Surface Activity of Amphiphilic Helical β-Peptides from Molecular Dynamics Simulation

Surface Activity of Amphiphilic Helical �-Peptides from MolecularDynamics 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 �-peptides is investigated using molecular simulations. The type and display of hydrophobicand hydrophilic groups on helical �-peptides is varied systematically. Peptides with 2/3 hydrophobic groups are foundto be surface active, and to adopt an orientation parallel to the air-water interface. For select �-peptides, we alsodetermine the potential of mean force required to bring a peptide to the air-water interface. Facially amphiphilichelices with 2/3 hydrophobic groups are found to exhibit the lowest free energy of adsorption. The adsorption processis driven by a favorable energetic term and opposed by negative entropic changes. The temperature dependence ofadsorption is also investigated; facially amphiphilic helices are found to adopt orientations that are largely independentof temperature, while nonfacially amphiphilic helices sample a broader range of interfacial orientations at elevatedtemperatures. The thermodynamics of adsorption of �-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 ofthe entropy of adsorption, which is negative for �- and R-peptides and positive for traditional surfactants such asoctanol.

Introduction

Antimicrobial molecules are thought to act upon bacterial andfungal cells in a variety of ways.1,2 Many of the proposedmechanisms of action involve some interaction with the cellularmembrane. A majority of naturally occurring antimicrobialpeptides are amphiphilic in nature, having hydrophobic andhydrophilic side chains.3 Amphiphilicity has in fact been usedto guide the development of new molecules that are active againstmicrobes.

Synthetic �-peptides4,5 offer an attractive class of materialsin the search for new antimicrobial peptides. As oligomers of�-amino acids, �-peptides have been shown to exhibit antibacte-rial6,7 and antifungal properties.8 Their efficiency as antimicrobialagents capitalizes on their resistance to enzymatic degradation;9

only one enzyme has been discovered that degrades pure �- andmixedR,�-peptides.10 When compared toR-peptides, the helicalstates of �-peptides have been found to be more structurallystable.11 This structural stability, and the ability to manipulatesequence synthetically, enable considerable control over the typeand spatial presentation of functional groups in �-peptide helices.The type of helix we consider here, the so-called 14-helix, has3 residues per turn and presents three distinct faces. In contrast,a traditional R-helix contains 3.6 residues per turn and the

segregation of hydrophobic and hydrophilic groups is not aspronounced. Taken together, all of these attributes make�-peptides an intriguing class of antimicrobial molecules fordetailed, molecular-level studies.

The overall goal of our work is to characterize at a fundamentallevel the behavior of �-peptide molecules in contact with modelcell membranes. At the experimental level, Langmuir mono-layers12 provide a useful means to quantify several aspects ofthe interaction of surface active molecules with lipids. In orderto interpret the results of Langmuir trough experiments, and todistinguish between increases in surface pressure due topreferential association with the lipid monolayer or self-association at the air-water interface, it is important to beginby characterizing the behavior of �-peptides at the air-waterinterface. More generally, the air-water interface is importantfrom the perspective of amphiphilicity, which is generallyaccompanied by surface activity at the air-water interface. Thiswork presents a computational study of a variety of �-peptidesat that interface.

The surface activity of amphiphilic peptides can be measuredusing static or dynamic surface tension (or surface pressure)measurements. Langmuir films of amphiphilic R-peptides havebeen studied using surface pressure-area isotherms, surfacepotential measurements, or Langmuir-Blodgett films. There area number of studies on the interaction of amphiphilic peptideswith interfaces; a recent review of the surface activity of peptidesthat form �-sheets and R-helices can be found by Rappaport.13

Maget-Dana et al.14 used dynamic surface tension measurementsand compression isotherms with peptides containing differentarrangements of lysine and leucine side chains. They found thatR-helices diffused and adsorbed faster to the air-water interfacethan �-sheets. They also determined the free energy of adsorption,∆Gads ≈ -1.2 × 10-24 kcal/residue, from the surface pressure-area isotherms for all the peptides they studied. Colfer, Kelly,

* 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.

(12) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109–140.(13) Rappaport, H. Supramol. Chem. 2006, 18, 445–454.(14) Maget-Dana, R.; Lelievre, D.; Brack, A. Biopolymers 1999, 49, 415–423.

2811Langmuir 2009, 25, 2811-2823

10.1021/la802973e CCC: $40.75 2009 American Chemical SocietyPublished on Web 01/27/2009

and Powers15,16 used surface pressure-area isotherms, fluores-cence microscopy, and Langmuir-Blodgett films of an am-phiphilic peptide forming a �-hairpin and observed the self-assembly of the peptide at the air-water interface. Ambroggioet al.17 measured the surface pressure-area isotherms ofR-helical,antibiotic (and amphiphilic) peptides maculatin and citropininteracting with both the air-water interface and lipid monolayers.They observed a ∆Gads of -8.1 kcal/mol for both peptides, andfound that both molecules interact more favorably with lipidscontaining anionic head groups than zwitterionic head groups.Lakshmanan and Dhathathreyan18 investigated systematic muta-tions of laminin-derived peptides interacting with air-waterinterfaces and lipid monolayers. They found that modifying atyrosine side chain to more hydrophobic side chains led toincreased surface activity and lipid interaction.

