Direct Visualization of the Alamethicin Pore Formed in a Planar Phospholipid Matrix

Direct visualization of the alamethicin pore formed in a planar phospholipid matrix

Piotr Pietaa,b, Jeff Mirzaa, and Jacek Lipkowskia,1

aDepartment of Chemistry, University of Guelph, Guelph, ON, Canada N1G 2W1; and bDepartment of Physical Chemistry of Supramolecular Complexes, Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland

Edited by Royce W. Murray, University of North Carolina, Chapel Hill, NC, and approved November 7, 2012 (received for review September 9, 2012) We present direct visualization of pores formed by alamethicin a certain critical value (P/L*) (13), which depends on tem- (Alm) in a matrix of phospholipids using electrochemical scanning perature and on the lipid nature (11, 13). For most lipids, at tunneling microscopy (EC-STM). High-resolution EC-STM images a concentration of P/L = 1:15 or higher, all peptides are in the show individual peptide molecules forming channels. The channels Istate(14). are not dispersed randomly in the monolayer but agglomerate The dipole moment of Alm was determined to be 60–79 D, forming 2D nanocrystals with a hexagonal lattice in which the corresponding to a net +1/2 charge at the N- and a −1/2 charge average channel–channel distance is 1.90 ± 0.1 nm. The STM (elementary charge units) at the C terminus of the helix (15). Due images suggest that each Alm is shared between the two adjacent to the large dipole moment, the insertion of Alm into the mem- channels. Every channel consists of six Alm molecules. Three or brane can be controlled by the transmembrane potential (16, 17). four of these molecules have the hydrophilic group oriented to- Upon insertion, Alm molecules aggregate to form voltage-gated ward the center of the channel allowing for water column forma- channels described by the barrel-stave model (18). In that model, tion inside the channel. The dimensions of the central pore in the Alm forms a cylindrical array of parallel helices with the hy- images are consistent with the dimension of the water column in drophilic sides of the helices oriented to the interior, creating a a model of hexameric pore proposed in the literature. The images water-ﬁlled and ion-conducting central lumen, whereas the hy- obtained in this work validate the barrel-stave model of the drophobic sides of helices are oriented toward the hydrophobic pore formed in phospholipid membranes by amphiphatic pep- portion of the membrane (6, 7). CHEMISTRY tides. They also provide direct evidence for cluster formation by The presence of multiple conductance levels observed for Alm such pores. in bilayer lipid membranes (BLMs) suggests formation of pores with various aggregation numbers of Alm molecules (11). The antimicrobial | interface | Langmuir-Blodgett number of Alm monomers per channel was estimated to vary between 6 and 12 (7, 19). The pore size varies with hydration and ntimicrobial peptides (AMPs), including alamethicin (Alm), with lipid composition (12). For example, in bilayer of 1,2- Aare small (6–100 amino acids) molecules produced by many dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) the pores n = –

