Membrane structure and conformational changesof the antibiotic heterodimeric peptide distinctinby solid-state NMR spectroscopyJarbas M. Resendea,b,1, Cleria Mendonca Moraesa,b,1, Victor H. O. Munhozb, Christopher Aisenbreya, Rodrigo M. Verlyb,Philippe Bertania, Amary Cesarb, Dorila Pilo-Velosob, and Burkhard Bechingera,2

aUniversite de Strasbourg, Centre National de la Recherche Scientifique, UMR7177, Institut de Chimie, 4 rue Blaise Pascal, 67070 Strasbourg, France;and bDepartamento de Química, Universidade Federal de Minas Gerais, Avenida Presidente Antonio Carlos, 6627, 31270-901, Belo Horizonte, MG, Brazil

Edited by Jean-Marie P. Lehn, Universite de Strasbourg - ISIS, Strasbourg Cedex, France, and approved August 3, 2009 (received for review May 10, 2009)

The heterodimeric antimicrobial peptide distinctin is composed of2 linear peptide chains of 22- and 25-aa residues that are connectedby a single intermolecular S-S bond. This heterodimer has beenconsidered to be a unique example of a previously unrecorded classof bioactive peptides. Here the 2 distinctin chains were prepared bychemical peptide synthesis in quantitative amounts and labeledwith 15N, as well as 15N and 2H, at selected residues, respectively,and the heterodimer was formed by oxidation. CD spectroscopyindicates a high content of helical secondary structures whenassociated with POPC/POPG 3:1 vesicles or in membrane-mimeticenvironments. The propensity for helix formation follows theorder heterodimer >chain 2 >chain 1, suggesting that peptide-peptide and peptide-lipid interactions both help in stabilizing thissecondary structure. In a subsequent step the peptides werereconstituted into oriented phospholipid bilayers and investigatedby 2H and proton-decoupled 15N solid-state NMR spectroscopy.Whereas chain 2 stably inserts into the membrane at orientationsclose to perfectly parallel to the membrane surface in the presenceor absence of chain 1, the latter adopts a more tilted alignment,which further increases in the heterodimer. The data suggest thatmembrane interactions result in considerable conformational re-arrangements of the heterodimer. Therefore, chain 2 stably an-chors the heterodimer in the membrane, whereas chain 1 interactsmore loosely with the bilayer. These structural observations areconsistent with the antimicrobial activities when the individualchains are compared to the dimer.

amphipathic alpha-helix � membrane insertion � membrane proteinstructure determination � peptide-peptide interactions � synergistic activity

As more and more pathogens develop resistance against manycommonly used antibiotics, urgent actions are needed to

counteract this resistance and new bactericidal and fungicidalcompounds have to be developed. Both plants and animalsproduce, store, and secrete antibiotic peptides in exposed tissues,or synthesize such compounds upon induction, and indeed manyantibiotic peptides have been isolated from natural sources (1,2). The availability of these molecules establishes a defensesystem that can be set into action immediately when infectionsoccur, and several antimicrobial peptides have been found toalso exhibit virucidal and tumorcidal activities (reviewed in refs.3 and 4). The lack of sequence homology, the activity of chiralanalogues, the amphipathic properties, and biophysical measure-ments all suggest that the bacterial membranes rather than chiralreceptors are the targets of many of these peptides (1, 3),although more recent findings suggest that at least some of themalso have internal targets after crossing the membrane (5, 6).

The heterodimeric peptide distinctin has been isolated fromPhyllomedusa distincta, a frog living in the southeast Atlanticforests of Brazil (7). The distinctin is composed of 2 linearpeptide chains, one of 22- and the other of 25-aa residues thatare connected by a single intermolecular S-S bond. Antimicro-bial assays demonstrate that distinctin, as well as its individual

