Effects of amphipathic polypeptides on membrane organization

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Effects of amphipathic polypeptides on membrane organization inferred from studies using bicellar lipid mixtures Chris Miranda, Valerie Booth, and Michael R. Morrow Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02257 • Publication Date (Web): 08 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018

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Effects of amphipathic polypeptides on membrane organization inferred from studies using bicellar lipid mixtures Chris Miranda, Valerie K. Booth, and Michael R. Morrow∗ Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada, A1B 3X7 E-mail: [email protected]

Abstract SP-B63−78 , a lung surfactant protein fragment, and magainin 2, an antimicrobial peptide, are amphipathic peptides with the same overall charge but different biological functions. Deuterium nuclear magnetic resonance has been used to compare the interactions of these peptides with dispersions of DMPC/DHPC (4:1) and DMPC/DMPG/DHPC (3:1:1), two mixtures of long-chain and short-chain lipids that display bicellar behavior. This study exploited the sensitivity of bicellar system structural organization to factors that modify partitioning of their lipid components between different environments. In small bicelle particles formed at low temperatures, short-chain components preferentially occupy curved rim environments around bilayer discs of the long-chain components. Changes in chain order and lipid mixing, on heating, can drive transitions to more extended assemblies including a magnetically orientable phase at intermediate temperature. In this work, neither peptide had a substantial effect on behavior of the zwitterionic DMPC/DHPC mixture. For bicellar mixtures containing the anionic lipid ∗ To

whom correspondence should be addressed

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DMPG, the peptide SP-B63−78 lowered the temperature at which magnetically orientable particles coalesced into more extended lamellar structures. SP-B63−78 did not promote partitioning of the zwitterionic and anionic long-chain lipid components into different environments. Magainin 2, on the other hand, was found to promote separation of the anionic lipid, DMPG, and the zwitterionic lipid, DMPC, into different environments for temperatures above 34◦ C. The contrast between the effects of these two peptides on the lipid mixtures studied appears to be consistent with their functional roles in biological systems.

Introduction Amphipathic alpha-helices are ubiquitous in nature and the biological functions of a large fraction of such peptides involve interactions with membranes. 1 Much work has gone into investigating the mechanism(s) by which different AMPs disrupt membranes. 2–4 Top contenders include the toroidal pore mechanism in which AMPs induce positive curvature in the bilayer leading to defects lined by one or more AMPs, as well as the carpet mechanism, in which the AMP causes a detergentlike disintegration of the membrane. Two questions that arise with regard to structure-function studies of these membrane-active helices are (1) "To what extent does function arise from relatively non-specific features such as hydrophobic moment and overall charge?" and (2) "To what extent does function rely on specific structural motifs or amino acids?" To help address such questions, we have carried out studies with two membrane-active peptides having the same overall charge but different biological functions. SP-B63−78 is one of several fragments of the essential lung surfactant protein SP-B that have been found to retain a substantial fraction of the function of the full length protein. 5,6 Magainin 2 is an antimicrobial peptide (AMP), which kills bacteria and fungi by permeabilizing their membranes. 7–12 Both peptides, illustrated in Figure 1, carry a charge of +4 in their amidated form, at neutral pH. Both peptides are also implicated in the modification of lipid assembly organization and efforts have been made to develop synthetic analogues of both lung surfactant proteins 13–15 and antimicrobial polypeptides 16,17 in order to address very different health-related challenges. One property shared by lung surfactant and bacterial membranes is 2


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the presence of a significant fraction of anionic lipid. 9,11,18 An important issue that thus arises is the extent to which the specific interactions of amphipathic, cationic polypeptides with such lipid structures are dependent on details of the peptide structure rather than overall charge and amphipathicity.

SP-B63-78: GRMLPQLVCRLVLRCS- NH2

Magainin 2: GIGKWLHSAKKFGKAFVGEIMNS- NH2

Figure 1: Sequences and electrostatic surfaces for SP-B63−78 and magainin 2. For SP-B63−78 the structure was experimentally determined by NMR in complex with SDS micelles 19 (PDBID 1rg3). For magainin 2 the structure was determined in DPC micelles by NMR 20 (PDGID 2mag). The electrostatic surface is blue in more positively charged regions, red in negatively charged regions, white in neutral regions and was produced using pymol. 21 The highlighting of the primary sequences is green for aromatic sidechains, cyan for positively charged moieties, red for negatively charged amino acids, and yellow for histidine which normally has a pKa at ∼6 and so is expected by be largely in its neutral form at neutral pH. Surfactant protein B (SP-B) is essential to the formation and maintenance of a functional surfactant layer at the air-water interface in lungs. In vivo, SP-B assembles into homodimers stabilized by disulfide-linked cysteines. 22,23 An SP-B monomer comprises 79 residues, over half of which are hydrophobic, carries a net charge of +7, and contains at least four α-helical regions. 22 Six of its seven cysteines are involved in forming three disulfide bonds that help to stabilize its tertiary