living organisms (1, 2). AMPs are biocidally active against Gram- are made of 8 9 monomers, with the inner and outer pore BIOPHYSICS AND negative and Gram-positive bacteria, fungi, enveloped viruses, radii of ∼0.9 and 2.0 nm, whereas in bilayer of diphytanoyl phos- eukaryotic parasites, and even tumor cells (3). Importantly, phatidylcholine (DPhPC) the pores are made of n = 11 monomers, COMPUTATIONAL BIOLOGY AMPs are effective against strains of antibiotic-resistant bacteria with the inner and outer pore radii of ∼1.3 and 2.5 nm, respectively (2). Their membranolytic activity involves nonspecific pore for- (12). For 1,2-dioleoyl-sn-glycero-phosphatidylcholine (DOPC) and mation mechanisms. Several models of the pores formed by 1,2-dierucoyl-sn-glycero-phosphatidylcholine (diC22:1PC) bilayers AMPS such as barrel-stave, torroidal or worm-like pore and was reported to be equal to 5 and 9 with the outer pores radii carpet models have been proposed (4). However, the mechanism 1.36 and 1.96 nm, respectively (20). The aggregation of Alm is of membrane disruption by AMPs is still not quite understood. affected by the phase transition of the lipid (21). In plane neutron Alm is a 20-residue peptide isolated from the Trichoderma and X-ray scattering experiments performed on multiple bilayers viride fungus (4). It has been thoroughly studied as a model of indicate that at temperatures below the phase transition and at channel formation in biological membranes (5), due to its activity low water content, lateral distribution of Alm pores is correlated against Gram-positive bacteria and fungi (4, 6). Alm has a linear and the pores are arranged in a 2D crystalline superstructure sequence of residues with the C-terminal being phenylalaninol (21–23). The immiscibility of Alm with phospholipids was observed (Pheol) and the N terminus being acetylated (6). Structural in monolayers at the air/water interface, indicating a tendency analysis of Alm revealed by X-ray crystallography (7) indicates of the peptide toward 2D crystallization (3). The segregation of a that the molecule is predominantly α-helical. In the helical mixture of hydrophobic helical peptides and phospholipid mol- conformation, the length of the molecule is ∼3.2 nm. The pres- ecules into separate peptide and lipid phases was also predicted ence of α-helical conformation of Alm in organic solutions and in by theoretical calculations (24). The elucidation of the lateral membrane environments was observed by NMR (8), circular distribution of Alm in the plane of the phospholipid membrane is dichroism (CD) (9), Raman (9), and FTIR (10) measurements. still under debate and it is essential to understand the mechanisms In bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) of its action (22). Alm is helical from residues 1–12 in the liquid crystalline state, There is a wealth of information about the behavior of Alm whereas in the gel state, the helix extends from residues 1–16 (9). in bilayers composed of lipids with no net charge (e.g., typical of Alm helix is amphiphatic with one face more hydrophilic than mammalian cells). However, the bactericidal action of Alm can the other (11). The amphiphatic properties influence the in- be better understood by studying bilayers composed of negatively teraction of the helix in phospholipid bilayers. Depending on experimental conditions, Alm can be oriented with its helical axis parallel to the plane of the bilayer (surface or S state) or can be Author contributions: J.L. designed research; P.P. and J.M. performed research; P.P. and inserted into the lipid bilayer with the helical axis pointing in the J.L. analyzed data; and P.P. and J.L. wrote the paper. vertical direction (inserted or I state) (12). The S state is observed The authors declare no conflict of interest. at low peptide to lipid ratios (P/L), whereas insertion of Alm into This article is a PNAS Direct Submission. the membrane takes place at higher peptide concentration, above 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1201559110 PNAS | December 26, 2012 | vol. 109 | no. 52 | 21223–21227 Downloaded by guest on September 27, 2021 charged lipids such as those found in bacteria. A model mem- compression isotherms for the DMPC/egPG (1:1) mixture and brane composed of neutral DMPC and negatively charged the pure Alm, respectively. Curve 1 is shifted toward higher phosphatidylglycerol (PG) lipids was used to replicate the lipid values of molecular areas in comparison with curve 2, for the membrane of Gram-positive bacteria (25). PG lipids are a major DMPC/egPG (1:1) mixture without Alm. It displays a step − membrane component (up to 58%) in Gram-positive bacteria at ∼31 mN·m 1 whose position coincides with the collapse (26). Negatively charged heads of PG give rise to an electrostatic pressure observed in the isotherm of the pure Alm. Such be- surface potential that promotes the insertion of Alm into the havior suggests that Alm is not miscible with the mixed lipids. membrane with the N terminus embedded into the bilayer and For immiscible monolayer of noninteracting molecules, the the C terminus directed out of the membrane (21). mean molecular area of the three-component mixture should The objective of this work is to provide molecular resolution be equal to: images of the Alm pore in a phospholipid matrix using the electrochemical scanning tunneling microscopy (STM). Re- A1 = ð1 − xÞ A2 + x A3; [1] cently, we obtained molecular resolution images of a gramicidin,