chains, exhibit marked antimicrobial activity against both gram-positive and gram-negative bacteria (7, 8). The distinctin het-erodimer has been considered as a unique example of a previ-ously unrecorded class of bioactive peptides from frog skin.Multidimensional solution NMR spectroscopy has been used todetermine the 3-dimensional structure of the distinctin het-erodimer in aqueous solution (9). The peptide forms a predom-inantly helical structure where the 2 chains of the heterodimerare in close contact with each other. To screen the hydrophobicsurfaces formed by the 2 amphipathic helices, 2 heterodimersassociate in aqueous solution and form a compact 4-helixarrangement that is better protected from proteolysis whencompared to the individual chains or the homodimers (8). Invitro and in vivo assays showed that distinctin, as well as mixturesof distinctin and some commonly used antibiotics, prevent thedetrimental effects of Staphylococus aureus in central venouscatheters (10) or after infections (11). It has also been shown thatdistinctin and its individual chains exhibit considerable pore-forming and membrane-interactive properties in agreement withthe amphipathic nature of the 2-peptide helices (8). Althoughthe structure of distinctin in aqueous solution has been deter-mined (9), little is known about the membrane interactions andthe mechanism of pore formation of distinctin. To better un-derstand the interactions of distinctin with the membranes andto evaluate some of the suggested models (e.g., ref. 9 or fig. 3Ein ref. 3), more structural and topological information of theheterodimer in membrane environments are required.

Whereas CD spectroscopy is a well-established technique toinvestigate the secondary structure of proteins and peptides,solid-state NMR spectroscopy on uniaxially aligned samples hasproven highly valuable during the analysis of the conformationof membrane-associated peptides, of helix tilt angles, and ofpeptide topologies (3). In particular, because of a combinationof favorable properties of the anisotropic 15N chemical shiftinteractions, the approximate orientation of a helix is directlyobtained from the 15N chemical shift of 15N-labeled peptidebonds (12). Whereas transmembrane helical peptides exhibit 15Nchemical shifts in the 200 ppm range, those that align parallel tothe surface resonate at �100 ppm.

Additional information on the molecular alignment is ob-tained from the deuterium quadrupolar splitting, which ideallycomplements the orientational restraints obtained from the 15N

Author contributions: D.P.-V. and B.B. designed research; J.M.R., C.M.M., V.H.O.M., C.A.,R.M.V., and P.B. performed research; J.M.R., C.A., and A.C. analyzed data; and B.B. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1J.M.R. and C.M.M. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: bechinger@chimie.u-strasbg.fr.

This article contains supporting information online at www.pnas.org/cgi/content/full/0905069106/DCSupplemental.

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chemical shift analysis, and when taken together allow for amuch more accurate determination of both the helix tilt androtational pitch angles (13). Because the deuterium NMR spec-tra are particularly sensitive to changes in the angle defined bythe C�-C� bond of alanines and the magnetic field direction, thedeuterium spectral-line shapes are also very sensitive indicatorsof even small orientational changes or of the mosaic spread ofthe peptide within oriented membranes (13).

In the present work, oriented solid-state NMR- and CD-spectroscopies are used to investigate the secondary structures,the membrane interactions, and alignments of distinctin and itsindividual chains. By systematically comparing the structures,membrane interactions, and biological activities of the distinctinindividual chains, as well as the heterodimer, the importance ofpeptide-peptide- and peptide-membrane interactions have beeninvestigated. This study thereby provides unique insights into themolecular mechanisms underlying the action of antimicrobialpeptides, as well as the role of dimer formation and inter-peptidecontacts within the membrane.

ResultsFor the present study, quantitative amounts of peptides wereprepared by solid-phase peptide synthesis, labeled with stableisotopes, oxidized to form the heterodimer, and purified. Thepeptide-membrane interactions of the individual chains and ofthe heterodimers were investigated using oriented solid-stateNMR- (Figs. 1–3) and CD- spectroscopies [see supportinginformation (SI) Figs. S1 and S2]. Fig. 1 A and B show proton-decoupled 15N solid-state NMR spectra of 1 mole% of theisolated chain 1 and chain 2, respectively, after reconstitution

into oriented POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes, whereas the 31P NMR spectra ofthe same samples are represented in Fig. S3. The site of the 15Nlabels and the chemical shift positions are summarized in Table1. Whereas for Ala-11 of chain 1 a 15N chemical shift of 96 ppmis observed (see Fig. 1 A), the 15N at the leucine-12 position ofchain 1 resonates at 69 ppm (not shown) and that of chain 2labeled with 15N at the Leu-16 position at 68 ppm (see Fig. 1B).These chemical shift values are in agreement with helix align-ments approximately parallel to the membrane surface.