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structure 23,24 There is evidence that SP-B plays a crucial role in maintaining contact between, and facilitiating transfer between, the surfactant layer and reservoirs of lipid-rich surfactant material as lung volume cycles. 18,25–27 It has been shown that a number of SP-B fragments, including SPB63−78 , have the capacity to facilitate respiration to varying degrees. 6 SP-B63−78 has been shown to take on an orientation parallel to the membrane surface in a variety of oriented bilayers. 28 The abilities of different AMPs to compromise bacteria may reflect a variety of mechanisms but it is likely that all involve some degree of membrane penetration or membrane disruption by the peptides. Magainin 2, is an AMP from the skin of Xenopus laevis and has broad spectrum activity against Gram(-) bacteria, Gram(+) bacteria and fungi. 9,11 It was one of the earliest AMPs to be identified. It has consequently undergone intense scrutiny 7,8,10,12,29–31 and has been used as the basis for a variety of synthetic analogs including 32 MSI-78. Magainin 2 and peptides derived from it act by perturbing the membranes of target cells. 11,33 Magainin 2 is known to orient parallel to the to the membrane surface 34,35 and has been observed to induce lipid flip-flop, which was suggested to be associated with pore formation. 36 Its analogs MSI-78, MSI-367, MSI- 594, and MSI-843 have been studied by solid state NMR and proposed to function by perturbing membranes via a detergent or carpet-like mechanism. 16,33 Amphipathic peptides interact with lipid assembly surfaces close to the hydrophilic/hydrophobic interface. Depending on details of the peptide/lipid system, such interactions can lead to a range of results. Changes in the average separation of lipid headgroups can affect lipid chain orientational order and bilayer thickness. Preferential peptide-lipid interactions might lead to non-random lateral distributions of lipid species. Such peptide-induced perturbations can affect bilayer stability or interactions with adjacent surfaces. Among the model membrane systems that can be used to investigate peptide-induced perturbations are appropriately-proportioned mixtures of long-chain and short-chain lipids. Such lipid mixtures, known as bicellar systems, are useful for studying membrane perturbation because they undergo easily detected transitions between differently organized states in response to factors that affect chain orientational order and/or mixing of their long-chain and short-chain lipid components. The sensitivity of bicellar

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lipid mixture properties to peptide-induced perturbation has previously been used to study the effects of a neuropeptide, Met-enkephalin, and an antibiotic, gramicidin A, on model membrane systems. 37,38 In one earlier study, 39 deuterium nuclear magnetic resonance (2 H-NMR) was used to assess the effect of the SP-B fragment, SP-B63−78 , on the temperature dependence of particle aggregation in a bicellar system comprising a 3:1:1 mixture of chain-perdeuterated 1,2-dimyristoylsn-glycero-3-phophocholine (DMPC-d54 ), 1,2-dimyristoyl-sn-glycero-3-phopho-(10 -rac-glycerol) (DMPG), and 1,2-dihexanoyl-sn-glycero-3-phophocholine (DHPC). On heating from below the chain-melting transition temperature of the long-chain lipid component, bicellar lipid mixtures typically pass from a state in which the mixture forms rapidly tumbling small disc-like particles to a magnetically orientable phase and then to an extended lamellar or multilamellar vesicle phase. The capacity to easily distinguish these states by 2 H NMR makes bicellar mixtures attractive systems in which to study peptide-lipid interactions to which these transitions might be sensitive. In the small disc-like particles formed at lower temperatures, the more planar surfaces are enriched in longer chain lipids and the edges are enriched in the shorter chain lipids. 40–42 Bicelle particles in this phase are characterized by fast, isotropic reorientation 43 which, for deuterated samples, gives rise to a narrow, unsplit 2 H NMR resonance. 44,45 For suitably proportioned bicellar mixtures, warming above the chain-melting transition of the long-chain lipid component results in coalescence of bicellar particles into structures that can orient in a magnetic field, 44–47 a property has generated considerable interest in such systems as platforms for NMR studies of membrane associated proteins and peptides. 37,43,48–53 Depending on mixture composition, magnetically orientable lipid assemblies in the intermediate phase have been reported to be worm-like or cylindrical micelles 54–58 or perforated lamellae. 54,59–62 As temperature is raised further, mixing of the long-chain and short-chain lipid components leads to further coalesence into the multilamellar vesicle or extended lamellar phase. 45,60 Mixtures such as DMPC/DHPC give a characteristic sequence of 2 H NMR spectra 44,45,63,64 or

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is raised and the sample passes from the isotropically reorienting phase, through the magnetically orientable phase, to the higher temperature extended lamellar phase. Phase diagrams summariz-

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ing the dependences of some bicellar mixture morphologies on composition and temperature have been reported. 51,56,63,65,66 In the work reported here, 2 H-NMR has been used to compare and contrast the effects of SPB63−78 and magainin 2 on the phase behavior and lipid mixing in bicellar mixtures in which the long-chain lipid component is either zwitterionic (DMPC) or a mixture of zwitterionic and anionic (DMPC/DMPG). For lipid mixtures containing a mixture of long-chain lipid components, separate observations have been made with either DMPC or DMPG chain perdeuterated in order to compare environments of the zwitterionic and anionic lipid components in the presence of the peptides and to test for the possibility of preferential interaction between the peptides and one or the other longchain lipid component. Neither peptide is found to substantially affect the behavior of bicellar mixtures containing only zwitterionic lipids. For bicellar mixtures including DMPG, however, magainin 2 is found to promote separation of the long-chain lipids into one environment that is highly enriched in the anionic lipid component and one that appears to accommodate DMPG and DMPC together.

