetta Di Napoli , C...
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Langmuir 2009, 25, 2811-2823

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Surface Activity of Amphiphilic Helical β-Peptides from Molecular Dynamics Simulation Clark A. Miller, Nicholas L. Abbott, and Juan J. de Pablo* Department of Chemical and Biological Engineering, UniVersity of Wisconsin-Madison, Madison, Wisconsin 53706-1691 ReceiVed September 9, 2008. ReVised Manuscript ReceiVed NoVember 24, 2008 The surface activity of β-peptides is investigated using molecular simulations. The type and display of hydrophobic and hydrophilic groups on helical β-peptides is varied systematically. Peptides with 2/3 hydrophobic groups are found to be surface active, and to adopt an orientation parallel to the air-water interface. For select β-peptides, we also determine the potential of mean force required to bring a peptide to the air-water interface. Facially amphiphilic helices with 2/3 hydrophobic groups are found to exhibit the lowest free energy of adsorption. The adsorption process is driven by a favorable energetic term and opposed by negative entropic changes. The temperature dependence of adsorption is also investigated; facially amphiphilic helices are found to adopt orientations that are largely independent of temperature, while nonfacially amphiphilic helices sample a broader range of interfacial orientations at elevated temperatures. The thermodynamics of adsorption of β-peptides is compared to that of 1-octanol, a well-known surfactant, and ovispirin, a naturally occurring antimicrobial peptide. It is found that the essential difference lies in the sign of the entropy of adsorption, which is negative for β- and R-peptides and positive for traditional surfactants such as octanol.

Introduction Antimicrobial molecules are thought to act upon bacterial and fungal cells in a variety of ways.1,2 Many of the proposed mechanisms of action involve some interaction with the cellular membrane. A majority of naturally occurring antimicrobial peptides are amphiphilic in nature, having hydrophobic and hydrophilic side chains.3 Amphiphilicity has in fact been used to guide the development of new molecules that are active against microbes. Synthetic β-peptides4,5 offer an attractive class of materials in the search for new antimicrobial peptides. As oligomers of β-amino acids, β-peptides have been shown to exhibit antibacterial6,7 and antifungal properties.8 Their efficiency as antimicrobial agents capitalizes on their resistance to enzymatic degradation;9 only one enzyme has been discovered that degrades pure β- and mixed R,β-peptides.10 When compared to R-peptides, the helical states of β-peptides have been found to be more structurally stable.11 This structural stability, and the ability to manipulate sequence synthetically, enable considerable control over the type and spatial presentation of functional groups in β-peptide helices. The type of helix we consider here, the so-called 14-helix, has 3 residues per turn and presents three distinct faces. In contrast, a traditional R-helix contains 3.6 residues per turn and the * Corresponding author. E-mail: [email protected]. (1) Shai, Y. Biochim. Biophys. Acta: Biomembr. 1999, 1462, 55–70. (2) Theis, T.; Stahl, U. Cell. Mol. Life Sci. 2004, 61, 437–455. (3) Zasloff, M. Nature 2002, 415, 389–395. (4) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001, 101, 3219–3232. (5) Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. BiodiVersity 2004, 1, 1111–1239. (6) Epand, R. F.; Raguse, T. L.; Gellman, S. H.; Epand, R. M. Biochemistry 2004, 43, 9527–9535. (7) Koyack, M. J.; Cheng, R. P. Methods Mol. Biol. 2006, 340, 95–109. (8) Karlsson, A. J.; Pomerantz, W. C.; Weisblum, B.; Gellman, S. H.; Palecek, S. P. J. Am. Chem. Soc. 2006, 128, 12630–12631. (9) Hintermann, T.; Seebach, D. Chimia 1997, 51, 244. (10) Geueke, B.; Heck, T.; Limbach, M.; Nesatyy, V.; Seebach, D.; Kohler, H.-P. E. FEBS J. 2006, 273, 5261–5272. (11) Rathore, N.; Gellman, S. H.; de Pablo, J. J. Biophys. J. 2006, 91, 3425– 3435.

segregation of hydrophobic and hydrophilic groups is not as pronounced. Taken together, all of these attributes make β-peptides an intriguing class of antimicrobial molecules for detailed, molecular-level studies. The overall goal of our work is to characterize at a fundamental level the behavior of β-peptide molecules in contact with model cell membranes. At the experimental level, Langmuir monolayers12 provide a useful means to quantify several aspects of the interaction of surface active molecules with lipids. In order to interpret the results of Langmuir trough experiments, and to distinguish between increases in surface pressure due to preferential association with the lipid monolayer or selfassociation at the air-water interface, it is important to begin by characterizing the behavior of β-peptides at the air-water interface. More generally, the air-water interface is important from the perspective of amphiphilicity, which is generally accompanied by surface activity at the air-water interface. This work presents a computational study of a variety of β-peptides at that interface. The surface activity of amphiphilic peptides can be measured using static or dynamic surface tension (or surface pressure) measurements. Langmuir films of amphiphilic R-peptides have been studied using surface pressure-area isotherms, surface potential measurements, or Langmuir-Blodgett films. There are a number of studies on the interaction of amphiphilic peptides with interfaces; a recent review of the surface activity of peptides that form β-sheets and R-helices can be found by Rappaport.13 Maget-Dana et al.14 used dynamic surface tension measurements and compression isotherms with peptides containing different arrangements of lysine and leucine side chains. They found that R-helices diffused and adsorbed faster to the air-water interface than β-sheets. They also determined the free energy of adsorption, ∆Gads ≈ -1.2 × 10-24 kcal/residue, from the surface pressurearea isotherms for all the peptides they studied. Colfer, Kelly, (12) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109–140. (13) Rappaport, H. Supramol. Chem. 2006, 18, 445–454. (14) Maget-Dana, R.; Lelie`vre, D.; Brack, A. Biopolymers 1999, 49, 415–423.

10.1021/la802973e CCC: $40.75  2009 American Chemical Society Published on Web 01/27/2009