Several simulation studies have examined the surface activityof amphiphillic molecules; a recent review of relevant simulationmethods is found in ref 19. Past studies20 and more recentinvestigations21-24 of surface activity have considered a varietyof molecules. Shin and Abbott20 used molecular dynamics (MD)simulations and transition-state theory to obtain the free energyof adsorption (∆Gads ) -9.5 kcal/mol) and the desorption ratefor 1-decanol. Canneaux et al.24 obtained free energy profiles ofethanol, acetone, and benzaldehyde as they progressed throughthe air-water interface at 298 and 273 K. They found that ∆Gads

was favorable for each of these molecules and became lessnegative at the lower temperature. For example, ethanol changedfrom-2.3 kcal/mol at 298 K to-1.6 kcal/mol at 273 K. Minofaret al.22 performed experiments and simulations of sodium formate,acetate, benzoate, and phenolate molecules to investigate changesin surface pressure with concentration. They found that sodiumformate increased the surface tension with concentration, similarto electrolytes at surfaces, while the other three decreased thesurface tension with concentration, especially the more hydro-phobic anions. Carignano et al.23 calculated the free energy ofadsorbing and solvating ammonia at 277 K. They found afavorable ∆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 decomposingthe energetic term into the water-water (sol-sol) andwater-ammonia (sol-NH3) interactions, they found that ∆Usol-sol

< 0 and ∆Usol-NH3> 0. In other words, a decrease in water-waterinteraction energy favors the adsorption of ammonia at theair-water interface. Gu et al.21 applied a solvation model topredict the surface activity and ∆Gads of systematically substitutedpeptides. They achieved a 50% success rate for the peptides thatwere expected to be surface active (as later verified in experi-ments).

The simulations in this work seek to explore not only thesurface activity of �-peptides, but also how amphiphilic peptidesin general behave at the air-water interface. In this regard,�-peptides are of interest because of their well-defined structure

and their ability to control the placement of specific hydrophilicand hydrophobic groups along the folded molecule. With regardto 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 thethermodynamic driving forces for that process. For peptides thatexhibit a thermodynamic preference for the air-water interface,we also examine their orientation (and that of distinct chemicalgroups) relative to the interface. And, given the tendency ofR-peptides to change structure upon adsorption,1,12 we alsodetermine whether the structural stability of �-peptides is alteredat the interface.

In the sections that follow, we describe our choices for sidechain functional groups, the simulation methods and modelsemployed in our work, and the properties that are measured toquantify the adsorption process. By dissecting the free energyof adsorption into energetic and entropic components, we proposeand test hypotheses regarding the temperature dependence of thefree energy of adsorption. We conclude our manuscript with aseries of general considerations for the design of peptides thatadsorb at the air-water interface.

MethodsPeptides to Test. In order to understand the roles of hydrophobicity

and amphilicity in driving adsorption at the air-water interface, wehave identified a series of �-peptides that vary in the type andpresentation of side chains. All the �-peptides considered here adopta common helical structure called the 14-helix. The 14-helix is formedby hydrogen bonds between atoms CdO(i) and H-N(i-2) on thebackbone and has approximately three residues per turn. With thisknowledge, we can select the location of the side chains on the threedistinct faces of the helix. All of the peptides we have studied (seeTable 1) consist of 10 residues and include a tyrosine-like side chain(�3-hTyr) at the N-terminus and at least three hydrophilic lysine-likeside chains (�3-hLys). The majority of the peptides that we simulatecontain the cyclic residue, trans-2-aminocyclohexanecarboxylic acid(ACHC), which has been shown to stabilize the 14-helix conforma-tion.25

Peptide 1a has three hydrophilic �3-homolysine side chains andsix hydrophobic ACHC side chains. This particular sequence leadsto a facial segregation of the hydrophilic and hydrophobic groups,with one hydrophilic face and two hydrophobic groups. Peptide 1balso has three hydrophilic �3-homolysine side chains and sixhydrophobic ACHC side chains, but differs from 1a by the sequenceof the side chains, and therefore the presentation of the hydrophilicand hydrophobic groups. We refer to this display of side chains as“scrambled” amphiphilicity, because one hydrophilic side chain ispresented on each face. Peptides 2a and 2b have three hydrophilic�3-homolysine side chains, three hydrophobic ACHC side chains,and three hydrophobic �3-homophenylalanine side chains. The

(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.(25) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman,

S. H. J. Am. Chem. Soc. 1996, 118, 13071–13072.

Table 1. �-Peptides Examined for Surface Activity in ThisWorka

peptide sequence fphobic

1a �3-hTyr-[ACHC-ACHC-(�3-hLys)]3 0.671b �3-hTyr-[ACHC-ACHC-(�3-hLys)]3 scram 0.682a �3-hTyr-[ACHC-(�3-hPhe)-(�3-hLys)]3 0.682b �3-hTyr-[ACHC-(�3-hPhe)-(�3-hLys)]3 scram 0.693 �3-hTyr-[ACHC-(�3-hLys)-(�3-hLys)]3 0.544 �3-hTyr-[(�3-hLys)-(�3-hLys)-(�3-hLys)]3 0.455 �3-hTyr-[(�3-hLys)]3-[ACHC]6 0.67

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

2812 Langmuir, Vol. 25, No. 5, 2009 Miller et al.

sequences of these two peptides were chosen to result in facialamphilicity for sequence 2a, and scrambled amphiphilicity forsequence 2b. Peptide 3 has six hydrophilic �3-homolysine side chainsand three hydrophobic ACHC side chains with facial amphiphilicity,and peptide 4 has nine hydrophilic �3-homolysine side chains andno hydrophobic ACHC side chains. Peptide 5 is similar to 1a and1b in the side chains selected, but the sequence is chosen with thehydrophilic �3-homolysine side chains near the positively chargedN-terminus. This display of hydrophilic groups is referred to as“end” amphiphilicity.

By selecting these particular side chains and sequence, a directcomparison 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 associationprocesses27 that provide a foundation for the adsorption studiespresented in this work.

Simulation. Biased and unbiased MD simulations are used toinvestigate the behavior of these peptides at the air-water interface.The CHARMm2728,29 all-atom force field was used to model our�-peptides. For a complete description of the parameters employedin our work, readers are referred to our previous simulations of�-peptides.11,26,27,30 A 1-3 exclusion principle was used fornonbonded interactions and the 1-4 Coulombic interactions werescaled by a factor of 0.4 to be consistent with the CHARMm forcefield. The TIP3P model31 of water was selected because it iscompatible with the CHARMm force field. In solution, these peptidesare expected to have protonated �3-homolysine residues andN-termini, with each peptide having a positive charge. To counterthe positive charge of each peptide, chloride counterions wereincluded in the simulation cell.