Fig. 2A shows the 2H solid-state NMR spectrum of chain 1labeled with 2H3 at the methyl group of alanine-11 for a samplealignment of the membrane normal parallel to the magnetic fieldof the NMR spectrometer (Bo). The spectrum is characterized bya quadrupolar splitting of 24 kHz, and this parameter definesadditional orientational constraints that will allow the determi-nation of accurate tilt and rotational pitch angles (13, 14). When

Fig. 1. Proton-decoupled 15N solid-state NMR spectra of (A) 1 mole%(15N-Ala-11)-chain 1, (B) 1 mole% (15N-Leu-16)-chain 2, (C) (15N-Ala-11)-chain1 and 15N-Leu-16)-chain 2 distinctin, and (D) (15N-Leu-12)-chain 1 distinctinreconstituted at about 0.4 mole% into oriented POPC membranes. All spectrawere recorded for sample orientations with the membrane normal parallel toBo. For clarity, only the 15N labels are indicated.

Table 1. Summary of solid-state NMR data obtained from various distinctin preparations reconstituted into oriented POPC at samplealignments with the normal parallel to the magnetic field direction

Chain 1 Chain 1 in dimer Chain 2 Chain 2 in dimer

15N labels and chemical shift (ppm) � 2 ppm 15N labels and chemical shift (ppm) � 2 ppm

Ala 11 96 108 Leu 16 68 70Leu 12 69 72

2H3 labels and quadrupolar splitting (kHz) �1 kHz 2H3 labels and quadrupolar splitting (kHz) �1 kHzAla 11 24 27/�57 Ala 9 52 54

Fig. 2. 2H solid-state NMR spectra of 0.5 mol% (3,3,3-2H3-Ala-11)-chain 1 (Aand C), 1 mole% (3,3,3-2H3-Ala-9)-chain 2 (B and D), 0.4 mole% (3,3,3-2H3-Ala-11)-chain 1 distinctin (E and G), and (3,3,3-2H3-Ala-9)-chain 2 distinctin (F)reconstituted into oriented POPC membranes. (A), (B), (E), and (F) wererecorded for sample orientations with the membrane normal parallel to Bo.(C), (D), and (G) were recorded for sample alignments with the membranenormal perpendicular to Bo. (H) The simulated spectrum for sample orienta-tions where the C�-C� bond is at 25o� 2o relative to Bo. For clarity, only the 2Hlabels are indicated.

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the oriented membrane sample encompassing chain 2 labeledwith 2H3-alanine at position 9 is investigated, the 2H solid-stateNMR spectrum shown in Fig. 2B is obtained. A deuteriumquadrupolar splitting of 52 kHz is observed. When compared tochain 2, the 15N and 2H solid-state NMR spectra of chain 1 arecharacterized by broader lines, a more heterogenous alignment,and additional contributions at increased quadrupolar splittings(see Figs. 1 A and 2A).

When the samples are tilted by 90o (membrane normalperpendicular to Bo), the spectra shown in Figs. 2 C and D areobtained. The quadrupolar splittings observed for chain 2 de-crease to about half the value (see Fig. 2D), indicating fastrotational diffusion around the membrane normal (15). Al-though the tilted spectrum of chain 1 exhibits on average adecreased quadrupolar splitting, the more heterogeneous align-ment of this peptide chain in combination with the strong signalintensity of the naturally abundant deuterons of membrane-associated water (central lines exhibiting a quadrupolar splittingof about 1 kHz) hamper a detailed analysis of the tilted spectrum(see Fig. 2C).

The deuterium and the proton-decoupled 15N solid-stateNMR spectra of the distinctin heterodimer are shown in Figs. 1C and D and 2 E to G, and the data are summarized in Table 1.Two heterodimers were synthesized for the present study, withdifferent isotopic labeling patterns. The first dimer was preparedfrom (15N-Ala-11)-chain 1 and (3,3,3-2H3-Ala-9, 15N-Leu-16)-chain 2 (see Figs. 1C and 2F), and the second polypeptide carriedlabels only at chain 1 at the 3,3,3-2H3-Ala-11 and the 15N-Leu-12positions. Notably, the 15N chemical shifts and deuterium qua-drupolar splitting observed for chain 2 resemble those of theindividual chain (see Table 1), where the deuterium line shapeand quadrupolar splitting obtained for alanine-9 (54 kHz) (seeFig. 2F) are indicative of an alignment of the C�-C� bond at 25o,with a mosaic spread of about � 2o (see Fig. 2H).