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and Powers15,16 used surface pressure-area isotherms, fluorescence microscopy, and Langmuir-Blodgett films of an amphiphilic peptide forming a β-hairpin and observed the selfassembly of the peptide at the air-water interface. Ambroggio et al.17 measured the surface pressure-area isotherms of R-helical, antibiotic (and amphiphilic) peptides maculatin and citropin interacting with both the air-water interface and lipid monolayers. They observed a ∆Gads of -8.1 kcal/mol for both peptides, and found that both molecules interact more favorably with lipids containing anionic head groups than zwitterionic head groups. Lakshmanan and Dhathathreyan18 investigated systematic mutations of laminin-derived peptides interacting with air-water interfaces and lipid monolayers. They found that modifying a tyrosine side chain to more hydrophobic side chains led to increased surface activity and lipid interaction. Several simulation studies have examined the surface activity of amphiphillic molecules; a recent review of relevant simulation methods is found in ref 19. Past studies20 and more recent investigations21-24 of surface activity have considered a variety of molecules. Shin and Abbott20 used molecular dynamics (MD) simulations and transition-state theory to obtain the free energy of adsorption (∆Gads ) -9.5 kcal/mol) and the desorption rate for 1-decanol. Canneaux et al.24 obtained free energy profiles of ethanol, acetone, and benzaldehyde as they progressed through the air-water interface at 298 and 273 K. They found that ∆Gads was favorable for each of these molecules and became less negative at the lower temperature. For example, ethanol changed from -2.3 kcal/mol at 298 K to -1.6 kcal/mol at 273 K. Minofar et al.22 performed experiments and simulations of sodium formate, acetate, benzoate, and phenolate molecules to investigate changes in surface pressure with concentration. They found that sodium formate increased the surface tension with concentration, similar to electrolytes at surfaces, while the other three decreased the surface tension with concentration, especially the more hydrophobic anions. Carignano et al.23 calculated the free energy of adsorbing and solvating ammonia at 277 K. They found a favorable ∆Gads of -0.7 kJ/mol and a ∆Uads of -2.4 kJ/mol, which corresponds to a T∆Sads of -1.7 kJ/mol. By decomposing the energetic term into the water-water (sol-sol) and water-ammonia (sol-NH3) interactions, they found that ∆Usol-sol < 0 and ∆Usol-NH3 > 0. In other words, a decrease in water-water interaction energy favors the adsorption of ammonia at the air-water interface. Gu et al.21 applied a solvation model to predict the surface activity and ∆Gads of systematically substituted peptides. They achieved a 50% success rate for the peptides that were expected to be surface active (as later verified in experiments). The simulations in this work seek to explore not only the surface activity of β-peptides, but also how amphiphilic peptides in general behave at the air-water interface. In this regard, β-peptides are of interest because of their well-defined structure (15) Powers, E. T.; Kelly, J. W. J. Am. Chem. Soc. 2001, 123, 775–776. (16) Colfer, S.; Kelly, J. W.; Powers, E. T. Langmuir 2003, 19, 1312–1318. (17) Ambroggio, E. E.; Separovic, F.; Bowie, J.; Fidelio, G. D. Biochim. Biophys. Acta 2004, 1664, 31–37. (18) Lakshmanan, M.; Dhathathreyan, A. J. Colloid Interface Sci. 2006, 302, 95–102. (19) Garrett, B. C.; Schenter, G. K.; Morita, A. Chem. ReV. 2006, 106, 1355– 1374. (20) Shin, J. Y.; Abbott, N. L. Langmuir 2001, 17, 8434–8443. (21) Gu, C.; Lustig, S.; Jackson, C.; Trout, B. L. J. Phys. Chem. B 2008, 112, 2970–2980. (22) Minofar, B.; Jungwirth, P.; Das, M. R.; Kunz, W.; Mahiuddin, S. J. Phys. Chem. C 2007, 111, 8242–8247. (23) Carignano, M. A.; Jacob, M. M.; Avila, E. E. J. Phys. Chem. A 2008, 111, 3676–367. (24) Canneaux, S.; Soetens, J.-C.; Henon, E.; Bohr, F. Chem. Phys. 2006, 327, 512–517.

Miller et al. Table 1. β-Peptides Examined for Surface Activity in This Worka peptide 1a 1b 2a 2b 3 4 5

sequence

fphobic

β3-hTyr-[ACHC-ACHC-(β3-hLys)]3 β3-hTyr-[ACHC-ACHC-(β3-hLys)]3 scram β3-hTyr-[ACHC-(β3-hPhe)-(β3-hLys)]3 β3-hTyr-[ACHC-(β3-hPhe)-(β3-hLys)]3 scram β3-hTyr-[ACHC-(β3-hLys)-(β3-hLys)]3 β3-hTyr-[(β3-hLys)-(β3-hLys)-(β3-hLys)]3 β3-hTyr-[(β3-hLys)]3-[ACHC]6

0.67 0.68 0.68 0.69 0.54 0.45 0.67

a We’ve also tabulated the fraction of hydrophobic surface area of the peptides from our calculations.

and their ability to control the placement of specific hydrophilic and hydrophobic groups along the folded molecule. With regard to surface activity, we seek to address two questions: (1) What is the role of hydrophobicity in surface activity? (2) What is the role of global amphiphilicity in surface activity? Using molecular simulations, we determine which peptides are likely to adsorb at the air-water interface, and what are the thermodynamic driving forces for that process. For peptides that exhibit a thermodynamic preference for the air-water interface, we also examine their orientation (and that of distinct chemical groups) relative to the interface. And, given the tendency of R-peptides to change structure upon adsorption,1,12 we also determine whether the structural stability of β-peptides is altered at the interface. In the sections that follow, we describe our choices for side chain functional groups, the simulation methods and models employed in our work, and the properties that are measured to quantify the adsorption process. By dissecting the free energy of adsorption into energetic and entropic components, we propose and test hypotheses regarding the temperature dependence of the free energy of adsorption. We conclude our manuscript with a series of general considerations for the design of peptides that adsorb at the air-water interface.

Methods Peptides to Test. In order to understand the roles of hydrophobicity and amphilicity in driving adsorption at the air-water interface, we have identified a series of β-peptides that vary in the type and presentation of side chains. All the β-peptides considered here adopt a common helical structure called the 14-helix. The 14-helix is formed by hydrogen bonds between atoms CdO(i) and H-N(i-2) on the backbone and has approximately three residues per turn. With this knowledge, we can select the location of the side chains on the three distinct faces of the helix. All of the peptides we have studied (see Table 1) consist of 10 residues and include a tyrosine-like side chain (β3-hTyr) at the N-terminus and at least three hydrophilic lysine-like side chains (β3-hLys). The majority of the peptides that we simulate contain the cyclic residue, trans-2-aminocyclohexanecarboxylic acid (ACHC), which has been shown to stabilize the 14-helix conformation.25 Peptide 1a has three hydrophilic β3-homolysine side chains and six hydrophobic ACHC side chains. This particular sequence leads to a facial segregation of the hydrophilic and hydrophobic groups, with one hydrophilic face and two hydrophobic groups. Peptide 1b also has three hydrophilic β3-homolysine side chains and six hydrophobic ACHC side chains, but differs from 1a by the sequence of the side chains, and therefore the presentation of the hydrophilic and hydrophobic groups. We refer to this display of side chains as “scrambled” amphiphilicity, because one hydrophilic side chain is presented on each face. Peptides 2a and 2b have three hydrophilic β3-homolysine side chains, three hydrophobic ACHC side chains, and three hydrophobic β3-homophenylalanine side chains. The (25) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071–13072.