Materials and Experimental Methods Lipids (chain-perdeuterated and non-deuterated) were obtained from Avanti Polar Lipids (Alabaster, Alabama). C-terminally amidated peptides SP-B63−78 (GRMLPQLVCRLVLRCS-NH2 ) and magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS-NH2 ) were obtained from solid phase synthesis using O-fluorenylmethylcarbonyl (Fmoc) chemistry and purified using methods described previously. 19 The peptides were purified to 90% purity via HPLC, the sizes confirmed by mass spectrometry, and residual TFA removed by dialysis first against 5% acetic acid followed by dialysis against distilled water. To facilitate the comparison of peptide effects on bicellar dispersions with and without a longchain anionic lipid component and the comparison of peptide effects on both the zwitterionic and anionic long-chain lipid components in mixed bicellar systems, the lipid compositions used

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were DMPC-d54 /DMPC/DHPC (3:1:1), DMPC-d54 /DMPG/DHPC (3:1:1), and DMPC/DMPGd54 /DHPC (3:1:1). Each of these samples is a bicellar lipid mixture with q = 4 where q is the ratio of long-chain lipid component to short-chain lipid or detergent component. Each sample was prepared with either 20-25 mg of DMPC-d54 or 10-12 mg of DMPG-d54 . The weights of other lipids in each sample were chosen to yield the target composition. Because of its hygroscopic character, all of the DHPC required for the planned series of experiments was dissolved in chloroform/methanol (2:1) and divided into aliquots for future use. To ensure consistency of corresponding lipid mixtures prepared with and without peptide, sample pairs intended for specific comparisons were prepared in tandem. The long-chain lipids for a given pair of samples were weighed, as dry powder, and then dissolved in choloroform/methanol (2:1). Volumes of each lipid solution appropriate to the target compositions were then transferred to round bottom flasks. Peptide (SP-B63−78 or magainin 2) corresponding to 10% of the total lipid mass in one sample (e.g. for SP-B63−78 in DMPC-d54 /DMPC/DHPC (3:1:1) this corresponds to 3.5 mol% and for Magainin 2 in DMPC-d54 /DMPC/DHPC (3:1:1) this corresponds to 2.8 mol%) was then weighed and added to one flask. Solvent was then removed by rotary evaporation followed by evacuation, using a liquid nitrogen cooled trap, for 4-5 hours. The dried mixtures were then hydrated in 10 mM HEPES buffer (pH = 7.0) using 15 minutes of sonication at room temperature followed by five cycles of freezing in liquid nitrogen, thawing in a 40◦ C water bath, and vortexing. The weight of lipid in each sample was 10% of the weight of water in which it was suspended. Hydrated samples were transferred to a 400 µL NMR tube which was sealed with a teflon stopper. 2H

NMR spectra were obtained with a 9.4 T superconducting magnet and a locally-assembled

spectrometer using a quadrupole echo sequence 67 with 4-5 µs π/2 pulses separated by 35µs. Free induction decays were oversampled with a dwell time of 1 µs and then collapsed to an effective dwell time of 4µs using an algorithm described previously. 68 For samples containing DMPC-d54 or DMPG-d54 , the free induction decays used to obtain a spectrum were the result of averaging either 1000-2000 or 3000-6000 echoes respectively. The recovery time between echo sequences (recycle time) was 0.9 s. No line broadening was applied. Spectra were normalized before plotting.

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Temperatures of samples in the NMR probe were controlled to ±0.1◦ using a Lakeshore Model 325 temperature controller (Lakeshore Cryotronics, Westerville, Ohio).

Results and Discussion Spectra for DMPC-d54 /DMPC/DHPC (3:1:1) The 2 H NMR spectra of deuterons on reorienting lipid chains are doublets split by the residual interaction between the deuteron quadrupole moment and the electric field gradient of the carbondeuterium bond after averaging by motions with correlation times shorter than about 10−5 s. The orientation-dependence of the quadrupole interaction makes 2 H-NMR a useful probe of bicellar dispersion phase behaviour. This is illustrated by Figure 2 (a) which shows a temperature series of 2 H NMR spectra for a 10% (w/w) suspension of DMPC-d54 /DMPC/DHPC (3:1:1) in 10 mM HEPES buffer (pH=7.0). The series of phases observed are those expected 45,63 for a DMPCd54 /DHPC mixture at q ∼ 4. Below 20◦ C, fast isotropic reorientation of the bicelle particles averages doublet splittings to zero and leaves a narrow resonance at the spectral centre. As the temperature is raised into the vicinity of the chain-melting transition temperature for the long-chain lipid component, the planar areas of the small particles can no longer accommodate increasing disorder of the long-chain component. At about 20◦ C, the particles begin to coalesce into more extended assemblies and the reorientation becomes anisotropic. This is reflected by the emergence of spectral intensity broadly distributed between about ±17 kHz and disappearance of the narrow unsplit central feature (blue arrow on Figure 2 (a)). This is thought to reflect the formation of worm-like micelles 54–58 or perforated lamellae 54,59–62 with the short-chain lipid component still primarily confined to edge regions of high surface curvature. At the onset of anisotropic lipid reorientation, chains are still reorienting about the bilayer normal with correlation times characteristic of the long-chain lipid gel phase as evidenced by the absence of resolved doublets. We will use "intermediate phase" to refer to the state of aggregation existing from the temperature at which anisotropic reorientation emerges to the temperature at which mixing of long-chain and 8


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short-chain lipid components leads to further aggregation into a multilamellar vesicle or extended lamellar phase. DMPC-d54/DMPC/DHPC (3:1:1)