Simulations were performed and analyzed using the GRO-MACS32-34 simulation package. Lennard-Jones (LJ) interactionswere truncated with a twin-range scheme at 10 and 15 Å. Theelectrostatic interactions were calculated using a particle mesh Ewaldtechnique35 with a short-range cutoff of 10 Å, a maximum relativeerror of 10-5, and a fourth-order spline. A time step of 0.002 ps wasused along with LINCS36 to constrain all bond lengths to theirequilibrium value. The procedure and setup of the simulation cellis described below.

NVT Simulation and System Setup. The molecular simulationswere started by solvating a single charged, helical �-peptide in TIP3Pwater (>1800 molecules) using the GROMACS utility editconf ina cubic box having dimensions of 4 nm. Then, the correct numberof chloride counterions (to maintain electroneutrality) were placedat the most electrostatically favorable positions using genion. Aftera brief energy minimization of 500 steps using the steepest descentalgorithm, we performed an NPT-ensemble equilibration run of 1ns using isotropic pressure scaling and a Berendsen thermostat andbarostat37 at P ) 1 bar and T ) 300 K. The simulation cell was then

adjusted by recentering the peptide at the center of the box andapplying periodic boundary conditions in all directions. Thesimulation box size was then changed in the z-dimension to 12 nm.The initial density distribution of the system and a snapshot of thestarting configuration of peptide 1a are given in Figure 1. Havingcompleted the initialization process just outlined, a 10 ns simulationwas performed in the NVT ensemble at 300 K.

Potential of Mean Force Calculation. In order to obtainquantitative estimates of the free energy of adsorption, we calculatedthe potential of mean force (PMF) required to move the center ofmass (COM) of the peptide across the film of water, from the middleof the box to the air-water interface. The reaction coordinate, �,is defined as the z-distance from the COM of the peptide to the COMof the water film. The free energy (or PMF) was determined usingthe constraint force (CF) method,38,39 which makes use of thefollowing:

w(�))∫�0

�⟨f(�′)⟩�′d�′+C (1)

Here, w is the PMF, � is the reaction coordinate, and f�′ is the forcerequired to constrain the reaction coordinate. The integration constantC can be chosen such that the free energy vanishes when the reactioncoordinate is zero, � ) |zpep - zwat| ) 0. By doing so, the PMFrepresents the difference between the free energy when the peptideis in the bulk phase and that at the interface.

The simulation was initialized by taking the starting configurationfrom the unbiased NVT simulations and pulling the COM of thepeptide with a constant velocity (0.025 nm/ps) over 120 ps in theNVT ensemble. Configurations were saved every 4 ps and used asstarting points for the PMF calculation. In the NVT ensemble at 300K, we performed simulations at each value of � ranging from 0.0to 2.9 nm and spaced every 0.1 nm. A force was applied at everytime step to constrain the peptide at each particular choice of thereaction coordinate. The value of this force was saved every 0.05ps and then averaged and used in eq 1 to obtain the PMF. Thesimulations ran for 2 ns, and data were accumulated over the last1.5 ns. Configurations and energies were saved every 1 ps to calculate

(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.; Hernandez-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.

(38) Trzesniak, D.; Kunz, A.-P. E.; van Gunsteren, W. F. ChemPhysChem2007, 8, 162–169.

(39) Mastny, E. A.; Miller, C. A.; de Pablo, J. J. J. Chem. Phys. 2008, 129,034701.

Figure 1. MD simulations are set up with the peptide at the center ofa rectangular box along with a water layer of approximately 4 nm. Thebottom part of the figure shows the initial density distribution of peptide1a. The simulation cell is periodic in all directions. The density data wasobtained from the first 500 ps, over which the peptide is not seen toinsert at the interface (see Figure 2).

Surface ActiVity of �-Peptides Langmuir, Vol. 25, No. 5, 2009 2813

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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 100random samples having the same length of the data, reintegratingeq 1, and determining the value of two standard deviations at eachvalue of the reaction coordinate.

Peptide-Water Properties. In order to characterize thepeptide-interface interactions more closely, we calculated the solventaccessible surface area, potential energies, angle with the interface,and location of the peptide and interface. We also determined thestructural stability of the peptide and the number of hydrogen bondsin the system.

The peptide stability was determined by calculating the end-to-end distance and helicity of the molecules. The end-to-end distancewas defined as the distance from the nitrogen of the N-terminalresidue to the carbonyl carbon of the C-terminal residue. In orderto avoid the fluctuations inherent to the first and last residues, thesecond and penultimate residues were selected. The helicity wascalculated from the dihedral angles φ (C(dO)-N-C�-CR) and ψ(C�-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 quantityHφ is defined as

Hφ) { 1 if |φ- φo| e a

1-|φ- φo|-a

b- aif a < |φ- φo| e b

0 if |φ- φo| > b

(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 potentialenergy and its various contributions, including LJ (ULJ) andelectrostatic energies (Ucoul). We also determined the intermolecularinteractions between peptide-peptide atoms (Upp), peptide-solventatoms (Ups), and solvent-solvent atoms (Uss).

The angle of the peptide with the interface was obtained by takingthe dot product of the end-to-end vector (defined as described above)with the normal vector to the interface (the z-axis). The solventaccessible surface area of the peptide was determined using theGROMACS utility g_sas, which uses a method reported by

Eisenhabaer et al.40 Hydrophobic atoms were defined as having anabsolute charge less than 0.2e, where e is the electronic charge. Theposition of the interface was determined by first obtaining a densityprofile (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 thez-position where the water density was 500 kg/m3. The position ofthe peptide, zpep, was defined by the z-coordinate of the COM of thepeptide atoms.