Surface ActiVity of β-Peptides sequences of these two peptides were chosen to result in facial amphilicity for sequence 2a, and scrambled amphiphilicity for sequence 2b. Peptide 3 has six hydrophilic β3-homolysine side chains and three hydrophobic ACHC side chains with facial amphiphilicity, and peptide 4 has nine hydrophilic β3-homolysine side chains and no hydrophobic ACHC side chains. Peptide 5 is similar to 1a and 1b in the side chains selected, but the sequence is chosen with the hydrophilic β3-homolysine side chains near the positively charged N-terminus. This display of hydrophilic groups is referred to as “end” amphiphilicity. By selecting these particular side chains and sequence, a direct comparison of the following features becomes possible: • Facial (1a) versus scrambled (1b) amphiphilicity versus end (5) amphiphilicity; • 2/3 (1a) versus 1/3 (3) versus 0 (4) hydrophobicity; • ACHC (1a, 1b) versus phenylalanine (2a, 2b) hydrophobicity. We note that peptides 1a, 1b, 2a, and 2b were the subject of previous simulations of the mechanical stability26 and association processes27 that provide a foundation for the adsorption studies presented in this work. Simulation. Biased and unbiased MD simulations are used to investigate the behavior of these peptides at the air-water interface. The CHARMm2728,29 all-atom force field was used to model our β-peptides. For a complete description of the parameters employed in our work, readers are referred to our previous simulations of β-peptides.11,26,27,30 A 1-3 exclusion principle was used for nonbonded interactions and the 1-4 Coulombic interactions were scaled by a factor of 0.4 to be consistent with the CHARMm force field. The TIP3P model31 of water was selected because it is compatible with the CHARMm force field. In solution, these peptides are expected to have protonated β3-homolysine residues and N-termini, with each peptide having a positive charge. To counter the positive charge of each peptide, chloride counterions were included in the simulation cell. Simulations were performed and analyzed using the GROMACS32-34 simulation package. Lennard-Jones (LJ) interactions were truncated with a twin-range scheme at 10 and 15 Å. The electrostatic interactions were calculated using a particle mesh Ewald technique35 with a short-range cutoff of 10 Å, a maximum relative error of 10-5, and a fourth-order spline. A time step of 0.002 ps was used along with LINCS36 to constrain all bond lengths to their equilibrium value. The procedure and setup of the simulation cell is described below. NVT Simulation and System Setup. The molecular simulations were started by solvating a single charged, helical β-peptide in TIP3P water (>1800 molecules) using the GROMACS utility editconf in a cubic box having dimensions of 4 nm. Then, the correct number of chloride counterions (to maintain electroneutrality) were placed at the most electrostatically favorable positions using genion. After a brief energy minimization of 500 steps using the steepest descent algorithm, we performed an NPT-ensemble equilibration run of 1 ns using isotropic pressure scaling and a Berendsen thermostat and barostat37 at P ) 1 bar and T ) 300 K. The simulation cell was then (26) Miller, C. A.; Gellman, S. H.; Abbott, N. L.; de Pablo, J. J. Biophys. J., in press. (27) Miller, C. A.; Abbott, N. L.; Gellman, S. H.; de Pablo, J. J. Biophys. J., submitted for publication. (28) Foloppe, N.; MacKerell, A. D., Jr J. Comput. Chem. 2000, 21, 86–104. (29) MacKerell, A. D., Jr J. Phys. Chem. B 1998, 102, 3586–3617. (30) Miller, C. A.; Herna´ndez-Ortiz, J. P.; Abbott, N. L.; Gellman, S. H.; de Pablo, J. J. J. Chem. Phys. 2008, 129, 015102. (31) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926–935. (32) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7, 306–317. (33) van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701–1718. (34) Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. Comput. Phys. Commun. 1995, 91, 43–56. (35) Essman, U.; Perela, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 193, 8577–8592. (36) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1997, 18, 1463–1472. (37) Berendsen, H. J. C.; Pstma, J. P. M.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684–3690.

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Figure 1. MD simulations are set up with the peptide at the center of a rectangular box along with a water layer of approximately 4 nm. The bottom part of the figure shows the initial density distribution of peptide 1a. The simulation cell is periodic in all directions. The density data was obtained from the first 500 ps, over which the peptide is not seen to insert at the interface (see Figure 2).

adjusted by recentering the peptide at the center of the box and applying periodic boundary conditions in all directions. The simulation box size was then changed in the z-dimension to 12 nm. The initial density distribution of the system and a snapshot of the starting configuration of peptide 1a are given in Figure 1. Having completed the initialization process just outlined, a 10 ns simulation was performed in the NVT ensemble at 300 K. Potential of Mean Force Calculation. In order to obtain quantitative estimates of the free energy of adsorption, we calculated the potential of mean force (PMF) required to move the center of mass (COM) of the peptide across the film of water, from the middle of the box to the air-water interface. The reaction coordinate, ξ, is defined as the z-distance from the COM of the peptide to the COM of the water film. The free energy (or PMF) was determined using the constraint force (CF) method,38,39 which makes use of the following:

w(ξ) )

∫ξξ 〈f(ξ)〉ξ dξ + C 0



(1)

Here, w is the PMF, ξ is the reaction coordinate, and fξ′ is the force required to constrain the reaction coordinate. The integration constant C can be chosen such that the free energy vanishes when the reaction coordinate is zero, ξ ) |zpep - zwat| ) 0. By doing so, the PMF represents the difference between the free energy when the peptide is in the bulk phase and that at the interface. The simulation was initialized by taking the starting configuration from the unbiased NVT simulations and pulling the COM of the peptide with a constant velocity (0.025 nm/ps) over 120 ps in the NVT ensemble. Configurations were saved every 4 ps and used as starting points for the PMF calculation. In the NVT ensemble at 300 K, we performed simulations at each value of ξ ranging from 0.0 to 2.9 nm and spaced every 0.1 nm. A force was applied at every time step to constrain the peptide at each particular choice of the reaction coordinate. The value of this force was saved every 0.05 ps and then averaged and used in eq 1 to obtain the PMF. The simulations ran for 2 ns, and data were accumulated over the last 1.5 ns. Configurations and energies were saved every 1 ps to calculate (38) Trzesniak, D.; Kunz, A.-P. E.; van Gunsteren, W. F. ChemPhysChem 2007, 8, 162–169. (39) Mastny, E. A.; Miller, C. A.; de Pablo, J. J. J. Chem. Phys. 2008, 129, 034701.

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Miller et al.

Figure 2. MD simulations of peptides 1-5 showed some of the peptides going to the air-water interface and staying there for the remainder of the simulation. The top set of graphs corresponds to the z-distance from the center of the water layer. The bottom set of graphs corresponds to the angle of the peptide with the z-axis. The shaded region in the top plot shows the approximate location of the interfacial region.

the properties of the system. We also performed longer simulations (10 ns) for peptide 1a and found no significant change in the results. Error estimates were obtained by taking from the force data 100 random samples having the same length of the data, reintegrating eq 1, and determining the value of two standard deviations at each value of the reaction coordinate. Peptide-Water Properties. In order to characterize the peptide-interface interactions more closely, we calculated the solvent accessible surface area, potential energies, angle with the interface, and location of the peptide and interface. We also determined the structural stability of the peptide and the number of hydrogen bonds in the system. The peptide stability was determined by calculating the end-toend distance and helicity of the molecules. The end-to-end distance was defined as the distance from the nitrogen of the N-terminal residue to the carbonyl carbon of the C-terminal residue. In order to avoid the fluctuations inherent to the first and last residues, the second and penultimate residues were selected. The helicity was calculated from the dihedral angles φ (C(dO)-N-Cβ-CR) and ψ (Cβ-CR-C(dO)-N) using the expression

Hdih )