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Figure 2: (a) 2 H NMR spectra for a DMPC-d54 /DMPC/DHPC (3:1:1) bicellar lipid mixture suspended at a concentration of 10% (w/w) in 10 mM HEPES buffer (pH=7.0) for a series of increasing temperatures. (b) 2 H NMR spectra for the same lipid suspension but with the addition of 10% SP-B63−78 peptide. (c) and (d) First spectral moments (M1 ) versus temperature for the spectra shown in panels (a) and (b) respectively. The uncertainty in the determination of M1 from a given spectrum is estimated to be about ±3%. Blue arrows indicate the temperature at which a DMPC-d54 spectral component characteristic of anisotropic reorientation emerges on heating of the dispersions. Red arrows indicate the temperatures of the first spectrum above a discontinuity in first spectral moment for each mixture. These are also the temperatures at which a small spectral component characteristic of nearly isotropic DMPC-d54 reorientation appears on heating. As temperature is increased further, reorientation about the bilayer normal becomes fast enough to be axially symmetric on the ∼ 10−5 s time scale characteristic of the 2 H NMR experiment. The spectrum becomes a superposition of doublets with splittings proportional to the orientational order parameter, SCD =

1

3 cos2 θCD − 1 , 2 9


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corresponding to each deuterated acyl chain segment. In Equation 1, θCD is the instantaneous angle between the carbon-deuterium (CD) bond direction and the direction of the motional symmetry axis and the average is over motions that modulate the quadrupole interaction on the timescale of the 2 H NMR experiment. Orientational order parameters, and thus spectral splittings, are larger for segments near the headgroup end of the chain, where motions are more constrained, and smaller for segments closer to the methyl end of the chain. The dependence of orientational order parameter on position along the acyl chain can be represented as an orientational order parameter profile. For perdeuterated acyl chains in fluid bilayer phases, orientational order parameters for chain segments closest to the headgroup change more slowly with position than for other chain segments. The resulting plateau in the order parameter profile is reflected in the spectrum by prominent spectal edges resulting from an enhanced concentration of doublets having similar quadrupole splittings. 69–72 The sharp doublet with the smallest quadrupole splitting, typically less than ∼4 kHz in a fluid bilayer phase, arises from deuterated methyl groups at the acyl chain ends. For deuterons on a particular chain segment in a fluid bilayer phase, the observed quadrupole splitting is also proportional to 12 3 cos2 β − 1 where β is the angle between the applied magnetic field and the symmetry axis for axially symmetric reorientation, the bilayer normal. For randomly oriented bilayers, the resulting distribution of intensity arising from a given deuterated acyl chain segment is a Pake doublet. 70 In Figure 2 (a), the spectra that emerge as the sample is warmed past 30◦ C are not superpositions of Pake doublets. They are, rather, superpositions of doublets in which the distribution of intensity reflects a preferential orientation of bilayer normals in directions perpendicular to the magnetic field (β = 90◦ ). This reflects the onset of magnetic orientation in the intermediate phase of such bicellar mixtures. As temperature is raised further, mixing of the short and long chain lipid components results in further coalescence of the magnetically-orientable assemblies into larger structures that can be less susceptible to magnetic orientation. The sample undergoes a transition from the intermediate phase to the extended lamellar or multilamellar vesicle phase. While 2 H NMR spectral changes associated with this transition can range from subtle to marked, depending on the mixture composition,

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the defining aspect of this transition is the onset of mixing and the coalescence of smaller structures typical of the intermediate phase, into more extended lipid assemblies (multilamellar vesicles or perforated lamellae depending on mixture composition). To reflect this aspect of the higher temperature transition, we choose to refer to the higher temperature bicellar mixture phase as an extended lamellar phase. For DMPC-d54 /DMPC/DHPC (3:1:1), spectral changes associated with the transition from the magnetically orientable phase to the higher temperature extended lamellar phase are subtle. For a completely random distribution of bilayer normal orientations, each deuteron would give rise to a Pake doublet spectral component. In Figure 2 (a), at 40◦ C, the doublets for deuterons on a particular chain segment begin to display distributions of intensity corresponding to broader ranges of β values and thus reflect a more random distribution of bilayer normal orientations. This implies the onset of a morphology that is less susceptible to magnetic orientation. The higher temperature phase is also characterized by the emergence of a narrow unsplit spectral component. In Figure 2 (a), this component begins to grow noticeably at about 44◦ C. The emergence of this narrow central component is indicated by the red arrow on Figure 2 (a). While it has been suggested that this might reflect the formation of small, isotropically reorienting particles, 45 it has also been shown that quadrupole splittings for deuterons on the short-chain component of such mixtures begin to increase signficantly at this transition. 45,73 That observation implies that the bicellar mixture transition from a magnetically orientable phase to the more extended lamellar phase is associated with enhanced mixing of the long-chain and short-chain lipid components. If so, the emergence of a narrow feature in the spectra of a long-chain lipid component may also reflect mixing that results in some of the long-chain lipid component sharing highly curved environments with the short-chain lipid component. Another interesting feature of the highest temperature spectra in panels (a) and (b) of Figure 2 is the separation of the prominent spectral edge around ±12kHz, the "plateau" feature, into two doublets with slightly different splittings. This may indicate that in the extended lamellar phase of DMPC-d54 /DMPC/DHPC bicellar dispersion, conformations near the headgroup end of one chain are more constrained than in a fluid phase multilamellar vesicle. While the change in the 2 H spectral shape at the upper transition for DMPC-d54 /DMPC/DHPC