In contrast, the broadened signal intensity at 108 ppm (see Fig.1C), which is assigned to 15N-alanine-11 of chain 1, has movedto a significantly higher chemical shift value when compared tothe isolated chain 1 (see Table 1), thereby indicating a change inthe orientation of this polypeptide upon covalent associationwith chain 2. The 2H solid-state NMR spectrum of the dimer(3,3,3-2H3 alanine-11) confirms a rather heterogeneous align-ment or conformation at this site, with at least 2 spectralcontributions, one exhibiting maxima defining a quadrupolarsplitting of about 27 kHz and another with a distribution ofsplittings �57 kHz (see Fig. 2 E and G). When the sample wastilted by 90o, a plateau of quadrupolar splittings �32 kHz isobserved (see Fig. 2G), indicating that motional averaging is slowon the 10�5 s time scale (15).

To characterize the membrane interactions and to determinethe resulting topology of the chains individually and in thecontext of the heterodimer, orientational restraints were calcu-lated from the 15N chemical shift and the 2H quadrupolarsplittings. The tilt and rotational pitch angular pairs that agreewith the experimental data are presented by the contours in Fig.3. Whereas Fig. 3A analyzes the experimental data in terms ofmembrane topologies of the individual chain 1, Fig. 3B repre-sents the measurements on chain 2. The traces in black andturquoise describe angular pairs that agree with the measuredexperimental 15N chemical shifts within � 2 ppm; the traces inred represent the topologies that are derived from the experi-mental 2H quadrupolar splitting within � 1 kHz.

Whereas for chain 1 the 15N-Ala-11 chemical shift alone (96ppm) limits the possible tilt angles to 52o to 80o and 104o to 128o,consideration of an additional experimental parameter (�nQ �24 kHz for the 3,3,3-2H3-Ala-11 site) limits the possible orien-tations of chain 1 to the 6 configurations that correspond to theintersections in the contour plots. These topologies are repre-sented as structural models in Fig. 3E. Additional orientational

restraints were obtained from the second peptide preparation,which exhibits a 15N chemical shift of 69 ppm for Leu-12 of chain1. These are shown in Fig. 3A by the contours in turquoise andare only in agreement with configurations II and III. Amongthese, orientation II (tilt/rotational pitch angular pair 71o/107o)represents the energetically most favorable positioning of hy-drophilic and hydrophobic residues within the membrane inter-face. The helix alignment of chain 1 thereby represents asignificant deviation (19o) from a perfect in-plane orientation.

Fig. 3B represents all possible alignments of chain 2 as afunction of the tilt and rotational pitch angles. Again, the blacktraces describe the angular restraints obtained from the exper-imental 15N chemical shifts (68 ppm � 2 ppm) and the red tracesthe restraints obtained from the 2H quadrupolar splitting (52kHz � 1 kHz). Only a few tilt/rotational pitch angular pairscorroborate both parameters simultaneously and are highlightedby the circles in Fig. 3B. The corresponding arrangements arerepresented in Fig. 3F and among these, orientation III (tilt/rotational pitch angles 88o/157o) indicates the optimal insertionof hydrophilic and hydrophobic residues of chain 2 into thebilayer interface. The almost perfect in-plane arrangement oftopology III allows the insertion of the hydrophobic residues inthe membrane interior, while the hydrophilic residues are incontact with the polar interface.