Results

Surface Adsorption in Unbiased MD Simulation. Weperformed MD simulations of all the peptides (1-5) starting inthe center of the water layer for 10 ns, and determined whetherthey migrate to the interface. The z-coordinate of the center ofmass of the peptide over time is shown in the top row of Figure2. Because the simulation contains two interfaces, the peptidesmay go to one or the other interface. By taking the absolute valueof the z-distance, we obtain a more concise presentation of theresults. We observe that only peptides 3 and 4 fail to reach theinterface. The MD simulations of 3 and 4 were continued for 15ns, and did not reveal any evidence for adsorption at the interface.In contrast, all the peptides that do adsorb at the interface remainthere for the duration of the simulation.

Figure 2 shows the angle the peptide makes with the z-axisas described in the Methods section. The peptides adopt highlyspecific orientations at the interface. In contrast, the peptides inthe bulk water phase explore a much wider range of orientations.This behavior is especially apparent for peptides that never reachthe interface during the simulation time, 3 and 4. Once adsorbed,the peptides make an approximately 90° angle with the z-axisand 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 allcases, the peptides are positioned near the water side of theinterface; by taking the absolute value, we remove the effect ofhaving two interfaces. Probability distributions of the distancebetween the peptide and the interface are shown in Figure 3 foreach peptide that adsorbs at the interface. The peptides that areclosest to the interface are the facially amphiphilic peptides 1aand 2a. Amphiphilic peptides that do not display facialamphiphilicity (1b and 2b) are 1-2 Å deeper into the waterphase. 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.

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 ofthe 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 theangle of the peptide with the z-axis. The shaded region in the top plot shows the approximate location of the interfacial region.



With regard to the degree of hydrophobicity, we determinedthe fraction of hydrophobic surface area of the peptides from thefirst 2 ns of the simulation. These results are summarized inTable 1. Each of the peptides with 2/3 hydrophobic residues alsodisplayed a surface area that was approximately 2/3 hydrophobic.Adding three more hydrophilic �3-homolysines (3) brought thehydrophobic fraction to 0.54, and having six more �3-homolysines(4) brought the hydrophobic fraction to 0.45. As expected,hydrophobicity plays a role in governing whether the peptidesadsorb at the interface. Our results indicate that 2/3 of the sidechains must be hydrophobic for favorable adsorption at theair-water interface. Furthermore, we see little difference betweenthe adsorption of peptides whether ACHC (1) or �3-homophe-nylalanine (2) residues are used for the hydrophobic groups.Amphiphilicity also plays a role in the observed depth of thepeptide relative to the interface, and, as shown below, influencesthe angle of the peptide with the z-axis.

Free Energy of Adsorption. While the fact that the peptidesgo to the surface during our short MD simulation provides someinsight into the adsorption process, it is of interest to quantifythe thermodynamic driving forces that give rise to that process.This information can be extracted from the PMF required tomove the peptides from the bulk water phase through the interfaceand beyond. Figure 4 shows the PMF for peptides 1a, 1b, 3, and5. 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 appearanceof a driving force that localizes the peptide at some optimal

position at the interface. Region III is characterized by an increasein the free energy beyond that corresponding to the bulk water.Only peptide 3 exhibits a slight maximum as it approaches theinterface from the water side. This maximum is reminiscent ofbut much weaker than that reported by Shin and Abbott20 fordecanol.

The PMF can be decomposed into energetic and entropic termsaccording to w ) ∆U - T∆S. The energetic and entropic termsare given in Figure 5 and Table 2. The change in energy, ∆Uis obtained by calculating a time average of the overall potentialenergy at each position and then taking the difference relativeto the peptide being at the center of the water layer. The errorwas estimated by taking 1000 random samples of the time seriesof potential energies, determining the averages of those 1000samples, and then reporting two standard deviations of theensemble of the averages as the error. The entropy was obtainedusing the formula, T∆S ) ∆U - w. The error reported for theentropy is taken as the largest error associated with the twoquantities, ∆U and w. The estimated error of the PMF is less than1 kcal/mol, while the estimated errors of the energy and entropyare 3 kcal/mol. Smaller variations in energy and entropy exhibitedduring adsorption are not statistically significant. Note, however,that the overall trends observed in our simulations are significant,particularly regarding whether the entropy or energy changesare positive or negative. Peptide 1a exhibits the largest ∆Gads;it also has the largest ∆Uads and a relatively small T∆Sads. Incontrast, peptide 3, which has the lowest ∆Gads, does not havethe smallest ∆Uads. Its ∆Uads is in fact relatively favorable, at-10.6 kcal/mol. The energetic term is largely offset by a largeunfavorable entropic term T∆Sads (-9.24 kcal/mol). The peptidewith the smallest ∆Uads is 1b, which also happens to exhibit thesmallest T∆Sads (with a value near zero). The adsorption of 1ais largely driven by energy (it has a significant ∆Uads and a smallT∆Sads). Peptide 5 exhibits a moderately favorable value of ∆Uads

and a large negative value of T∆Sads that leads to a favorable freeenergy of adsorption. Peptides 1b and 5 both have one hydrophilicside chain on each face, but placing all of the hydrophilic sidechains at the N-terminus (as in 5) makes the adsorption moreenergetically favorable. We also note that the minimum valuein the energy or entropy does not correspond to the minimumin the free energy. Taking 1a as an example, the free energyminimum occurs at 1.8 nm; the minima in energy and entropyoccur at 1.5 nm and at 1.2 nm, respectively. Similar behavioris also observed in the other peptides.