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(3:1:1) is subtle, the redistribution of intensity from sharp doublets characteristic of magnetic orientation to more Pake-like doublets characteristic of a less oriented, extended lamellar phase does affect the average quadrupole splitting which is proportional to the first spectral moment, R∞

ωS(ω)dω , M1 = R0 ∞ 0 S(ω)dω

(2)

where S(ω) is the intensity at frequency ω measured from the spectrum centre. In a liquid crystalline phase bilayer sample with randomly oriented bilayer normals, M1 is proportional to average orientational order of the deuterated acyl chain segments. Changes in M1 can also indicate devitations from a random distribution of bilayer normal orientations. For a given spectrum, the primary source of uncertainty in the determination of M1 is the selection of points, beyond the spectrum, from which the integration baseline is obtained. For the spectra reported here, uncertainty in the determination of M1 from a given spectrum is estimated to be about ±3%. Figure 2 (c) shows the temperature dependence of first spectral moments (M1 ) for the DMPC-d54 /DMPC/DHPC (3:1:1) spectra shown in Figure 2 (a). The small discontinuity indicated by the red arrow corresponds with the change in spectral shape, in part due to a change in the distribution of bilayer normal orientations, at the transition to the extended lamellar phase.

Effects of SP-B63−78 Figure 2, panels (b) and (d), show 2 H NMR spectra and first spectral moments, respectively, for DMPC-d54 /DMPC/DHPC (3:1:1) with 10% (w/w) SP-B63−78 added. The temperature for the transition from the magnetically orientable phase to the extended lamellar phase is lowered by about 4◦ , but the phase behavior of the DMPC/DHPC lipid mixture is otherwise unchanged by SP-B63−78 . This may indicate that SP-B63−78 slightly lowers the barrier for coalescence of the magnetically orientable particles into more extended structures but that it otherwise has little effect on the properties of the bicellar lipid dispersion containing only zwitterionic lipids. To probe the effects of SP-B63−78 on lipid structures containing an anionic lipid component,

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Figure 3: (a) 2 H NMR spectra for a DMPC-d54 /DMPG/DHPC (3:1:1) bicellar lipid mixture suspended at a concentration of 10% (w/w) in 10 mM HEPES buffer (pH=7.0) for a series of increasing temperatures. (b) 2 H NMR spectra for the same DMPC-d54 /DMPG/DHPC (3:1:1) bicellar lipid suspension but with the addition of 10% SP-B63−78 peptide. (c) 2 H NMR spectra for a 10% (w/w) suspension of DMPC/DMPG-d54 /DHPC (3:1:1) bicellar lipid mixture suspended at a concentration of 10% (w/w) in 10 mM HEPES buffer (pH=7.0) for a series of increasing temperatures. (d) 2 H NMR spectra for same DMPC/DMPG-d54 /DHPC (3:1:1) bicellar lipid suspension but with the addition of 10% SP-B63−78 peptide. Blue arrows indicate the temperature at which a DMPCd54 (panels a or b) or DMPG-d54 (panels c and d) spectral component characteristic of anisotropic reorientation emerges on heating of the dispersions. Red arrows indicate the temperatures above which a small spectral component characteristic of nearly isotropic DMPC-d54 (panels a or b) or DMPG-d54 (panels c and d) reorientation begins to grows in intensity on heating.

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2H

NMR spectra were acquired for a 10% (w/w) suspension of DMPC-d54 /DMPG/DHPC (3:1:1)

in 10 mM HEPES buffer (pH=7.0) (Figure 3 (a)). As was reported earlier, 64,74 the transition from the magnetically orientable phase to the extended lamellar phase, indicated by the red arrow, is very clear when this q = 4 bicellar dispersion contains an anionic long-chain lipid component. In particular, the spectrum at the transition is a superposition of spectral components from the two coexisting phases. In an earlier study using DMPC−d54 /DMPG/DHPC (3:1:1), it was reported that SP-B63−78 lowers the temperature for the transition from the magnetically orientable phase to the extended lamellar phase in lipid dispersions with this composition. 39 The spectra of DMPCd54 /DMPG/DHPC (3:1:1) with 10% (w/w) SP-B63−78 shown in Figure 3 (b) are consistent with this observation. The peptide-induced lowering of the magnetically-orientable to extended lamellar phase transition has been interpreted as indicating that, for bicellar mixtures with an anionic longchain lipid component, SP-B63−78 promotes coalescence to the more extended lamellar phase at a lower temperature than in the absence of peptide. The spectra in Figure 3 (b) also show that, for DMPC-d54 /DMPG/DHPC (3:1:1), SP-B63−78 promotes persistence of the isotropically reorienting phase to higher temperature. In the present study, examination of the effects of SP-B63−78 on DMPC/DMPG/DHPC (3:1:1) has been extended to samples with deuteron labels on the anionic lipid in order to compare the environments of the zwitterionic and anionic long-chain components of the bicellar mixture in the presence and absence of the peptide. Panels (c) and (d) of Figure 3 show 2 H NMR spectra for a 10% (w/w) suspension of DMPC/DMPG-d54 /DHPC (3:1:1) in (c) the absence of and (d) the presence of SP-B63−78 . In comparing the behaviors of the mixtures with one or the other longchain lipid component deuterated, it should be noted that the degree of deuteration can affect the phase behavior of lipid bilayers. For example complete deuteration of DMPC can lower the gel to liquid crystal transition temperature of DMPC-d54 bilayers by ∼ 4 − 5◦ C relative to that of undeuterated DMPC. For DMPC/DMPG/DHPC (3:1:1), changing the deuterated component from DMPC-d54 to DMPG-d54 corresponds to changing the long chain lipid from 75% chain deuterated to 25% chain deuterated.