∑ φ,ψ HφHψ Nφ + Nψ

(2)

where Nφ and Nψ are the number of φ and ψ angles. The quantity Hφ is defined as

{

if |φ - φo| e a |φ - φo|-a if a < |φ - φo| e b Hφ ) 1 b-a 0 if |φ - φo| > b 1

(3)

and a similar definition was used for Hψ. The parameters a, b, φo, and ψo depend on the type of helix. For the 14-helix, a ) 20°, b ) 39°, φo ) -135°, and ψo ) -140°, while for the 12-helix, a ) 20°, b ) 39°, φo ) 95°, and ψo ) 103°. It was particularly instructive to evaluate the overall potential energy and its various contributions, including LJ (ULJ) and electrostatic energies (Ucoul). We also determined the intermolecular interactions between peptide-peptide atoms (Upp), peptide-solvent atoms (Ups), and solvent-solvent atoms (Uss). The angle of the peptide with the interface was obtained by taking the dot product of the end-to-end vector (defined as described above) with the normal vector to the interface (the z-axis). The solvent accessible surface area of the peptide was determined using the GROMACS utility g_sas, which uses a method reported by

Eisenhabaer et al.40 Hydrophobic atoms were defined as having an absolute charge less than 0.2e, where e is the electronic charge. The position of the interface was determined by first obtaining a density profile (of the water atoms) along the z-axis with bins of 0.1 nm. The location of the interface, zint, was found by interpolating to the z-position where the water density was 500 kg/m3. The position of the peptide, zpep, was defined by the z-coordinate of the COM of the peptide atoms.

Results Surface Adsorption in Unbiased MD Simulation. We performed MD simulations of all the peptides (1-5) starting in the center of the water layer for 10 ns, and determined whether they migrate to the interface. The z-coordinate of the center of mass of the peptide over time is shown in the top row of Figure 2. Because the simulation contains two interfaces, the peptides may go to one or the other interface. By taking the absolute value of the z-distance, we obtain a more concise presentation of the results. We observe that only peptides 3 and 4 fail to reach the interface. The MD simulations of 3 and 4 were continued for 15 ns, and did not reveal any evidence for adsorption at the interface. In contrast, all the peptides that do adsorb at the interface remain there for the duration of the simulation. Figure 2 shows the angle the peptide makes with the z-axis as described in the Methods section. The peptides adopt highly specific orientations at the interface. In contrast, the peptides in the bulk water phase explore a much wider range of orientations. This behavior is especially apparent for peptides that never reach the interface during the simulation time, 3 and 4. Once adsorbed, the peptides make an approximately 90° angle with the z-axis and lie parallel to the interface. The position of the interface (zint) and the COM of the peptide (zpep) were determined for the last 2 ns of each simulation. In all cases, the peptides are positioned near the water side of the interface; by taking the absolute value, we remove the effect of having two interfaces. Probability distributions of the distance between the peptide and the interface are shown in Figure 3 for each peptide that adsorbs at the interface. The peptides that are closest to the interface are the facially amphiphilic peptides 1a and 2a. Amphiphilic peptides that do not display facial amphiphilicity (1b and 2b) are 1-2 Å deeper into the water phase. The end-amphiphilic peptide 5 is just below them. (40) Eisenhaber, F.; Lijnzaad, P.; Argos, P.; Sander, C.; Scharf, M. J. Comput. Chem. 1995, 16, 273–284.

Surface ActiVity of β-Peptides

Figure 3. The probability distribution of the z-distance of the peptide relative to the air-water interface from MD simulations of peptides 1-5. Shown here are only those peptides that go to the air-water interface and stay there for the remainder of the simulation. Data for this plot came from the last 2 ns of the simulations in Figure 2.

Figure 4. Free energy profile for selected β-peptide sequences moving from the bulk water phase through the air-water interface calculated from MD simulations. Each profile contains a minimum between 1.5 and 2 nm where the peptide is located closest to the interface.

With regard to the degree of hydrophobicity, we determined the fraction of hydrophobic surface area of the peptides from the first 2 ns of the simulation. These results are summarized in Table 1. Each of the peptides with 2/3 hydrophobic residues also displayed a surface area that was approximately 2/3 hydrophobic. Adding three more hydrophilic β3-homolysines (3) brought the hydrophobic fraction to 0.54, and having six more β3-homolysines (4) brought the hydrophobic fraction to 0.45. As expected, hydrophobicity plays a role in governing whether the peptides adsorb at the interface. Our results indicate that 2/3 of the side chains must be hydrophobic for favorable adsorption at the air-water interface. Furthermore, we see little difference between the adsorption of peptides whether ACHC (1) or β3-homophenylalanine (2) residues are used for the hydrophobic groups. Amphiphilicity also plays a role in the observed depth of the peptide relative to the interface, and, as shown below, influences the angle of the peptide with the z-axis. Free Energy of Adsorption. While the fact that the peptides go to the surface during our short MD simulation provides some insight into the adsorption process, it is of interest to quantify the thermodynamic driving forces that give rise to that process. This information can be extracted from the PMF required to move the peptides from the bulk water phase through the interface and beyond. Figure 4 shows the PMF for peptides 1a, 1b, 3, and 5. The PMF for each peptide exhibits three characteristic regions: (I) bulk water region; (II) interfacial region; (III) increase from the optimal position. Region I is characterized by a flat free energy value. Region II corresponds to a decrease in the free energy or the appearance of a driving force that localizes the peptide at some optimal

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position at the interface. Region III is characterized by an increase in the free energy beyond that corresponding to the bulk water. Only peptide 3 exhibits a slight maximum as it approaches the interface from the water side. This maximum is reminiscent of but much weaker than that reported by Shin and Abbott20 for decanol. The PMF can be decomposed into energetic and entropic terms according to w ) ∆U - T∆S. The energetic and entropic terms are given in Figure 5 and Table 2. The change in energy, ∆U is obtained by calculating a time average of the overall potential energy at each position and then taking the difference relative to the peptide being at the center of the water layer. The error was estimated by taking 1000 random samples of the time series of potential energies, determining the averages of those 1000 samples, and then reporting two standard deviations of the ensemble of the averages as the error. The entropy was obtained using the formula, T∆S ) ∆U - w. The error reported for the entropy is taken as the largest error associated with the two quantities, ∆U and w. The estimated error of the PMF is less than 1 kcal/mol, while the estimated errors of the energy and entropy are 3 kcal/mol. Smaller variations in energy and entropy exhibited during adsorption are not statistically significant. Note, however, that the overall trends observed in our simulations are significant, particularly regarding whether the entropy or energy changes are positive or negative. Peptide 1a exhibits the largest ∆Gads; it also has the largest ∆Uads and a relatively small T∆Sads. In contrast, peptide 3, which has the lowest ∆Gads, does not have the smallest ∆Uads. Its ∆Uads is in fact relatively favorable, at -10.6 kcal/mol. The energetic term is largely offset by a large unfavorable entropic term T∆Sads (-9.24 kcal/mol). The peptide with the smallest ∆Uads is 1b, which also happens to exhibit the smallest T∆Sads (with a value near zero). The adsorption of 1a is largely driven by energy (it has a significant ∆Uads and a small T∆Sads). Peptide 5 exhibits a moderately favorable value of ∆Uads and a large negative value of T∆Sads that leads to a favorable free energy of adsorption. Peptides 1b and 5 both have one hydrophilic side chain on each face, but placing all of the hydrophilic side chains at the N-terminus (as in 5) makes the adsorption more energetically favorable. We also note that the minimum value in the energy or entropy does not correspond to the minimum in the free energy. Taking 1a as an example, the free energy minimum occurs at 1.8 nm; the minima in energy and entropy occur at 1.5 nm and at 1.2 nm, respectively. Similar behavior is also observed in the other peptides. As mentioned above, peptides 1a, 3, and 5 exhibit a decrease in entropy upon adsorption at the air-water interface. Previous simulations23 of ammonia at the air-water interface have also revealed a decrease in entropy.23 This drop in entropy, or increase in order, could have two origins: the entropy of the peptide or the entropy of the solvent. Our unbiased MD simulations suggest that the peptides in the water phase can explore a variety of orientations, but they adopt a well-defined orientation at the air-water interface. As the peptides move to the air-water interface, their orientational entropy decreases. Similarly, peptide translation becomes restricted in one dimension, but not in the other two. The translational entropy of the peptide is therefore expected to decrease as well. In previous work,26 we observed that the solvent entropy makes an important contribution to the mechanical stability of β-peptides. This solvent entropy can be attributed to the solvation of a hydrophobic surface. In a similar manner, the solvent entropy plays a role in the adsorption process. We hypothesize that by moving the hydrophobic surface from the bulk water to the interface, the order of the solvent decreases and the entropy increases.