As mentioned above, peptides 1a, 3, and 5 exhibit a decreasein entropy upon adsorption at the air-water interface. Previoussimulations23 of ammonia at the air-water interface have alsorevealed a decrease in entropy.23 This drop in entropy, or increasein order, could have two origins: the entropy of the peptide orthe entropy of the solvent. Our unbiased MD simulations suggestthat the peptides in the water phase can explore a variety oforientations, but they adopt a well-defined orientation at theair-water interface. As the peptides move to the air-waterinterface, their orientational entropy decreases. Similarly, peptidetranslation becomes restricted in one dimension, but not in theother two. The translational entropy of the peptide is thereforeexpected to decrease as well. In previous work,26 we observedthat the solvent entropy makes an important contribution to themechanical stability of �-peptides. This solvent entropy can beattributed to the solvation of a hydrophobic surface. In a similarmanner, the solvent entropy plays a role in the adsorption process.We hypothesize that by moving the hydrophobic surface fromthe bulk water to the interface, the order of the solvent decreasesand the entropy increases.

Figure 3. The probability distribution of the z-distance of the peptiderelative to the air-water interface from MD simulations of peptides1-5. Shown here are only those peptides that go to the air-water interfaceand stay there for the remainder of the simulation. Data for this plotcame from the last 2 ns of the simulations in Figure 2.

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


http://pubs.acs.org/action/showImage?doi=10.1021/la802973e&iName=master.img-002.png&w=179&h=120


Another possible origin of the change in water entropy is thestructure of the water at the liquid-vapor interface. Recenttheoretical studies41,42 of the contributions to vibrational sum-frequency spectroscopy suggests that, near the interface, thespectrum is largely a result of water molecules with two or threehydrogen bonds. This suggests that the entropy of the interfacialwater molecules may actually decrease when they are displacedby the peptide and pushed into the bulk water phase. Thishypothesis is strengthened by the observed increase inwater-water hydrogen bonds upon adsorption of the peptideshown in Figure 9. It is difficult to know whether the overalleffect of hydrogen bond formation may lead to positive or negativeentropy changes upon adsorption of the peptides. We surmisethat there are two competing influences on entropy;a decreasein the peptide entropy and an increase in the solvententropy;during the adsorption of amphiphilic molecules at theair-water interface. In the case of peptide 1b, these two forcesare balanced in the same manner as when the peptide is in thebulk water. For the other three peptides, the change in entropyis negative, which suggests that the decrease in peptide entropyis 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

curves from the PMF calculation are plotted in Figure 6 for thefour �-peptides considered here. In all cases, the LJ energygenerally increases as the peptide approaches the interface andhas a regular slope beyond 1 nm from the center of the waterlayer. The LJ energy increases as the peptide leaves the waterbecause there are less atoms in close contact. The LJ energybegins to change from its bulk value even when the peptide iswithin 1 nm of the interface. At this depth, it is unlikely that thepeptide atoms have begun to leave the water layer since the sizeof the peptides is roughly 1 nm. The Coulombic energy decreasesas the peptide approaches the minimum in free energy but thenincreases again. The minimum in total potential energy occursbecause the minimum in Coulombic energy is greater than theincrease in LJ energy. This suggests that the favorable energeticterm is largely due to favorable electrostatic forces. We maythink of this in terms of two competing effects. First, theunfavorable electrostatic nature of solvating nonpolar, hydro-phobic groups in a polar solvent, and second, the favorableelectrostatic nature of solvating polar, hydrophilic groups.

The nonbonded energy can also be decomposed (Figure 7) interms of peptide-peptide energy (∆Upp), peptide-solvent energy(∆Ups), and solvent-solvent energy (∆Uss). Here we observethat the peptide-peptide energy does not vary much as the peptideapproaches the interface. As expected, the peptide-solvent energyincreases as the peptide leaves the water phase by at least 1 order

(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.

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 averagepotential energy of the simulations while the entropy comes from w ) ∆U - T∆S. When the peptide is near the interface, both contributions reacha minimum value.

Figure 6. The Lennard-Jones and Coulombic contributions to the nonbonded potential energy of the system from the free energy calculations ofthe peptides moving through the interface. Only the Coulombic contribution contains a minimum in the potential energy.

Table 2. Thermodynamics of Adsorption of �-Peptides at the Air-Water Interfacea

peptide ∆Gads ∆Uads T∆Sads ∆Uads, pp ∆Uads, ps ∆Uads, ss

1a -15.0 (0.92) -17.9 (2.68) -2.7 (2.68) 1.57 (0.38) 37.1 (1.14) -57.7 (3.3)1b -6.48 (0.81) -6.38 (2.46) 0.10 (2.46) 9.85 (0.38) 21.6 (1.10) -39.0 (2.9)3 -0.75 (0.99) -10.6 (2.60) -9.24 (2.60) -3.65 (0.33) 44.5 (1.16) -52.6 (3.1)5 -4.73 (0.99) -13.3 (2.76) -8.53 (2.76) -1.23 (0.27) 43.4 (0.60) -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 tothe minimum in the free energy are shown above. Energies are in reported in kilocalories per mole. The numbers in parentheses represent error estimatesof two standard deviations from the mean. These results are obtained from the minimum in free energy from the PMF calculation.




of magnitude compared with the peptide-peptide energy. Thesolvent-solvent energy decreases significantly as the peptidesapproach the interface, and then levels off once the peptide haspassed the interface. Beyond the interface, the water atoms areno longer affected by the peptide and reach a somewhat constantlevel. These potential energy contributions to the value at theminimum in free energy are given in Table 2. Previoussimulations23 of ammonia at the air-water interface also reporteda favorable solvent-solvent energy (∆Uss < 0) and unfavorablesolvent-solute energy (∆Us-NH3

> 0) at the interface.We emphasize that adsorption of these �-peptides is largely

driven by a favorable energetic term. We noted that it is theelectrostatic energy that decreases and not the LJ energy uponadsorption. We have also seen how the peptides adsorbing at theinterfaceisaccompaniedbyafavorabledecreaseinsolvent-solventenergy. Taken together, these results indicate that the peptideadsorption is largely driven by favorable solvent-solventinteractions that could be electrostatic in nature. These electrostaticinteractions may be related to a change in the number of hydrogenbonds of the system.