The alignment of chain 1 within the distinctin heterodimer wasdetermined in an analogous manner (see Fig. 3C). Among theintersections, only orientations II and III agree with the 3experimental measurements and the first of these, orientation II(tilt/rotational pitch angles 66o/101o), represents the energeti-cally most favorable interaction with the membrane interface.When compared to the isolated polypeptide, a slightly moretilted arrangement is observed for chain 1 in the dimer. Thetopology of the chain 2 within the distinctin heterodimer isrepresented by the 5 intersections in Fig. 3D. Among thoseintersections, topology III (88o/156o tilt/rotational pitch angularpair) represents best the hydrophilic/hydrophobic properties ofthe bilayer interface. Once the arrangement of both chains in thedimer have been defined from experimental data and energeticconsiderations, it is possible to determine the orientation of thedistictin heterodimer with respect to the lipid bilayer by ap-proaching the 2 chains to satisfy the formation of the disulfidebond. The resulting model of distinctin in POPC lipid bilayers ispresented in Fig. 4C, where tilt/rotational pitch angular pairs of66o/101o and 88o/156o are observed for the distinctin chain 1 andchain 2, respectively. The 2 chains are shown in a shifted ratherthan a side-by-side alignment, because the latter forces theinsertion of one of the hydrophilic faces of chain 1 or -2 insidethe hydrophobic core of the lipid bilayer.

DiscussionHere we have prepared the heterodimeric peptide distinctin andits individual chains and labeled selected sites of the peptidechains with 2H and 15N for solid-state NMR investigations andfor CD spectroscopy. Whereas the CD experimental data indi-cate that the distinctin heterodimer adopts helical conformationsin membrane environments, in agreement with recent investi-gation of distinctin using FTIR spectroscopy (8), the solid-stateNMR experiments show that the peptide helices are orientedapproximately parallel to the membrane surface, thereby re-flecting the amphipathic distribution of apolar and hydrophilicamino acid side chains (see Fig. 3). The alignment of chain 2 iswithin experimental error, perfectly in-plane, and does notchange when this polypeptide associates with chain 1. Thisobservation suggests that helix 2 stably anchors the individualchain as well as the dimer in the membrane interface. In contrast,chain 1 adopts a more tilted arrangement that deviates fromperfect in-plane alignments by about 19o for the individual chain,and further augments to about 24o when covalently linked to

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chain 2. Closer inspection of the models shown in Figs. 3E and4C shows that this positioning of helix 1 removes the lysine-20side chains from close contact with the hydrophobic portions ofthe membrane interior. These findings agree with previousattenuated total reflectance FTIR investigations where themaximal average tilt angles of the dimer are altered whencompared to the monomers (8). Notably, the membrane-associated chain 1 not only undergoes a change in topology upon

attachment to chain 2, but the observed line broadening alsoindicates a more heterogeneous conformation and alignment atthe labeled Ala-11-site (see Figs. 1 C and D, and 2 E and G). Theline broadening and the additional intensities observed in thespectra shown in Figs. 1C and 2E agree, among other possibil-ities, with a distribution of rotational pitch angles (Fig. S4).Conformational and topological variants have also been de-tected for phospholamban, sarcolipin, and cytochrome b5 using

Fig. 3. Possible alignments of chain 1 (A) and chain 2 (B) individually or of chain 1 (C) and chain 2 (D) in the distinctin herterodimer reconstituted into POPClipid bilayers as a function of the tilt and rotational pitch angles. The black and turquoise traces represent orientations that are in agreement with theexperimental 15N chemical shift within � 2 ppm; the red traces represent alignments that agree with experimental deuterium quadrupolar splittings within �1 kHz. The intersections between these traces are marked by blue circles (I to VI) and correspond to topologies that agree with both experimental measurements.(E and F) Molecular models based on PDB 1XKM (9) that agree with the measured topologies of the isolated chain 1 (see text for details) and chain 2, respectively.The hydrophilic peptide residues are represented in green and the hydrophobic residues in blue. Lysine-20 of chain 1 is highlighted for configuration II (E). Thegray zones schematically represent the hydrophobic membrane interior. The side chains are shown as they occur in 1XKM to illustrate the distribution ofhydrophobic and hydrophilic residues along the helix, but these and the flexible termini may rearrange to optimize the interactions within the phospholipidbilayers.

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solid-state NMR spectroscopy (16–18). In addition, when theCD data of chain 1 and chain 2 are compared to each other, itbecomes obvious that the latter exhibits a more pronouncedrandom coil�helix transition (see SI Text). The data suggest thatchain 1 is more loosely associated with the membrane whencompared to chain 2.