a hydrophobic channel forming peptide, dispersed in a mono- where A1 and A2 are the mean molecular areas of the compres- layer of DMPC (27). The goal of the present study was to use sion isotherms 1 and 2, A3 is the mean molecular area of the pure similar methodology to provide complementary information Alm monolayer, and x is the mole fraction of Alm in the three- about the structure of the pore formed by the amphiphatic component mixture. Fig. 1B compares the area per Alm mole- peptide such as alamethicin. The Alm molecules were in- cule in the three-component monolayer, calculated as: corporated into the 1:1 mixture of DMPC and egg-PG (egPG). ½A − ð − xÞ A The choice of egg-PG was serendipitous. However, it gave good A = 1 1 2 ; [2] results. The negative charge of egg-PG molecules helped to in- 3 x sert Alm with N terminus facing the gold surface and C terminus oriented toward the solution. We succeeded in obtaining first to the area per molecule in the pure Alm monolayer. The molecular resolution images of Alm aggregates in the DMPC/ differences between the two curves are small but measurable. egg-PG matrix. The images revealed that aggregates of Alm form The areas per Alm molecule in the mixture are about 15% 2D nanocrystals of variable size. The size of one pore in the smaller than in the pure Alm monolayer, suggesting that nanocrystal is in reasonable agreement with models of hexameric molecules in the three-component mixture are slightly com- pore proposed in the literature. The presence of a variable pressed. However, in the range of surface pressures between ∼5 − number of pores in a nanocrystal correlates well with the ob- and ∼30 mN·m 1 there is no significant change in the area per servation of multiple conduction states for Alm incorporated molecule whose value is ∼3nm2/molecule. This value is in good into a BLM. At present, high resolution STM images of a film of agreement with the area for Alm molecule oriented with its phospholipid in solution can be acquired for a monolayer only. helical axis parallel to the interface (S state). These results are However, molecular resolution STM images of a phospholipid consistent with the literature which reported that Alm is immis- bilayer supported at a conductive surface in air have already cible with phospholipids in monolayers spread at the air/water been reported (28–30). With further methodological improve- interface and the Alm molecules are oriented with axis of the ments, imaging of the supported bilayer in a solution should also helix parallel to the interface (3, 31, 32). be possible in the future. This study illustrates how significant The mixed monolayer of Alm/(DMPC/egPG) (P/L = 1:15) was information for the understanding of peptide aggregation in lipid transferred onto the Au(111) electrode surface at surface pres- −1 matrixes the STM experiments may provide. sure 27 mN·m (below the Alm collapse pressure), using the Langmuir–Blodgett technique. The data reported in Fig. 1 in- Results dicate that this monolayer consisted of phase segregated phos- Before the transfer of the monolayer onto the gold electrode pholipids and Alm molecules that were oriented with helical axis surface its properties were characterized at the air–solution in- in plane of the monolayer. This monolayer was dried at ∼17 °C terface. Fig. 1A shows compression isotherms recorded at the overnight. Next the gold electrode covered by the monolayer was air–solution interface of the Langmuir trough. Curve 1 plots the assembled into the electrolyte-filled cell of the electrochemical isotherm of the mixed monolayer containing Alm and DMPC/ STM and the monolayer-covered surface of gold was imaged in egPG at 1:15 mol ratio. For comparison, curves 2 and 3 plot the solution. Fig. 2 A and B shows STM images of this monolayer.

Fig. 1. (A) Surface pressure/area isotherm of Alm/(DMPC/ePG) (1:15) (curve 1), DMPC/ePG (1:1) (curve 2), and Alm (curve 3), spread on aqueous subphase at 15 °C. Dotted line indicates the value of surface pressure at which monolayers were transferred onto the Au bead. (B) Molecular area vs. surface pressure curve obtained by subtracting the isotherm of the DMPC/egPG mixture (curve 2 in A) from the isotherm of the Alm/DMPG/egPG mixture (curve 1 in A).