The number of hydrogen bonds between the peptides andwater was characterized as described above, and the result is

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

Figure 7. The nonbonded energy divided into interactions between peptide and water atoms from the PMF calculation of four �-peptides. Thepeptide-peptide energy (∆Upp) changes little, the peptide-solvent energy (∆Ups) increases, and the solvent-solvent energy (∆Uss) decreases as thepeptides 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 betweenthe change in peptide-water hydrogen bonds and the change in peptide-water energy. The symbols come from the simulation data, while the linesare linear fits to the data for each peptide.

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





slope for peptide 1a corresponds to a value of 13 kcal/mol for1 hydrogen bond. In Table 3 we have tabulated ∆Nhbonds at theminimum in free energy and show a decrease of zero to fourhydrogen bonds upon adsorption at the air-water interface. Thenumber of hydrogen bonds between water is presented in Figure9 as a function of distance of the peptide from the water layer.The number of water-water hydrogen bonds are observed toincrease by between 8 and 12 hydrogen bonds when the peptidesare at the interface. This increase has been discussed previously,but may be due to a drop in the number of interfacial watermolecules, which have been shown41,42 to form less hydrogenbonds than bulk water molecules.

As with the unbiased MD simulations, we have also investigatedthe presentation of the peptides at the interface by examining thedepth relative to the interface and the angle the end-to-end vectormakes with the z-axis. Figure 10 shows the probability distributionof the angle from the simulation box at the minimum. Table 3lists the average angle at the minimum and the depth relative tothe position of the interface. Again, the peptides adopt close toa 90° angle with the z-axis (parallel to the interface). The peptidewith the largest free energy of adsorption, 1a, also exhibits theclosest approach to the interface. The peptide with the lowestfree energy of adsorption, 3, is also the furthest from the interface.It is interesting to examine the manner in which the peptideswithout facial amphilicity present themselves at the interface.The optimal location at the interface for peptides 1b and 5 isdeeper into the water than that of the facially amphiphilic isomer,1a. In Figure 11 we present a representative snapshot of thepeptides at the minimum configuration. Peptide 1a is observedto have the hydrophobic face sticking partially out of the waterphase. For peptides 1b and 5, the hydrophobic face that is turnedtoward the air contains lysine residues. This orientation can besatisfied in both of these peptides by having the positively chargedlysine side chains wrap around the peptide and into the waterphase, as seen in Figure 11, and, as noted in our prior discussion,

by not approaching the interface too closely. Because of thepresentation of the hydrophilic groups at the N-terminus, onemight have expected an orientation of peptide 5 perpendicularto the interface (peptide 5 is “end” amphiphilic). Our simulationsdo not suggest that peptide 5 can orient itself perpendicular tothe interface. It is of interest to point out that experimental evidenceforR-helices suggests that assembled monolayers at the air-waterinterface lie parallel to the interface, even when compressed tolarge surface pressures.43 In our work, we have only consideredinfinitely dilute surface concentrations of peptides and not themonolayers examined in ref 43. Peptide 5 may orient differentlyin a monolayer, which would be an interesting idea to considerin subsequent investigations.

Figure 12 shows the average helicity of the peptide as a functionof distance along the z-axis. All four peptides are structurallystable as they diffuse to the interface. Peptide 3 has a loweraverage helicity compared to the other three peptides, but itcontains only 1/3 ACHC residues (versus 2/3 ACHC for theothers). The decrease in helical content with a decrease of ACHCresidues is consistent with our previous work.26 The �-peptidestested here are stable, both in solution and at the interface; incontrast to some other antimicrobial peptides, we do not observemajor changes in secondary structure upon adsorption.12,1 Thestructure in solution is therefore a reliable predictor for thepresentation of side chains at the interface. Note that this resultis specific to ACHC-containing �-peptides and may differ whenother groups are considered.

The results of PMF calculations and the above analysis aresummarized in Table 2. Several general conclusions can be drawnfrom that Table. At least 2/3 hydrophobic residues are necessaryfor favorable air-water surface activity. When it is favorable forthe peptides to be at the interface, they adopt a parallel orientationto the interface, irrespective of the presentation of charged sidechains. Two competing entropic forces, peptide entropy andsolvent order influence the adsorption process. We also findevidence for the role of energetic forces, including favorableelectrostatic energy change upon adsorption and favorablenonbonded interactions between water atoms, which drives theadsorption at the interface. We find that peptides with hydrophilicside chains on each face can still exhibit a favorable free energyof adsorption by not approaching the interface too closely andby inserting all the positively charged side chains into the waterphase. Arranging the hydrophilic groups at the N-terminus doesnot induce the peptide to stand perpendicular to the interface indilute solution.

Temperature Effects. The energetic and entropic terms tothe free energy of adsorption suggest that energy is the favorablecomponent driving the adsorption of �-peptides. By assumingnegligible changes in ∆Uads and ∆Sads with temperature, anincrease in temperature should increase the magnitude of theT∆Sads term, and therefore decrease the magnitude of the freeenergy of adsorption, making it less favorable for the peptidesto be localized at the interface. We also saw above that theadsorption of 1a is largely an energetic process, with little relativeentropy gain compared to the other peptides. We thereforespeculate that there should be little or no change in the freeenergy of adsorption with temperature for this peptide. In orderto investigate how temperature affects the properties at theair-water interface, we performed unbiased MD simulations ofthe four peptides from PMF calculations at 270, 300, and 330K. Starting from the final minimum free energy configuration ofthe PMF calculation, the peptide-solvent system ran for 20 nsin the NVT ensemble.

(43) Boncheva, M.; Vogel, H. Biophys. J. 1997, 73, 1056–1072.

Figure 10. Angle of the peptide with the air-water interface. Thedistributions shown here are at the minimum in the free energy. Thepeptides adopt orientations near 90° with the normal to the interface (orparallel to the interface).