Interestingly, the CD data presented in Fig. S1 C to E indicatethat when compared to the individual chains, the dimer exhibitsan increased tendency for helix formation in both aqueous andin membrane environments. Therefore, the peptide-peptideinteractions not only cause the realignment of helix 1 relative tothe membrane surface by about 5o (topology II, see Fig. 3 A andC) but they also stabilize the helix conformation inherent in thesequences in the presence as well as in the absence of membranes(see Fig. S1 C–E). Whereas hydrophobic mismatch-dependenttopological alterations (19, 20) or interactions between trans-membrane helical peptides have been studied previously inconsiderably detail (21–23), the data shown here provide aunique example where helix-helix interactions are described atan interfacial bilayer location. A significant increase in helixconformations has also been observed when the concentration oflinear amphipathic antimicrobial peptides in aqueous buffer isincreased or their solubility reduced by, for example, a change inpH (24–26). These observations are indicative that interactionsof the peptide with both the membrane and with other peptidescan shift the conformational equilibria (27).

NMR studies in aqueous solution indicate the existence ofhydrophobic interchain interactions within the dimer structure(Fig. 4A). These results allied to size-exclusion chromatographysuggested that distincin is present in water as a homodimer (9).The chains arranged as a 4-helix bundle (Fig. 4B) optimize thehydrophobic interactions in the core of the complex and stabilizethe structure of distinctin in aqueous environments (9). How-ever, when interacting with membranes, conformational rear-rangements occur and our data point to a more open structure(see Fig. 4C) where the hydrophobic peptide-peptide interac-tions are replaced by peptide-membrane interactions within thebilayer interface. Related conformational changes have beendemonstrated for the pore-forming domains of colicins wheninteracting with phospholipid membranes (28) and for domainsof proteins of the Bcl-2 family (29).

Notably, the activity of naturally occurring combinations ofsingle-chain peptides, such as PGLa and magainin 2 (30, 31) orof covalently linked antiparallel dimers of magainin 2 and the

monomer of the same peptide (32), exhibit synergistic enhance-ment of antimicrobial activities. Homodimers formed by reduc-tion of a cysteine 22 variant of magainin 2 have also been shownmore efficient in carboxyfluorescine release from large unila-mellar vesicles and in antibacterial assays when compared to itsoxydized form (33). Furthermore, cecropin-melittin or cecropin-magainin hybrid peptides are more active by up to 2 orders ofmagnitude in antibiotic assays when compared to nativececropins, when at the same time these combinatorial peptidesretain the lack in hemolytic activity (34, 35). In contrast, thecovalent association of the distinctin chains is associated withonly a modest increase in biological activities (8). Therefore, ithas been suggested that the covalent association of 2 helices isof advantage to protect the peptides from proteolytic degrada-tion rather than to increase the membrane permeabilizationactivities (8, 9). This theory is consistent with our structuralobservations where the membrane interactions, structure, andtopology of chain 2 resemble those of other linear cationicpeptide antimicrobials (reviewed in ref. 3), but where chain 1 ismore loosely associated with the membrane.

The structural data from both individual chains, as well as theheterodimer, indicate that the amphipathic helices of distinctinare stably inserted into the membrane at alignments approxi-mately parallel to the membrane surface. Partitioning into themembrane interface at alignments parallel to the surface havebeen observed experimentally as well for other amphipathicsequences, such as dermaseptin (36), magainins (3), dermadis-tinctin K (37), or designed sequences (38). Our structural datasuggest that membrane permeabilization occurs in an analogousmanner as with other amphipathic peptides, which all intercalateinto the bilayer interface (see Fig. 3). The solid-state datatherefore favor a mechanism where, under the experimentalconditions of the present study, the membrane packing is locallydestabilized by the insertion of amphipathic molecules ratherthan models where the helices adopt transmembrane alignments(39). Therefore, the nature of the molecules, such as charge andhydrophobic volume, strongly contribute to the variety of actionsobserved and the lipid polymorphism induced. In addition, thistype of supramolecular polypeptide-lipid assemblies can exhibitmany different morphologies that are best described by phasediagrams (3, 39) where lysis, wormhole- (40) and carpet-model(41), or regions where the bilayer is unaffected or even stabilized,correspond to different regions in the diagram. The ‘‘phaseboundaries’’ depend on lipid composition, temperature, salt, pH,and other environmental parameters, and thereby reflect thevariable environments encountered by the peptides when inter-acting with the membranes of different species (39, 42).