21224 | www.pnas.org/cgi/doi/10.1073/pnas.1201559110 Pieta et al. Downloaded by guest on September 27, 2021 packed monolayers of phospholipids determined by STM and X- ray diffraction (27, 35, 36). They suggest that the contrast in Fig. 3A is formed by lipid molecules oriented with polar groups facing the gold surface and the acyl chains directed to the electrolyte (35, 36). Therefore, the stripes seen in Figs. 2A and 3A show rows of upright oriented lipid molecules and each spot in the stripes corresponds to the top view of a single acyl chain of a lipid molecule. Fig. 4A shows a high resolution image of the domain with the flower-like structure. The contrast is formed by pairs of spots that are arranged into a hexagonal lattice whose unit cell has two vectors equal to 1.90 ± 0.1 nm and the angle between these vectors equal to 60°. The average area of the cell is equal to 3.13 ± 0.3 nm2. Each cell of this lattice contains three pairs of Fig. 2. Electrochemical STM (EC-STM) images of a monolayer of Alm and spots with the pair–pair distance 0.8 ± 0.2 nm. The mean area of DMPC/egPG (1:15 molar ratio) deposited onto a Au(111) surface. Images the pair of spots is 0.6 ± 0.2 nm2. This is in agreement with the were acquired using tunneling currents of (A) 1.16 and (B) 0.78 nA. effective cross-section area of the Alm molecule in I state (7, 22). Fig. 4B is an image of the structure in which the shaded area The ability to image an insulating molecule by STM may be illustrates the hexagonal nature of the 2D lattice. A group of flower-like six split spots, enclosed in the shaded area shown explained in terms of a weak coupling between electronic states in Fig. 4B, resembles a barrel-stave model of Alm channel pro- in the adsorbate and in the substrate near the Fermi level that posed in the literature (7). The average radius of the circle gives the adsorbate a property of an antenna capable of receiving shown in Fig. 4B is equal to 1.0 ± 0.2 nm. This number corre- tunneling electrons (33, 34). The electrolyte screens the negative sponds to the sum of the radius of the central pore plus half of charge on PG lipids and assists in ordering of the monolayer. the dimension of the split spot (each split spot is shared by two Water molecules may play a role in tunneling through bio- neighboring circles). In the literature, the channels are modeled molecules. However, the role of solvent in STM imaging in so- as a column of water surrounded by cylinders of Alm molecules. lution is still poorly understood (33). Two significantly different fi For the model of six Alm molecules the radius of the water CHEMISTRY structures can clearly be distinguished in these images. The rst column is equal to 0.55 nm, whereas the effective radius of the fl consists of elliptical spots arranged into a ower-like pattern and individual cylinders of Alm molecules is equal to 0.5 nm (22). A the second structure consists of long parallel stripes. In Fig. 2 This is in good agreement with the dimension of the radius of the fl fi the ower-like aggregates are imbedded into the ordered lm circle shown in Fig. 4B. We can therefore safely assume that the B consisting of long stripes. Fig. 2 shows that the aggregates can flower-like domains are formed by Alm molecules oriented also be observed in disordered film, indicating that formation of perpendicular to the surface of the monolayer. This is a different these aggregates is not induced by the presence of the ordered orientation than in the monolayer at the air–solution interface. long stripe domains. The presence of two ordered structures The film that was transferred from the air–solution interface BIOPHYSICS AND within the same monolayer indicates that the phase separation onto the gold electrode surface changed its structure upon drying occurs in the film. overnight and subsequent rehydration after immersion into the COMPUTATIONAL BIOLOGY To identify the nature of the two phases, higher resolution solution. The STM image suggests that each Alm molecule is images of each phase were acquired. Fig. 3A shows a domain shared between the two adjacent channels. Such an arrangement where only parallel stripes are seen. The average distance be- is unusual in the Alm literature. All models describing Alm tween the two adjacent spots within the stripe is 0.45 ± 0.1 nm channels in lipid bilayers suggest formation of individual bundles (Fig. 3B) and the mean area of the spot is 0.23 ± 0.1 nm2. These of Alm arranged into the barrel-stave channel (22). In the pore dimensions are consistent with the average distance between two models, Alm molecules are arranged such that the interior of the lipid chains and cross-sectional area of a lipid chain in closely channel is hydrophilic, whereas the exterior is hydrophobic, a