Table 3. Properties of the �-Peptides at the Interfacea

peptide ∆Nhbonds |zpep - zint| θz

1a -1.8 (0.14) 0.281 95.251b 0.51 (0.14) 0.388 93.863 -3.4 (0.13) 0.566 110.415 -4.0 (0.11) 0.351 87.19

a Shown in the table are the change in number of peptide-water hydrogenbonds (compared to the bulk case), the depth of the minimum relative to thelocation of the interface, and the angle of the peptide with the z-axis. Theseresults are obtained from the minimum in free energy from the PMFcalculation. Distances are reported in nm and angles in degrees. The numbersin parentheses represent error estimates of two standard deviations from themean.



Figure 13 shows the probability distribution of the differencebetween the COM of the water film and the COM of the peptide,|zpep - zwat|. For peptides 1a and 3, we observe an outward shiftof the location of the peptide relative to the center of the waterlayer. Peptide 1b does not exhibit this outward shift, but seemsto place itself in the same position relative to the center of thewater layer at each temperature. We find a similar trend forpeptide 5 as we saw for 1b.

The density distribution and location of the air-water interfacechanges with temperature; at lower temperatures the bulk densityis higher, and the air-water interface moves closer to the centerof the water film. This shift in location of the interface is takeninto consideration measuring the position of the peptide relativeto the interface, |zpep - zint|; the results are shown in Figure 14.Consistent with an energetically driven process, the position ofpeptide 1a relative to the center of the water shifted outward, butthe position relative to the interface remained unchanged. Thispeptide exhibits only a slight broadening of the probabilitydistribution with increasing temperature. We observe a muchmore pronounced broadening of the probability distribution forpeptide 1b that is accompanied by an overall shift away fromthe interface as the temperature increases. Peptide 3 remains atthe same relative position, with a broadening of the probabilitydistribution. Peptide 5 exhibits a large shift away from the interfacefrom 270 to 300 K, but there is little difference between theprobability distributions at 300 and 330 K.

Figure 15 shows the probability distribution of angles with thez-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 andshifts closer to 90° with increasing temperature. The peptide isallowed more orientational flexibility at the interface at highertemperatures, but that is also coupled to a peptide position furtherfrom the interface. The angular distribution of peptide 3 is alsonarrow, with a distribution slightly off of 90° that does not varywith temperature. Peptide 5 exhibits an angular distribution thatbroadens considerably with increased temperature. In fact, someof the angle population indicates that the peptide begins to alignclose to 30° with the z-axis at 330 K, which corresponds to thepeptide aligning almost perpendicular to the interface. Our PMFcalculations at 300 K did not explore these conformations. Onthe basis of these temperature studies, it would appear that thistransition from parallel to perpendicular orientation is inducedby increasing temperature. Combining this result with theprobability distribution seen in Figure 14, we hypothesize thatby adopting the almost perpendicular orientation (at 330 K), thepeptide can position itself closer to the interface such that it hasa distribution similar to that at 300 K.

Peptide 1a adsorption is driven by energy and is not sensitiveto temperature. In contrast, the adsorption of peptides 1b, 3, and5 represents a balance between entropy and energy, and it doesexhibit a temperature dependence, with higher delocalization atelevated temperatures. The results here can be compared to thesimulations of Canneaux et al.24 on small amphiphilic moleculessuch as ethanol. They determined the free energy of adsorptionand found that increasing temperature leads to a more negative∆Gads. Here, we observe that increasing the temperature leadsto less negative ∆Gads. The influence of temperature is alsomanifest on the peptide’s angle distributions, which range fromlittle change (1a) to considerable change (5) depending on thepeptide sequence. Clearly, the effects of temperature are consistentwith the PMF calculation and indicate a complex relationshipbetween temperature, position at the interface, and angle at theinterface.

Comparison to Surfactant and r-Peptide Adsorption. It isof interest to compare the behavior of surface-active �-peptidesto that of other surfactant systems for which much more isknown.44,45 In general, the measured experimental thermody-namic behavior44 of the adsorption of surfactants at air-water

(44) Rosen, M. J. Surfactants and Interfacial Phenomena; John Wiley andSons: New York, 1989.

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 oneach 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 peptidemoves from the bulk (0 nm) through the interface (∼1.7 nm). The interfacehas little effect on the helicity of the peptides.




interfaces displays a favorable free energy of adsorption (∆Gads

< 0), a favorable enthalpy of adsorption (∆Hads < 0), and afavorable entropy of adsorption, ∆Sads>0. The increase in entropyof surfactants has been thought to arise from two sources, namelythe entropy of solvating the hydrophobic part of the moleculeand the configurational entropy of the hydrophobic tail itself.45

We consider, as an example, surfactants containing a longhydrophobic tail, such as sodium dodecyl sulfate (SDS) or a

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

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

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

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

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




using the approach described in the Methods section. We usedthe force field and charges suggested by MacCallum andTieleman46 in their work on water-octanol solutions. The freeenergy, energetic, and entropic terms are plotted in Figure 16.The trends reported from experiments for other surfactants areobserved here, namely, the favorable free energy and energy ofadsorption. We also find a favorable increase in entropy uponadsorption, 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 theangle of the octanol end-to-end vector with the z-axis at theinterface and in the bulk. At the interface, octanol is observedto adopt an angle near θ ) 90°, or parallel to the interface; thiswas also observed in previous simulations of decanol.20 Experi-ments of n-alkanol monolayers reveal a perpendicular orientationto the interface.47,48 However, this difference could be attributedto concentration effects; our simulations correspond to dilute

solution conditions. In contrast to the angle in the middle of thewater layer, the probability distribution exhibits a well-definedpeak. The orientational entropy of the octanol molecule in thewater phase is large and decreases when it adsorbs at the interface.It has also been suggested that the tail is free to adopt moreconfigurations in the oil or vapor phase. We calculated thedeuterium order parameter, SCD,49 of the hydrocarbon tails fromthese simulations, and found no difference between the bulk andinterfacial behavior (data not shown). This suggests that theincrease in entropy is not due to the tail adopting differentconfigurations in the oil or vapor phase. The remaining explanationfor the increase in entropy is a decrease in the order of the waterupon adsorption of the hydrocarbon tail at the interface.