14


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Langmuir

Comparison of panels (a) and (c) of Figure 3 shows that in the absence of peptide, the two longchain lipid components in the DMPC/DMPG/DHPC (3:1:1) dispersion are in similar environments over most of the temperature range studied. The small shift in apparent transition temperatures between the two samples is likely due to the difference in degree of chain deuteration as noted above. The lower degree of doublet resolution, at 30◦ C and 32◦ C, in Figure 3 (c) compared to Figure 3 (a) also suggests that magnetic orientation in the intermediate phase of DMPC/DMPG-d54 /DHPC (3:1:1) (Figure 3 (c)) may be slightly less complete than in DMPC-d54 /DMPG/DHPC (3:1:1) (Figure 3 (a)). It should be noted, though, that the extent of magnetic orientation in the intermediate phase is sensitive to details of the sample preparation. Significantly, spectra of the sample containing DMPG-d54 show no evidence of the anionic lipid partitioning into more than one environment, other than at the transition where phase coexistence over a small temperature range is not surprising. It is also notable that phase coexistence at the transition in the DMPC/DMPG/DHPC (3:1:1) dispersions is apparent regardless of which long-chain lipid component is deuterated. Figure 3 (d) shows 2 H NMR spectra for DMPC/DMPG-d54 /DHPC (3:1:1) containing 10% (w/w) SP-B63−78 . As is seen in panel (b) of Figure 3, addition of SP-B63−78 to this lipid mixture promotes persistence of the isotropically reorienting phase to higher temperature and promotes coalescence of the magnetically orientable particles to more extended lamellar structures at lower temperature. The temperature range between isotropic reorientation and extended lamellar is smaller in DMPC/DMPG-d54 /DHPC (3:1:1) than in DMPC-d54 /DMPG/DHPC (3:1:1). This may, in part, reflect different degrees of long-chain lipid deuteration but might also arise from slight differences in preparation or sample composition. Nevertheless, there is again no evidence of the long-chain anionic lipid component being partitioned between distinct coexisting environments in the presence of SP-B63−78 . Comparisons of behavior displayed by the series of spectra in Figures 2 and 3 suggest that, for the bicellar dispersion with an anionic long-chain lipid component, SP-B63−78 lowers the temperature at which magnetically orientable structures coalesce into more extended lamellae and raises the temperature to which isotropically reorienting bicelles can persist. They also suggest that, for

15


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DMPC/DMPG/DHPC (3:1:1), the zwitterionic and anionic long-chain lipid components occupy similar environments and SP-B63−78 does not promote partitioning of the anionic component between distinct lipid assembly environments.

Effects of magainin 2 A similar set of comparisons was carried out for the antibiotic polypeptide magainin 2 using fresh bicellar lipid preparations. Figure 4 shows 2 H NMR spectra and first spectral moments for DMPCd54 /DMPC/DHPC (3:1:1) with and without the addition of 10% (w/w) magainin 2. Comparison of Figure 4, panels (a) and (c), with the corresponding panels of Figure 2, show that the behavior of the DMPC-d54 /DMPC/DHPC (3:1:1) dispersion prepared for the magainin 2 series of experiments is very similar to that of the dispersion used in the SP-B63−78 series. Panels (b) and (d) of Figure 4 show the effect of magainin 2 on the bicellar lipid mixture in the absence of an anionic long-chain lipid component. The transition from the intermediate temperature phase to the higher temperature phases is again very subtle both in the absence of and in the presence of the peptide. The transition can be identified from the emergence of the narrow unsplit feature arising from increased occupation of highly curved environments by DMPC-d54 , as lipid mixing increases, and from a small discontinuity in the temperature dependence of first spectral moments. Aside from a 2◦ increase in the lower and upper transition temperatures, magainin 2 appears to have little effect on the behavior of this lipid mixture or on the environments of its long-chain, zwitterionic lipid component. Figure 5 shows spectra for DMPC-d54 /DMPG/DHPC (3:1:1) and for DMPC/DMPG-d54 /DHPC (3:1:1) in the absence of and presence of 10% (w/w) magainin 2. Comparisons of these spectral series show that magainin 2 significantly perturbs the behaviors of this mixture and promotes partitioning of the anionic long-chain lipid component into two environments that are distinct over the ∼ 10−5 s timescale characteristic of the 2 H NMR experiment. Panels (a) and (c) of Figure 5 show spectra for the lipid-only DMPC-d54 /DMPG/DHPC (3:1:1) and DMPC/DMPG-d54 /DHPC (3:1:1) dispersions prepared for this series of comparisons. The 16