Fig. 3. (A) EC-STM images of DMPC/egPG monolayers (1:1 molar ratio) deposited onto a Au(111) surface. The image was acquired using a constant tunneling current of 1.16 nA. (B) Cross-sectional proﬁle of the lipid stripes. The proﬁle was taken along the black line shown on the STM image.

Pieta et al. PNAS | December 26, 2012 | vol. 109 | no. 52 | 21225 Downloaded by guest on September 27, 2021 Fig. 4. (A) EC-STM image of the ﬂower-like structures on a Au(111) surface. The image was acquired using a constant tunneling current of 1.16 nA. The hexagonal lattice of the channels deﬁned by two base vectors of length a = 1.90 ± 0.1 nm with an angle γ = 60 ± 5° between these vectors is superimposed on the image. (B) Schematic arrangement of channels formed by Alm molecules.

result of the amphiphatic character of the peptide. In the wheel center. Consequently, two different effective radii of the chan- model of the Alm helix (20), the polar groups occupy ∼40% of nels are observed ∼0.25 and ∼0.18 nm for the four and the three the circumference of the Alm molecules. These polar groups Gln7 residues, respectively (Fig. 5B). The radius of the channel (the Gln7 and Glu18 side chains and the carbonyl oxygen atoms formed by four Gln7 residues is in good agreement with the min- of Aib10 and Gly11) are located along a strip parallel to the Alm imum radius of the water column predicted for the hexameric axis, whereas Glu19 is rotated by about 98° with respect to the bundle by molecular dynamics (MD) calculations (37). positions of Gln7 (11, 20). The Gly11 and Glu18 are rotated Conclusions about 33° and 44°, respectively, with respect to the position of Gln7. The models assume that hydration and formation of hy- We have provided unique direct images of Alm aggregates in drogen bonded network between the Gln7 residues play a pivotal a Alm/DMPC/egg-PG matrix. The Alm molecules formed well- role in the channel formation (7, 11). This information was used ordered 2D nanocrystals surrounded by the lipids. The phos- to build the model that explains the contrast of Alm domains in pholipids assist insertion of Alm molecules; however, they do not STM images shown in Fig. 5A. In Fig. 5B the same structure is template the nanocrystals’ formation. The nanocrystal size dis- shown without the background of the STM image. In this model, tribution is quite broad. The Alm molecules are arranged into fl fl hydrophilic groups of Alm molecules are directed toward the a ower-like pattern with hexagonal lattice. Each ower-like unit center of the channels and Gln7 residues form a hydrogen- has a central pore surrounded by six Alm molecules. This unit bonded annulus. In this arrangement, the interior of the channels resembles the barrel-stave model of a hexameric pore formed by is hydrophilic, whereas the Alm molecules from neighboring cells Alm in phospholipid bilayers. The radius of the central pore is of the 2D lattice are oriented to each other with the hydrophobic comparable to the minimum radius of a column of water in the groups. The hydrogen-bonded annulus formed by Gln7 residues barrel-stave model of Alm aggregates predicted by MD calcu- determines the narrower fragment of the channel and controls lations. The present data provide a unique direct visualization of the conductive behavior of the Alm channels. In a hexameric the barrel-stave aggregation of an amphiphatic peptide. We have bundle, the number of water molecules hydrogen bonded to six also provided direct evidence that individual channels may form Gln7 side chains is between 20 and 25, giving the minimum ra- large clusters (nanocrystals). Cluster formation has been sug- dius of water column inside the channel 0.20–0.25 nm (37, 38). In gested in the literature. However, it has not been proven. The our model each channel consists of six Alm molecules. However, variable number of pores in the nanocrystals may explain mul- only three or four Gln7 residues are oriented toward the channel tiple conduction states for Alm incorporated into a BLM. These results constitute significant contribution to the understanding of the pore formation by amphiphatic peptides. Experimental Methods The working electrode was a small Au bead formed by melting a gold wire (Alfa Aesar, 99.999% purity). The bead was welded to a gold plate. The atomically flat (111) facets at the bead surface were used for image acquisition. Before each experiment, the gold electrode was cleaned in hot (80 °C)