In-plane membrane alignments have been observed previ-ously for many amphipathic peptides, including antimicrobialcompounds. However, these previous investigations addressedthe membrane interactions of peptide antibiotics that lack acovalent bond for dimerization. To our knowledge, the studypresented here is unique in addressing the structural transitionsduring membrane association of a heterodimeric peptide usingoriented solid-state NMR spectroscopy. Upon membrane asso-ciation, distinctin refolds from a compact globular structure insolution to a more open configuration, where one in-planeoriented helix anchors the dimer in the membrane interfacewhile the other chain interacts more loosely with the bilayer.Furthermore, by comparing the structures of the monomers withthose of the heterodimer, important insights were gained on thecontributions of peptide-peptide interactions in membrane en-vironments. The structural and topological findings correlatewell with the biological activities of these polypeptide chains.

Materials and MethodsPhospholipids [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and1-palmitoyl-2-oleoyl–sn-glycero-3-phosphoglycerol (POPG)] were purchased

Fig. 4. NMR-structures of distinctin in aqueous solution [PDB 1XKM (9)]: (A)heterodimer and (B) dimer of heterodimers. (C) Structural arrangement ofdistinctin in POPC lipid bilayers.

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from Avanti Polar Lipids. The 2 individual chains of the distinctin peptide weresynthesized independently using solid-phase peptide synthesis and Fmoc(9-fluorenylmethyloxycarbonyl) chemistry. The sequences of the individualchains are:

chain 1: ENREV PPGFT ALIKT LRKCK II (net charge �3)chain 2: NLVSG LIEAR KYLEQ LHRKL KNCK V (net charge �4/�5).The 15N-labeled analogues of alanine or leucine studied here are shown in

bold and the (3,3,3-2H3)-alanine sited are highlighted by the italicized posi-tions. The Fmoc precursors carrying the isotopic labels were from CambridgeIsotope Laboratories, Inc.. The individual chains of distinctin were synthesizedindependently using solid-phase peptide synthesis and the heterodimericpeptide was prepared by air oxidation (43). First (15N-Ala-11)-chain 1; (3,3,3-2H3-Ala-9, 15N-Leu-16)-chain 2 distinctin were prepared. In a second synthesis,chain 1 was prepared carrying labels at 15N-Leu-12 and 3,3,3-2H3-Ala-11. Thispeptide was subsequently coupled to chain 2, which had been prepared in theabsence of isotopic enrichment.

Samples for solid-state NMR spectroscopy were prepared and the spectrawere acquired, as described previously (13) and as detailed in the SI Text.

Simulations were performed to derive the topology of individual chains, aswell as of the distinctin dimer relative to the lipid bilayers from the experi-mental 15N chemical shift and 2H quadrupolar splittings. The calculations wereperformed using a molecular model based on the published structure insolution (PDB 1XKM) (9), followed earlier protocols (13), and are described indetail in the SI Text.

ACKNOWLEDGMENTS. We thank the Institute of Supramolecular Chemistryof the University of Strasbourg for hosting the Bechinger laboratory. Thiswork was supported in part by a postdoctoral and 2 PhD grants from theConselho Nacional de Desenvolvimento Científico e Tecnologico (to J.M.R.,V.H.O.M., and R.M.V.); a postdoctoral grant from the Coordenacao de Aper-feicoamento de Pessoal de Nível Superior (to C.M.M.); Grant TG0501 fromVaincre la Mucovicidose; the Agence National de Recherche; the Fundação deAmparo à Pesquisa do Estado de Minas Gerais; the Brazilian-French programCoordenacao de Aperfeicoamento de Pessoal de Nível Superior-Comite Fran-cais pour l’evaluation de la Cooperation Universitaire avec le Bresil (487/05);and the NMR Network Grand-Est of the French Ministry of Science andEducation.

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