In contrast to the surfactants described above, the thermo-dynamic behavior of surface-active �-peptides at the air-waterinterface includes a favorable free energy of adsorption, ∆Gads

< 0, and enthalpy of adsorption, ∆Hads < 0, but an unfaVorableentropy of adsorption, ∆Sads < 0. Again, the entropy changecould come from three sources: water structure from solvating(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.(49) Tieleman, D. P.; Marrink, S. J.; Berendsen, H. J. C. Biochim. Biophys.

Acta 1997, 1331, 235–270.

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

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-endvector with the z-axis (normal to the interface).




hydrophobic groups, internal configurational entropy of the surf-actant, or orientational and translational entropy of the molecule.We have already observed that the helicity of the peptides islargely unchanged upon adsorption at the interface (see Figure12), which rules out the decrease in entropy that comes frominternal configurational entropy. We have also observed (seeFigure 10) how specific orientations are adopted by the peptidesupon adsorption, which leads to an increase in orientational orderand a decrease in entropy upon adsorption. If we assume thatsolvating the hydrophobic residues causes a similar increase inorder as that required to solvate the hydrocarbon tail of octanol,then the solvation entropy increases upon adsorption of �-peptides.We therefore surmise that the large change in orientational andtranslational entropy upon adsorption is the central reason forthe unfavorable (negative) ∆Sads for �-peptides and is a significantdeviation from the thermodynamic behavior of common sur-factants such as octanol. The change in sign for entropy causesa reversal of the effect of temperature upon adsorption. We findthat increasing temperature renders the adsorption of �-peptidesless favorable, whereas adsorption of octanol and other surfactantsbecomes more favorable with increasing temperature.

The thermodynamic differences between adsorption of helical�-peptides and more traditional surfactants such as octanol raisethe question of whether unfavorable entropic terms are uniqueto �-peptides, or whether they also arise in R-helical peptides.We address this issue by simulating the adsorption of ovispirinat the air-water interface. Ovispirin contains 18 residues and isfacially amphiphilic.50 It is largelyR-helical, with a length of 2.7nm. Compared to the 10-residue �-peptides considered in thiswork (1.54 nm long), ovispirin is almost twice as long. TheNMR structure of ovispirin is available (1hu5),50 and previoussimulations inside lipid micelles51-54 and in model membraneenvironments55,56 have appeared in the literature. For consistency,we have performed our own simulations using the approachdescribed in the Methods section using the GROMOS force fieldversion 53a6.57 The structure of ovispirin was taken from theProtein Data Bank,58 given a charge of +7, and solvated in SPCwater with chloride counterions.

Our results are presented in Figure 17. The figure includes thefree energy (w), energy (∆U) and entropy (-T∆S) along withthe probability distribution of the angle at the free energy minimumand in the center of the water layer. The free energy and theenergy 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 hydrophobicsurface area, fphobic was 0.66, which is comparable to that ofpeptide 1a (fphobic ) 0.67). The angle distributions shown inFigure 17 are narrower than those observed for octanol. Ovispirinadopts almost a 90° angle with the interface normal. These resultssuggest that the behavior of helical, facially amphiphilic �- andR-peptides is remarkably similar.

Conclusions

We have performed biased and unbiased MD simulations of�-peptides with different types and displays of hydrophobic andhydrophilic side chains. Favorable adsorption at the air-waterinterface occurs when �-peptides include at least 2/3 hydrophobicresidues and facial display of amphiphilicity. Using biased MDsimulations, we have determined the thermodynamics of adsorp-tion of �-peptides at the air-water interface. In particular, boththe free energy and energy of adsorption are favorable, while theentropic term is not. We have also found that adsorption isaccompanied by a decrease in electrostatic energy andsolvent-solvent energy. The helical stability of �-peptides ismaintained, even upon adsorption at the interface.

The data presented here were obtained using the CHARMmforce field and TIP3P model for water. Certainly, differencesmay appear when considering other force fields and water models.Of particular note would be the interfacial characteristics of watermodels. It has been shown that the surface tension of watervaries depending on the force field used.59,60 We also note thatthe data obtained for ovisprin was based on the GROMOS/SPCforce field parameters. While this is not definitive evidence, thisdoes suggest that the results may be independent of the parameterset.

Our results indicate that the presentation of hydrophilic groupson helices does influence the quantitative value of the free energyof adsorption and the depth of the peptide relative to the interface.In contrast, the presentation of hydrophilic side chains has no

(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.

(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.

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



significant effect on the angle with interface. When the �-peptidesdo adsorb at the interface, they adopt specific orientations thatare largely parallel to the interface. At higher temperatures, wehave found evidence that an end-amphiphilic �-peptide may orientperpendicular to the interface, which would be contrary to whathas been observed forR-helices.43 The thermodynamic propertiesof �-peptides at the air-water interface are almost orthogonalto those of traditional surfactants (such as octanol), but areremarkably similar to those of helical R-peptides (such asovispirin).

We hope that the results presented in this work will motivatefuture experiments aimed at investigating the surface activity ofthe �-peptides considered in this work, including measurementof the thermodynamics of adsorption. We note that associationof some of these �-peptides has been observed in solution59-61

and at gold surfaces.62 It would be of interest to pursue studiesof the two-dimensional assembly process at the air-waterinterface. Preliminary simulations of monolayers of �-peptides

indicate significant association of �-peptides at the interface,which may be observed in experiments using atomic forcemicroscopy of Langmuir-Blogett films. Future studies oflipid-�-peptide interactions, using both experiment and theory,might help us design more effective peptides for antimicrobialapplications.

Acknowledgment. This work is supported by the NationalScience Foundation (NSF) through the Nanoscale Science andEngineeringCenter(NSEC)attheUniversityofWisconsin-Madison.The authors acknowledge the use of considerable computationalresources 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.


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