piranha solution (concentrated H2SO4/30% H2O2 3:1 vol/vol) for 30 min and rinsed thoroughly with Milli-Q utrapure water. (CAUTION: piranha solution reacts violently with organic materials and should be handled with extreme care). The gold electrode was then ﬂame annealed and quenched in Milli-Q water before the experiment. The Kel-F parts of the STM electrochemical cell were cleaned in cool (20 °C) piranha solution. The STM images were acquired using a Nanoscope II EC-STM connected to a Nanoscope IIIa controller (Digital Instruments) with an A scanner. The constant current mode was used for imaging. The tungsten STM tips were electrochemically etched in 2 M NaOH Fig. 5. Proposed structural model for the Alm molecules assembly (A) and then coated with polyethylene to minimize the faradaic currents. The superimposed on the STM image and (B) without the STM image; image STM experiments were carried out at 18 ± 1 °C. For all experiments 0.1 M NaF dimensions 5.5 × 5.5 nm. (MV Laboratories) was used as the supporting electrolyte. Milli-Q ultrapure

21226 | www.pnas.org/cgi/doi/10.1073/pnas.1201559110 Pieta et al. Downloaded by guest on September 27, 2021 water (final resistivity ≥18.2 MΩ cm) was used to prepare all solutions. During mM in the solution. The final P/L molar ratio was 1:15, whereas DMPC/egPG the image acquisition, the electrode was kept at an open circuit potential was 1:1. A few drops of this solution were spread at the surface of a water- (OCP) of +0.2 V vs. Ag/AgCl with a bias voltage of −0.35 V. filled Langmuir–Blodgett trough (KSV) to form a monolayer. The trough was DMPC, egPG, both from Avanti, and alamethicin, from Sigma-Aldrich, controlled by a computer using KSV LB5000 v1.70 software. The monolayers of −1 were used without further purification to make 10 and 4 mg·mL stock Alm/(DMPC+eggPG) (P/L = 1:15) were transferred from the Langmuir trough solutions, respectively, in chloroform:trifluoroethanol (Sigma-Aldrich) (1:1, at a surface pressure of 27 mN/m by the Langmuir–Blodgett technique. During vol/vol) mixed solvent. DMPC, egPG, and Alm solutions were added to a test the compression the temperature of the aqueous subphase was kept at 15 °C. tube to obtain a 6.25% molar ratio of Alm with respect to the lipids. The After deposition, each sample was dried overnight at temperature ∼17 °C tube was then heated to about 40 °C for 1 h. During this time the solution and then transferred into the STM electrochemical cell. was mixed in a vortex (Fisher; Vortex Genie 2) and the solvent was evapo- rated under an argon stream. A thin film of the lipid–peptide mixture ACKNOWLEDGMENTS. P.P. thanks Dmitriy Soldatov (Department of Chem- remained on the wall of the test tube. Further drying was achieved by istry, University of Guelph) for help with interpretation of X-ray diffraction storing the test tube under vacuum for at least 12 h at room temperature. data and J.L. acknowledges a Canada Research Chair award. This work was The dry film in the test tube was dissolved in chloroform (Sigma-Aldrich) to supported by a Natural Sciences and Engineering Research Council (Canada) give the final concentration Alm 0.153 mM, DMPC 1.145 mM, and eggPG 1.145 Discovery grant.

1. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415(6870): 22. Constantin D, Brotons G, Jarre A, Li C, Salditt T (2007) Interaction of alamethicin pores 389–395. in DMPC bilayers. Biophys J 92(11):3978–3987. 2. Hancock REW (2001) Cationic peptides: Effectors in innate immunity and novel an- 23. He K, Ludtke SJ, Heller WT, Huang HW (1996) Mechanism of alamethicin insertion timicrobials. Lancet Infect Dis 1(3):156–164. into lipid bilayers. Biophys J 71(5):2669–2679. 3. Volinsky R, Kolusheva S, Berman A, Jelinek R (2006) Investigations of antimicrobial 24. Wang J, Pullman A (1991) Do helices in membranes prefer to form bundles or stay peptides in planar ﬁlm systems. Biochim Biophys Acta 1758(9):1393–1407. dispersed in the lipid phase? Biochim Biophys Acta 1070(2):493–496. 4. Meyer CE, Reusser F (1967) A polypeptide antibacterial agent isolated from Tricho- 25. Cheng JTJ, Hale JD, Elliot M, Hancock REW, Straus SK (2009) Effect of membrane derma viride. Experientia 23(2):85–86. composition on antimicrobial peptides aurein 2.2 and 2.3 from Australian southern 5. Marsh D (1996) Peptide models for membrane channels. Biochem J 315(Pt 2):345–361. bell frogs. Biophys J 96(2):552–565. 6. Leitgeb B, Szekeres A, Manczinger L, Vágvölgyi C, Kredics L (2007) The history of 26. Clejan S, Krulwich TA, Mondrus KR, Seto-Young D (1986) Membrane lipid composi- alamethicin: A review of the most extensively studied peptaibol. Chem Biodivers 4(6): tion of obligately and facultatively alkalophilic strains of Bacillus spp. J Bacteriol – 1027–1051. 168(1):334 340. 7. Fox RO, Jr., Richards FM (1982) A voltage-gated ion channel model inferred from the 27. Sek S, Laredo T, Dutcher JR, Lipkowski J (2009) Molecular resolution imaging of an – crystal structure of alamethicin at 1.5-A resolution. Nature 300(5890):325–330. antibiotic peptide in a lipid matrix. J Am Chem Soc 131(18):6439 6444. 28. Hörber JKH, Lang CA, Hänsch TW, Heckl WM, Möhwald H (1988) Scanning tunneling

8. Bertelsen K, et al. (2007) Membrane-bound conformation of peptaibols with methyl- CHEMISTRY ﬁ deuterated α-amino isobutyric acids by 2H magic angle spinning solid-state NMR microscopy of lipid lms and embedded biomolecules. Chem Phys Lett 145(2): – spectroscopy. J Am Chem Soc 129(47):14717–14723. 151 158. 9. Vogel H (1987) Comparison of the conformation and orientation of alamethicin and 29. Smith DPE, et al. (1987) Images of a lipid bilayer at molecular resolution by scanning tunneling microscopy. Proc Natl Acad Sci USA 84(4):969–972. melittin in lipid membranes. Biochemistry 26(14):4562–4572. 30. Gregory BW, Dluhy RA, Bottomley LA (1994) Structural characterization and nano- 10. Haris PI, Chapman D (1988) Fourier transform infrared spectra of the polypeptide meter-scale domain formation in a model phospholipid bilayer as determined by alamethicin and a possible structural similarity with bacteriorhodopsin. Biochim Bi- infrared spectroscopy and scanning tunneling microscopy. J Phys Chem 98(3): ophys Acta 943(2):375–380. 1010–1021. 11. Sansom MSP (1993) Alamethicin and related peptaibols—model ion channels. Eur 31. Ionov R, El-Abed A, Angelova A, Goldmann M, Peretti P (2000) Asymmetrical ion- Biophys J 22(2):105–124. channel model inferred from two-dimensional crystallization of a peptide antibiotic.