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Figure 5. The energetic (∆U) and entropic (T∆S) contribution to the calculated free energy profile in Figure 4. The energy is taken from the average potential energy of the simulations while the entropy comes from w ) ∆U - T∆S. When the peptide is near the interface, both contributions reach a minimum value.

Figure 6. The Lennard-Jones and Coulombic contributions to the nonbonded potential energy of the system from the free energy calculations of the peptides moving through the interface. Only the Coulombic contribution contains a minimum in the potential energy. Table 2. Thermodynamics of Adsorption of β-Peptides at the Air-Water Interfacea peptide 1a 1b 3 5

∆Gads

∆Uads

T∆Sads

∆Uads, pp

∆Uads, ps

∆Uads, ss

-15.0 (0.92) -6.48 (0.81) -0.75 (0.99) -4.73 (0.99)

-17.9 (2.68) -6.38 (2.46) -10.6 (2.60) -13.3 (2.76)

-2.7 (2.68) 0.10 (2.46) -9.24 (2.60) -8.53 (2.76)

1.57 (0.38) 9.85 (0.38) -3.65 (0.33) -1.23 (0.27)

37.1 (1.14) 21.6 (1.10) 44.5 (1.16) 43.4 (0.60)

-57.7 (3.3) -39.0 (2.9) -52.6 (3.1) -58.0 (3.3)

a Simulations were performed to obtain the PMF of moving from bulk water through the interface, and the difference in energies from the bulk water to the minimum in the free energy are shown above. Energies are in reported in kilocalories per mole. The numbers in parentheses represent error estimates of two standard deviations from the mean. These results are obtained from the minimum in free energy from the PMF calculation.

Another possible origin of the change in water entropy is the structure of the water at the liquid-vapor interface. Recent theoretical studies41,42 of the contributions to vibrational sumfrequency spectroscopy suggests that, near the interface, the spectrum is largely a result of water molecules with two or three hydrogen bonds. This suggests that the entropy of the interfacial water molecules may actually decrease when they are displaced by the peptide and pushed into the bulk water phase. This hypothesis is strengthened by the observed increase in water-water hydrogen bonds upon adsorption of the peptide shown in Figure 9. It is difficult to know whether the overall effect of hydrogen bond formation may lead to positive or negative entropy changes upon adsorption of the peptides. We surmise that there are two competing influences on entropy;a decrease in the peptide entropy and an increase in the solvent entropy;during the adsorption of amphiphilic molecules at the air-water interface. In the case of peptide 1b, these two forces are balanced in the same manner as when the peptide is in the bulk water. For the other three peptides, the change in entropy is negative, which suggests that the decrease in peptide entropy is greater than the increase in solvent entropy. The nonbonded energy can be decomposed into the LJ energy (∆ULJ) and the electrostatic energy (∆Ucoul). The corresponding (41) Auer, B. M.; Skinner, J. L. J. Chem. Phys., submitted for publication. (42) Auer, B. M.; Skinner, J. L. J. Phys. Chem., submitted for publication.

curves from the PMF calculation are plotted in Figure 6 for the four β-peptides considered here. In all cases, the LJ energy generally increases as the peptide approaches the interface and has a regular slope beyond 1 nm from the center of the water layer. The LJ energy increases as the peptide leaves the water because there are less atoms in close contact. The LJ energy begins to change from its bulk value even when the peptide is within 1 nm of the interface. At this depth, it is unlikely that the peptide atoms have begun to leave the water layer since the size of the peptides is roughly 1 nm. The Coulombic energy decreases as the peptide approaches the minimum in free energy but then increases again. The minimum in total potential energy occurs because the minimum in Coulombic energy is greater than the increase in LJ energy. This suggests that the favorable energetic term is largely due to favorable electrostatic forces. We may think of this in terms of two competing effects. First, the unfavorable electrostatic nature of solvating nonpolar, hydrophobic groups in a polar solvent, and second, the favorable electrostatic nature of solvating polar, hydrophilic groups. The nonbonded energy can also be decomposed (Figure 7) in terms of peptide-peptide energy (∆Upp), peptide-solvent energy (∆Ups), and solvent-solvent energy (∆Uss). Here we observe that the peptide-peptide energy does not vary much as the peptide approaches the interface. As expected, the peptide-solvent energy increases as the peptide leaves the water phase by at least 1 order

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Figure 7. The nonbonded energy divided into interactions between peptide and water atoms from the PMF calculation of four β-peptides. The peptide-peptide energy (∆Upp) changes little, the peptide-solvent energy (∆Ups) increases, and the solvent-solvent energy (∆Uss) decreases as the peptides move to the interface.

Figure 8. The change in number of hydrogen bonds between peptide and water from the PMF simulations. Also shown is the correlation between the change in peptide-water hydrogen bonds and the change in peptide-water energy. The symbols come from the simulation data, while the lines are linear fits to the data for each peptide.

of magnitude compared with the peptide-peptide energy. The solvent-solvent energy decreases significantly as the peptides approach the interface, and then levels off once the peptide has passed the interface. Beyond the interface, the water atoms are no longer affected by the peptide and reach a somewhat constant level. These potential energy contributions to the value at the minimum in free energy are given in Table 2. Previous simulations23 of ammonia at the air-water interface also reported a favorable solvent-solvent energy (∆Uss < 0) and unfavorable solvent-solute energy (∆Us-NH3 > 0) at the interface. We emphasize that adsorption of these β-peptides is largely driven by a favorable energetic term. We noted that it is the electrostatic energy that decreases and not the LJ energy upon adsorption. We have also seen how the peptides adsorbing at the interface is accompanied by a favorable decrease in solvent-solvent energy. Taken together, these results indicate that the peptide adsorption is largely driven by favorable solvent-solvent interactions that could be electrostatic in nature. These electrostatic interactions may be related to a change in the number of hydrogen bonds of the system. The number of hydrogen bonds between the peptides and water was characterized as described above, and the result is

Figure 9. The change in number of hydrogen bonds between water from the PMF simulations.