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Langmuir

DMPC-d54/DMPC/DHPC (3:1:1) lipids only

a

lipids + magainin 2

b

o

46 C o

o

46 C o

44 C

44 C

o

o

42 C

42 C

o

o

40 C

40 C

o

o

38 C

38 C

o

o

36 C

36 C

o

o

34 C

34 C

o

o

32 C

32 C

o

o

30 C

30 C

o

o

28 C

28 C

o

o

26 C

26 C

o

o

24 C

24 C o

o

22 C

22 C

o

o

20 C o

18 C o

x 0.3

-40

16 C


40

x 0.3

20 C o 18 C

x 0.3

16 C

-40

o


c

40

d

Figure 4: (a) 2 H NMR spectra for a DMPC-d54 /DMPC/DHPC (3:1:1) bicellar lipid mixture suspended at a concentration of 10% (w/w) in 10 mM HEPES buffer (pH=7.0) for a series of increasing temperatures. (b) 2 H NMR spectra for the same lipid suspension but with the addition of 10% magainin 2 peptide. (c) and (d) First spectral moments (M1 ) versus temperature for the spectra shown in panels (a) and (b) respectively. The uncertainty in the determination of M1 from a given spectrum is estimated to be about ±3%. Blue arrows indicate the temperature at which a DMPC-d54 spectral component characteristic of anisotropic reorientation emerges on heating of the dispersions. Red arrows indicate the temperatures of the first spectrum above a discontinuity in first spectral moment for each mixture. These are also the temperatures at which a small spectral component characteristic of nearly isotropic DMPC-d54 reorientation appears on heating.

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Page 18 of 36

DMPC-d54/DMPG/DHPC (3:1:1)

a

lipids only

b

o

44 C

lipids + Magainin 2 o

44 C

o

o

42 C

42 C

o

o

40 C

40 C

o

o

38 C

38 C

o

o

36 C

36 C

o

o

34 C

34 C

o

o

32 C

32 C

o

o

30 C

30 C

o

o

28 C

28 C

o

o

26 C

26 C

o

o

24 C

24 C

o

o

22 C

22 C

o

o

20 C o

o

18 C

18 C

x 0.3 x 0.3

o

o

16 C

16 C

o

x 0.3

-40

20 C

o

12 C


12 C

40

-40


40

DMPC/DMPG-d54/DHPC (3:1:1)

c

d

o

44 C o

o

44 C o

42 C

42 C

o

o

40 C

40 C

o

o

38 C

38 C

o

o

36 C

36 C

o

o

34 C

34 C

o

o

32 C

32 C

o

o

30 C

30 C

o

o

28 C

28 C

o

o

26 C

26 C

o

o

24 C

24 C

o

o

22 C

22 C o

x 0.3

20 C

x 0.3

18 C

o

o

18 C

o

x 0.3

16 C

x 0.3

12 C

-40

o

20 C

o


40

o

x 0.3

16 C

x 0.3

12 C

-40

o


40

Figure 5: (a) 2 H NMR spectra for a DMPC-d54 /DMPG/DHPC (3:1:1) bicellar lipid mixture suspended at a concentration of 10% (w/w) in 10 mM HEPES buffer (pH=7.0) for a series of increasing temperatures. (b) 2 H NMR spectra for the same DMPC-d54 /DMPG/DHPC (3:1:1) bicellar lipid suspension but with the addition of 10% magainin 2 peptide. (c) 2 H NMR spectra for a 10% (w/w) suspension of DMPC/DMPG-d54 /DHPC (3:1:1) bicellar lipid mixture suspended at a concentration of 10% (w/w) in 10 mM HEPES buffer (pH=7.0) for a series of increasing temperatures. (d) 2 H NMR spectra for same DMPC-d54 /DMPG-d54 /DHPC (3:1:1) bicellar lipid suspension but with the addition of 10% magainin 2 peptide. Blue arrows indicate the temperature at which a DMPC-d54 (panels a or b) or DMPG-d54 (panels c and d) spectral component characteristic of anisotropic reorientation emerges on heating of the dispersions. Red arrows on panels (a) and (c) indicate the temperatures of the first spectrum above which a small spectral component characteristic of nearly isotropic DMPC-d54 (panel a) or DMPG-d54 (panel c) reorientation emerges. The red arrow on panel (d) indicates the emergence of a second spectral component characteristic of higher DMPG-d54 chain orientational order.

18


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Langmuir

behavior displayed is effectively identical to that shown in panels (a) and (c) of Figure 3 for the corresponding mixtures. Notably, comparison of the series of spectra shown in panels (a) and (c) of Figure 5 shows that the DMPC-d54 and DMPG-d54 in the corresponding mixtures occupy equivalent environments. A striking feature of the series of spectra for DMPC-d54 /DMPG/DHPC (3:1:1) shown in Figure 5 (a) is the abrupt change from the oriented bilayer spectrum to the extended lamellar phase spectrum at 36◦ C. As noted previously, this behavior seems to be a direct consequence of substituting DMPG for a fraction of the DMPC in the q = 4 bicellar dispersions studied here. 64,74 In contrast, the series of spectra shown in Figure 5 (b) suggest that addition of 10% (w/w) magainin 2 effectively eliminates the higher temperature transition in DMPC-d54 /DMPG/DHPC (3:1:1). Indeed, up to about 40◦ C, the spectra for DMPC-d54 /DMPG/DHPC (3:1:1) plus 10% (w/w) magainin 2 are most similar to those observed for DMPC-d54 /DMPC/DHPC (3:1:1), the corresponding bicellar lipid mixture containing no anioinic lipid component (see Figures 2 (a) and 4 (a)). In effect, magainin 2 appears to have, at least in part, eliminated the effect of the anionic lipid on the phase behavior of the bicellar mixture. The resulting behavior is not identical to that of DMPCd54 /DMPC/DHPC (3:1:1), however, since the spectra of Figure 5 (b) suggest that the DMPC-d54 component in the DMPC/DMPG/DHPC plus magainin 2 sample remains in a magnetically orientable phase environment up to at least 50◦ C. Comparison of the spectra in panels (b) and (d) of Figure 5 shows that, in contrast to the DMPCd54 component in the mixture containing magainin 2, the DMPG-d54 component of DMPC/DMPGd54 /DHPC (3:1:1) appears to go through a transition at which part of the DMPG-d54 partitions into a more ordered environment that is distinct from that occupied by DMPC and the rest of the DMPG-d54 remains in the magnetically orientable phase. Above 32◦ C, the spectra of DMPC-d54 in Figure 5 (d) are superpositions of one spectral component that appears to be typical of the magnetically oriented phase (i.e. doublets with intensity concentrated at the β = 90◦ splittings) and another spectral component with a larger plateau segment quadrupole splitting and less sharply resolved doublets. This suggests a second environment in which DMPG-d54 acyl chains are more