12. He K, Ludtke SJ, Worcester DL, Huang HW (1996) Neutron scattering in the plane of BIOPHYSICS AND Biophys J 78(6):3026–3035. membranes: Structure of alamethicin pores. Biophys J 70(6):2659–2666.

32. Haefele T, Kita-Tokarczyk K, Meier W (2006) Phase behavior of mixed Langmuir COMPUTATIONAL BIOLOGY 13. Huang HW (2006) Molecular mechanism of antimicrobial peptides: The origin of monolayers from amphiphilic block copolymers and an antimicrobial peptide. Lang- cooperativity. Biochim Biophys Acta 1758(9):1292–1302. muir 22(3):1164–1172. 14. Huang HW, Wu Y (1991) Lipid-alamethicin interactions inﬂuence alamethicin orien- 33. Giancarlo LC, Flynn GW (1998) Scanning tunneling and atomic force microscopy – tation. Biophys J 60(5):1079 1087. probes of self-assembled, physisorbed monolayers: Peeking at the peaks. Annu Rev 15. Bechinger B (1997) Structure and functions of channel-forming peptides: Magainins, Phys Chem 49:297–336. – cecropins, melittin and alamethicin. J Membr Biol 156(3):197 211. 34. He YF, Ye T, Borguet E (2002) The role of hydrophobic chains in self-assembly at 16. Breed J, Kerr ID, Sankararamakrishnan R, Sansom MSP (1995) Packing interactions of electriﬁed interfaces: Observation of potential-induced transformations of two-di- Aib-containing helices: Molecular modeling of parallel dimers of simple hydrophobic mensional crystals of hexadecane by in-situ scanning tunneling microscopy. J Phys – helices and of alamethicin. Biopolymers 35(6):639 655. Chem B 106(43):11264–11271. 17. Woolley GA, Wallace BA (1992) Model ion channels: Gramicidin and alamethicin. J 35. Lewis RNAH, Zhang Y-P, McElhaney RN (2005) Calorimetric and spectroscopic studies of Membr Biol 129(2):109–136. the phase behavior and organization of lipid bilayer model membranes composed of 18. Mathew MK, Balaram P (1983) A helix dipole model for alamethicin and related binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol. transmembrane channels. FEBS Lett 157(1):1–5. Biochim Biophys Acta 1668(2):203–214. 19. Cascio M, Wallace BA (1988) Conformation of alamethicin in phospholipid vesicles: 36. Fischer A, Sackmann E (1984) Electron-microscopy and diffraction study of phos- Implications for insertion models. Proteins 4(2):89–98. pholipid monolayers transferred from water to solid substrates. J Phys (Paris) 45(3): 20. Pan J, Tristram-Nagle S, Nagle JF (2009) Alamethicin aggregation in lipid membranes. 517–527. J Membr Biol 231(1):11–27. 37. Tieleman DP, Berendsen HJC, Sansom MSP (1999) An alamethicin channel in a lipid 21. Salditt T, Li C, Spaar A (2006) Structure of antimicrobial peptides and lipid bilayer: Molecular dynamics simulations. Biophys J 76(4):1757–1769. membranes probed by interface-sensitive X-ray scattering. Biochim Biophys Acta 38. Tieleman DP, Hess B, Sansom MSP (2002) Analysis and evaluation of channel models: 1758(9):1483–1498. Simulations of alamethicin. Biophys J 83(5):2393–2407.

Pieta et al. PNAS | December 26, 2012 | vol. 109 | no. 52 | 21227 Downloaded by guest on September 27, 2021