given in Figure 8. The change in number of hydrogen bonds is plotted relative to the number of hydrogen bonds in bulk water, and is observed to decrease for all four β-peptides. We found a negative linear correlation between ∆Nhbonds and ∆Ups shown in Figure 8. As the peptide loses hydrogen bonds with the solvent, it also loses favorable peptide-solvent energy. The slopes for those correlations are very similar for all four peptides. The

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Figure 10. Angle of the peptide with the air-water interface. The distributions shown here are at the minimum in the free energy. The peptides adopt orientations near 90° with the normal to the interface (or parallel to the interface). Table 3. Properties of the β-Peptides at the Interfacea peptide 1a 1b 3 5

∆Nhbonds

|zpep - zint|

θz

-1.8 (0.14) 0.51 (0.14) -3.4 (0.13) -4.0 (0.11)

0.281 0.388 0.566 0.351

95.25 93.86 110.41 87.19

a Shown in the table are the change in number of peptide-water hydrogen bonds (compared to the bulk case), the depth of the minimum relative to the location of the interface, and the angle of the peptide with the z-axis. These results are obtained from the minimum in free energy from the PMF calculation. Distances are reported in nm and angles in degrees. The numbers in parentheses represent error estimates of two standard deviations from the mean.

slope for peptide 1a corresponds to a value of 13 kcal/mol for 1 hydrogen bond. In Table 3 we have tabulated ∆Nhbonds at the minimum in free energy and show a decrease of zero to four hydrogen bonds upon adsorption at the air-water interface. The number of hydrogen bonds between water is presented in Figure 9 as a function of distance of the peptide from the water layer. The number of water-water hydrogen bonds are observed to increase by between 8 and 12 hydrogen bonds when the peptides are at the interface. This increase has been discussed previously, but may be due to a drop in the number of interfacial water molecules, which have been shown41,42 to form less hydrogen bonds than bulk water molecules. As with the unbiased MD simulations, we have also investigated the presentation of the peptides at the interface by examining the depth relative to the interface and the angle the end-to-end vector makes with the z-axis. Figure 10 shows the probability distribution of the angle from the simulation box at the minimum. Table 3 lists the average angle at the minimum and the depth relative to the position of the interface. Again, the peptides adopt close to a 90° angle with the z-axis (parallel to the interface). The peptide with the largest free energy of adsorption, 1a, also exhibits the closest approach to the interface. The peptide with the lowest free energy of adsorption, 3, is also the furthest from the interface. It is interesting to examine the manner in which the peptides without facial amphilicity present themselves at the interface. The optimal location at the interface for peptides 1b and 5 is deeper into the water than that of the facially amphiphilic isomer, 1a. In Figure 11 we present a representative snapshot of the peptides at the minimum configuration. Peptide 1a is observed to have the hydrophobic face sticking partially out of the water phase. For peptides 1b and 5, the hydrophobic face that is turned toward the air contains lysine residues. This orientation can be satisfied in both of these peptides by having the positively charged lysine side chains wrap around the peptide and into the water phase, as seen in Figure 11, and, as noted in our prior discussion,

by not approaching the interface too closely. Because of the presentation of the hydrophilic groups at the N-terminus, one might have expected an orientation of peptide 5 perpendicular to the interface (peptide 5 is “end” amphiphilic). Our simulations do not suggest that peptide 5 can orient itself perpendicular to the interface. It is of interest to point out that experimental evidence for R-helices suggests that assembled monolayers at the air-water interface lie parallel to the interface, even when compressed to large surface pressures.43 In our work, we have only considered infinitely dilute surface concentrations of peptides and not the monolayers examined in ref 43. Peptide 5 may orient differently in a monolayer, which would be an interesting idea to consider in subsequent investigations. Figure 12 shows the average helicity of the peptide as a function of distance along the z-axis. All four peptides are structurally stable as they diffuse to the interface. Peptide 3 has a lower average helicity compared to the other three peptides, but it contains only 1/3 ACHC residues (versus 2/3 ACHC for the others). The decrease in helical content with a decrease of ACHC residues is consistent with our previous work.26 The β-peptides tested here are stable, both in solution and at the interface; in contrast to some other antimicrobial peptides, we do not observe major changes in secondary structure upon adsorption.12,1 The structure in solution is therefore a reliable predictor for the presentation of side chains at the interface. Note that this result is specific to ACHC-containing β-peptides and may differ when other groups are considered. The results of PMF calculations and the above analysis are summarized in Table 2. Several general conclusions can be drawn from that Table. At least 2/3 hydrophobic residues are necessary for favorable air-water surface activity. When it is favorable for the peptides to be at the interface, they adopt a parallel orientation to the interface, irrespective of the presentation of charged side chains. Two competing entropic forces, peptide entropy and solvent order influence the adsorption process. We also find evidence for the role of energetic forces, including favorable electrostatic energy change upon adsorption and favorable nonbonded interactions between water atoms, which drives the adsorption at the interface. We find that peptides with hydrophilic side chains on each face can still exhibit a favorable free energy of adsorption by not approaching the interface too closely and by inserting all the positively charged side chains into the water phase. Arranging the hydrophilic groups at the N-terminus does not induce the peptide to stand perpendicular to the interface in dilute solution. Temperature Effects. The energetic and entropic terms to the free energy of adsorption suggest that energy is the favorable component driving the adsorption of β-peptides. By assuming negligible changes in ∆Uads and ∆Sads with temperature, an increase in temperature should increase the magnitude of the T∆Sads term, and therefore decrease the magnitude of the free energy of adsorption, making it less favorable for the peptides to be localized at the interface. We also saw above that the adsorption of 1a is largely an energetic process, with little relative entropy gain compared to the other peptides. We therefore speculate that there should be little or no change in the free energy of adsorption with temperature for this peptide. In order to investigate how temperature affects the properties at the air-water interface, we performed unbiased MD simulations of the four peptides from PMF calculations at 270, 300, and 330 K. Starting from the final minimum free energy configuration of the PMF calculation, the peptide-solvent system ran for 20 ns in the NVT ensemble. (43) Boncheva, M.; Vogel, H. Biophys. J. 1997, 73, 1056–1072.

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Figure 11. Snapshots of the peptides at the minimum in the free energy. The three peptides have the same type of side chains and are 2/3 hydrophobic. They differ in their presentation of the hydrophilic residues. Peptide 1a is facially amphiphilic, 1b is “scrambled” with one hydrophilic residue on each face, and 5 is end amphiphilic. The hydrophilic residues are in blue with the hydrophobic residues in green.

Figure 12. The average helicity (14-helix) of the peptides as the peptide moves from the bulk (0 nm) through the interface (∼1.7 nm). The interface has little effect on the helicity of the peptides.

Figure 13 shows the probability distribution of the difference between the COM of the water film and the COM of the peptide, |zpep - zwat|. For peptides 1a and 3, we observe an outward shift of the location of the peptide relative to the center of the water layer. Peptide 1b does not exhibit this outward shift, but seems to place itself in the same position relative to the center of the water layer at each temperature. We find a similar trend for peptide 5 as we saw for 1b. The density distribution and location of the air-water interface changes with temperature; at lower temperatures the bulk density is higher, and the air-water interface moves closer to the center of the water film. This shift in location of the interface is taken into consideration measuring the position of the peptide relative to the interface, |zpep - zint|; the results are shown in Figure 14. Consistent with an energetically driven process, the position of peptide 1a relative to the center of the water shifted outward, but the position relative to the interface remained unchanged. This peptide exhibits only a slight broadening of the probability distribution with increasing temperature. We observe a much more pronounced broadening of the probability distribution for peptide 1b that is accompanied by an overall shift away from the interface as the temperature increases. Peptide 3 remains at the same relative position, with a broadening of the probability distribution. Peptide 5 exhibits a large shift away from the interface from 270 to 300 K, but there is little difference between the probability distributions at 300 and 330 K. Figure 15 shows the probability distribution of angles with the z-axis. Peptide 1a exhibits a narrow angular probability distribu-

tion near 90° that does not vary with temperature. In contrast, we find that the distribution of peptide 1b becomes broader and shifts closer to 90° with increasing temperature. The peptide is allowed more orientational flexibility at the interface at higher temperatures, but that is also coupled to a peptide position further from the interface. The angular distribution of peptide 3 is also narrow, with a distribution slightly off of 90° that does not vary with temperature. Peptide 5 exhibits an angular distribution that broadens considerably with increased temperature. In fact, some of the angle population indicates that the peptide begins to align close to 30° with the z-axis at 330 K, which corresponds to the peptide aligning almost perpendicular to the interface. Our PMF calculations at 300 K did not explore these conformations. On the basis of these temperature studies, it would appear that this transition from parallel to perpendicular orientation is induced by increasing temperature. Combining this result with the probability distribution seen in Figure 14, we hypothesize that by adopting the almost perpendicular orientation (at 330 K), the peptide can position itself closer to the interface such that it has a distribution similar to that at 300 K. Peptide 1a adsorption is driven by energy and is not sensitive to temperature. In contrast, the adsorption of peptides 1b, 3, and 5 represents a balance between entropy and energy, and it does exhibit a temperature dependence, with higher delocalization at elevated temperatures. The results here can be compared to the simulations of Canneaux et al.24 on small amphiphilic molecules such as ethanol. They determined the free energy of adsorption and found that increasing temperature leads to a more negative ∆Gads. Here, we observe that increasing the temperature leads to less negative ∆Gads. The influence of temperature is also manifest on the peptide’s angle distributions, which range from little change (1a) to considerable change (5) depending on the peptide sequence. Clearly, the effects of temperature are consistent with the PMF calculation and indicate a complex relationship between temperature, position at the interface, and angle at the interface. Comparison to Surfactant and r-Peptide Adsorption. It is of interest to compare the behavior of surface-active β-peptides to that of other surfactant systems for which much more is known.44,45 In general, the measured experimental thermodynamic behavior44 of the adsorption of surfactants at air-water (44) Rosen, M. J. Surfactants and Interfacial Phenomena; John Wiley and Sons: New York, 1989.

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Figure 13. The probability distribution of the z-distance between the peptide, zpep, and the center of the water layer, zwat, at various temperatures from unbiased NVT simulations.

Figure 14. The probability distribution of the z-distance between the peptide, zpep, and interface, zint, at various temperatures from unbiased NVT simulations.

interfaces displays a favorable free energy of adsorption (∆Gads < 0), a favorable enthalpy of adsorption (∆Hads < 0), and a favorable entropy of adsorption, ∆Sads > 0. The increase in entropy of surfactants has been thought to arise from two sources, namely the entropy of solvating the hydrophobic part of the molecule and the configurational entropy of the hydrophobic tail itself.45 We consider, as an example, surfactants containing a long hydrophobic tail, such as sodium dodecyl sulfate (SDS) or a

long-chain n-alchohol. In these cases, the solvation of the entire molecule induces a more ordered water structure surrounding the hydrophobic tail. Upon adsorption at the interface, the water structure is “released”, and the entropy increases. The tail itself is also thought to adopt more conformational flexibility in the oil or vapor phase, and so increases entropy as well. For many surfactants, increases in temperature have been shown to decrease ∆Gads, making adsorption more favorable.44

(45) Surfactants: Chemistry, Interfacial Properties, Applications; Fainerman, V. B., Mobius, D., Miller, R., Eds.; Elsevier: New York, 2001.

To make a direct comparison to the peptides examined here, we also simulated the free energy of adsorption for 1-octanol

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Figure 15. The probability distribution of the angle between the peptide end-to-end vector and the z-axis at various temperatures from unbiased NVT simulations.

Figure 16. The PMF, energy, and entropy of adsorption for 1-octanol at the air-water interface. Probability distribution of angle of octanol end-to-end vector with the z-axis (normal to the interface).

using the approach described in the Methods section. We used the force field and charges suggested by MacCallum and Tieleman46 in their work on water-octanol solutions. The free energy, energetic, and entropic terms are plotted in Figure 16. The trends reported from experiments for other surfactants are observed here, namely, the favorable free energy and energy of adsorption. We also find a favorable increase in entropy upon adsorption, confirming that octanol behaves like other “traditional” surfactants. To quantify the role of octanol orientational entropy, we also plot in Figure 16 the probability distributions of the angle of the octanol end-to-end vector with the z-axis at the interface and in the bulk. At the interface, octanol is observed to adopt an angle near θ ) 90°, or parallel to the interface; this was also observed in previous simulations of decanol.20 Experiments of n-alkanol monolayers reveal a perpendicular orientation to the interface.47,48 However, this difference could be attributed to concentration effects; our simulations correspond to dilute (46) MacCallum, J. L.; Tieleman, D. P. J. Am. Chem. Soc. 2002, 124, 15085– 15093. (47) Can, S. Z.; Mago, D. D.; Walker, R. A. Langmuir 2006, 22, 8043–8049. (48) Can, S. Z.; Mago, D. D.; Esenturk, O.; Walker, R. A. J. Phys. Chem. C 2007, 111, 8739–8748.

solution conditions. In contrast to the angle in the middle of the water layer, the probability distribution exhibits a well-defined peak. The orientational entropy of the octanol molecule in the water phase is large and decreases when it adsorbs at the interface. It has also been suggested that the tail is free to adopt more configurations in the oil or vapor phase. We calculated the deuterium order parameter, SCD,49 of the hydrocarbon tails from these simulations, and found no difference between the bulk and interfacial behavior (data not shown). This suggests that the increase in entropy is not due to the tail adopting different configurations in the oil or vapor phase. The remaining explanation for the increase in entropy is a decrease in the order of the water upon adsorption of the hydrocarbon tail at the interface. In contrast to the surfactants described above, the thermodynamic behavior of surface-active β-peptides at the air-water interface includes a favorable free energy of adsorption, ∆Gads < 0, and enthalpy of adsorption, ∆Hads < 0, but an unfaVorable entropy of adsorption, ∆Sads < 0. Again, the entropy change could come from three sources: water structure from solvating (49) Tieleman, D. P.; Marrink, S. J.; Berendsen, H. J. C. Biochim. Biophys. Acta 1997, 1331, 235–270.

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Figure 17. (Left) The PMF, energy, and entropy of adsorption of ovispirin at the air-water interface. (Right) Probability distribution of angle of ovispirin end-to-end vector with the z-axis (normal to the interface).

hydrophobic groups, internal configurational entropy of the surfactant, or orientational and translational entropy of the molecule. We have already observed that the helicity of the peptides is largely unchanged upon adsorption at the interface (see Figure 12), which rules out the decrease in entropy that comes from internal configurational entropy. We have also observed (see Figure 10) how specific orientations are adopted by the peptides upon adsorption, which leads to an increase in orientational order and a decrease in entropy upon adsorption. If we assume that solvating the hydrophobic residues causes a similar increase in order as that required to solvate the hydrocarbon tail of octanol, then the solvation entropy increases upon adsorption of β-peptides. We therefore surmise that the large change in orientational and translational entropy upon adsorption is the central reason for the unfavorable (negative) ∆Sads for β-peptides and is a significant deviation from the thermodynamic behavior of common surfactants such as octanol. The change in sign for entropy causes a reversal of the effect of temperature upon adsorption. We find that increasing temperature renders the adsorption of β-peptides less favorable, whereas adsorption of octanol and other surfactants becomes more favorable with increasing temperature. The thermodynamic differences between adsorption of helical β-peptides and more traditional surfactants such as octanol raise the question of whether unfavorable entropic terms are unique to β-peptides, or whether they also arise in R-helical peptides. We address this issue by simulating the adsorption of ovispirin at the air-water interface. Ovispirin contains 18 residues and is facially amphiphilic.50 It is largely R-helical, with a length of 2.7 nm. Compared to the 10-residue β-peptides considered in this work (1.54 nm long), ovispirin is almost twice as long. The NMR structure of ovispirin is available (1hu5),50 and previous simulations inside lipid micelles51-54 and in model membrane environments55,56 have appeared in the literature. For consistency, we have performed our own simulations using the approach described in the Methods section using the GROMOS force field version 53a6.57 The structure of ovispirin was taken from the Protein Data Bank,58 given a charge of +7, and solvated in SPC water with chloride counterions. (50) Sawai, M. V.; Waring, A. J.; Kearney, W. R.; McCray, Jr, P. B.; Forsyth, W. R.; Lehrer, R. I.; Tack, B. F. Protein Eng. 2002, 15, 225–232. (51) Khandelia, H.; Kaznessis, Y. J. Phys. Chem. B 2005, 109, 12990–12996. (52) Khandelia, H.; Kaznessis, Y. N. Peptides 2005, 26, 2037–2049. (53) Khandelia, H.; Langham, A. A.; Kaznessis, Y. N. Biochim. Biophys. Acta: Biomembr. 2006, 1758, 1224–1234. (54) Khandelia, H.; Kaznessis, Y. N. Peptides 2006, 27, 1192–1200. (55) Ulmschneider, M. B.; Ulmschneider, J. P.; Sansom, M. S. P.; Di Nola, A. Biophys. J. 2007, 92, 2338–2349. (56) Ulmschneider, M. B.; Sansom, M. S. P.; Di Nola, A. Biophys. J. 2006, 90, 1650–166. (57) Oostenbrink, C.; Villa, A.; Mark, A. E.; Gunsteren, W. F. V. J. Comput. Chem. 2004, 25, 1656–1676.

Our results are presented in Figure 17. The figure includes the free energy (w), energy (∆U) and entropy (-T∆S) along with the probability distribution of the angle at the free energy minimum and in the center of the water layer. The free energy and the energy of adsorption are favorable (-14.7 and -29.3 kcal/mol, respectively), but the entropic term is unfavorable (14.6 kcal/ mol). This behavior is qualitatively similar to that of peptide 1a, which is also amphiphilic. The calculated fraction of hydrophobic surface area, fphobic was 0.66, which is comparable to that of peptide 1a (fphobic ) 0.67). The angle distributions shown in Figure 17 are narrower than those observed for octanol. Ovispirin adopts almost a 90° angle with the interface normal. These results suggest that the behavior of helical, facially amphiphilic β- and R-peptides is remarkably similar.

Conclusions We have performed biased and unbiased MD simulations of β-peptides with different types and displays of hydrophobic and hydrophilic side chains. Favorable adsorption at the air-water interface occurs when β-peptides include at least 2/3 hydrophobic residues and facial display of amphiphilicity. Using biased MD simulations, we have determined the thermodynamics of adsorption of β-peptides at the air-water interface. In particular, both the free energy and energy of adsorption are favorable, while the entropic term is not. We have also found that adsorption is accompanied by a decrease in electrostatic energy and solvent-solvent energy. The helical stability of β-peptides is maintained, even upon adsorption at the interface. The data presented here were obtained using the CHARMm force field and TIP3P model for water. Certainly, differences may appear when considering other force fields and water models. Of particular note would be the interfacial characteristics of water models. It has been shown that the surface tension of water varies depending on the force field used.59,60 We also note that the data obtained for ovisprin was based on the GROMOS/SPC force field parameters. While this is not definitive evidence, this does suggest that the results may be independent of the parameter set. Our results indicate that the presentation of hydrophilic groups on helices does influence the quantitative value of the free energy of adsorption and the depth of the peptide relative to the interface. In contrast, the presentation of hydrophilic side chains has no (58) Bergman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235– 242. (59) Pomerantz, W. C.; Abbott, N. L.; Gellman, S. H. J. Am. Chem. Soc. 2006, 128, 8730–8731. (60) Pomerantz, W. C.; Yuwono, V. M.; Pizzey, C. L.; Hartgerink, J. D.; Abbott, N. L.; Gellman, S. H. Angew. Chem., Int. Ed. 2008, 47, 1241–1244.

Surface ActiVity of β-Peptides

significant effect on the angle with interface. When the β-peptides do adsorb at the interface, they adopt specific orientations that are largely parallel to the interface. At higher temperatures, we have found evidence that an end-amphiphilic β-peptide may orient perpendicular to the interface, which would be contrary to what has been observed for R-helices.43 The thermodynamic properties of β-peptides at the air-water interface are almost orthogonal to those of traditional surfactants (such as octanol), but are remarkably similar to those of helical R-peptides (such as ovispirin). We hope that the results presented in this work will motivate future experiments aimed at investigating the surface activity of the β-peptides considered in this work, including measurement of the thermodynamics of adsorption. We note that association of some of these β-peptides has been observed in solution59-61 and at gold surfaces.62 It would be of interest to pursue studies of the two-dimensional assembly process at the air-water interface. Preliminary simulations of monolayers of β-peptides

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indicate significant association of β-peptides at the interface, which may be observed in experiments using atomic force microscopy of Langmuir-Blogett films. Future studies of lipid-β-peptide interactions, using both experiment and theory, might help us design more effective peptides for antimicrobial applications. Acknowledgment. This work is supported by the National Science Foundation (NSF) through the Nanoscale Science and EngineeringCenter(NSEC)attheUniversityofWisconsin-Madison. The authors acknowledge the use of considerable computational resources provided through the Grid Laboratory of Wisconsin (GLOW) network, which is also supported by the NSF. LA802973E (61) Kritzer, J. A.; Stephens, O. M.; Guarracino, D. A.; Reznik, S. K.; Schepartz, A. Bioorg. Med. Chem. 2005, 13, 11–16. (62) Pomerantz, W. C.; Cadwell, K. D.; Hsu, Y.-J.; Gellman, S. H.; Abbott, N. L. Chem. Mater. 2007, 19, 4436–4441.