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highly ordered and less mobile. Unlike the small range of two phase coexistence observed in the DMPC/DMPG/DHPC dispersions at the transition from the ordered phase to the extended lamellar phase, the coexistence of two DMPG-d54 environments induced by the presence of magainin 2 persists to higher temperature. Taken together, comparisons of spectral series in Figure 5 suggest that, above about 32◦ C, magainin 2 preferentially extracts DMPG from the orientable phase of the DMPC/DMPG/DHPC bicellar mixture to leave a magnetically orientable phase that is depleted of DMPG and a more ordered bilayer environment that is enriched in DMPG.

Comparison of the effects of SP-B63−78 and magainin 2 on the PC and PG components of DMPC/DMPG/DHPC (3:1:1) Figures 3 (b) and 5 (b) show spectra of the DMPC-d54 components of the DMPC/DMPG/DHPC mixtures containing SP-B63−78 or magainin 2 respectively. Figures 3 (d) and 5 (d) show spectra of the DMPG-d54 component in the corresponding mixtures. One interesting difference between the effects of the two peptides is their effects on the stability of the isotropically reorienting phase in the lipid mixture containing DMPG. The onset of anisotropic lipid reorientation in bicellar dispersions likely reflects coalescence into larger particles, possible worm-like or ribbon-like micelles, as the area per long-chain lipid in the planar surfaces of bicelle particles becomes too large to be accommodated within the constraints imposed by the short-chain lipid "edges". Comparison of Figures 3 (b) and 5 (b) with Figures 3 (d) and 5 (d) suggests that SP-B63−78 tends to raise the temperature at which anisotropic reorientation emerges, while magainin 2 tends to have the opposite effect. This may reflect a greater tendency for magainin 2, relative to that of SP-B63−78 , to promote orientational disorder of the long-chain lipid components or reduced stability of the short-chain lipid bicelle "rim" with increasing temperature. Since the effects of both peptides on the mixtures containing long-chain anionic lipid are larger than their effects on bicellar mixtures containing only zwitterionic lipid, the former possibility seems more likely. The most striking difference between the effects of SP-B63−78 and magainin 2 on the disper20


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Langmuir

a b -30

c d -30

DMPC/DMPG-d54/ DHPC (3:1:1)

+ SP-B63-78 o

42 C DMPC-d54/DMPG/ DHPC (3:1:1)

+ SP-B63-78 o

42 C -20


DMPC/DMPG-d54/ DHPC (3:1:1)

20

30

+ magainin 2 o

42 C DMPC-d54/DMPG/ DHPC (3:1:1)

+ magainin 2 o

42 C -20


20

30

e

Figure 6: Comparison of peptide effects on the PC and PG components of DMPC/DMPG/DHPC (3:1:1) dispersions. 2 H NMR spectra at 42◦ C for (a) DMPC/DMPG-d54 /DHPC (3:1:1) and (b) DMPC-d54 /DMPG/DHPC (3:1:1), both with 10% SP-B63−74 , and for (c) DMPC/DMPGd54 /DHPC (3:1:1) and (d) DMPC-d54 /DMPG/DHPC (3:1:1), both with 10% Magainin 2. Vertical lines are drawn to facilitate comparison of sharp doublet splittings. (e) First spectral moments (M1 ) for (open circle) DMPC-d54 /DMPG/DHPC (3:1:1) and (filled circle) DMPC/DMPG-d54 /DHPC (3:1:1), both with 10% SP-B63−74 , and for (open triangle) DMPC-d54 /DMPG/DHPC (3:1:1) and (filled triangle) DMPC/DMPG-d54 /DHPC (3:1:1) both with 10% magainin 2. The uncertainty in the determination of M1 from a given spectrum is estimated to be about ±3%. 21


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sions of DMPC/DMPG/DHPC is their effects on mixing of the two long-chain lipid components in the higher temperature extended lamellar phase. Figure 6 compares DMPC-d54 and DMPG-d54 spectra for the DMPC/DMPG/DHPC (3:1:1) mixtures in the presence of SP-B63−78 or magainin 2 at 42◦ C. Comparisons of corresponding spectra at any temperature between 36◦ C and 50◦ C would be similar. The vertical lines between pairs of spectra in Figure 6 facilitate comparision of DMPGd54 and DMPC−d54 doublet splittings in the mixtures containing one peptide or the other. Figure 6 also shows first spectral moments for these mixtures between 31◦ C and 50◦ C. The transition from the magnetically-orientable intermediate temperature phase to the higher temperature extended lamellar phase is largely driven by increased mixing of the short-chain and long-chain lipid components as temperature is raised. 59 The observation that SP-B63−78 lowers the temperature for this transition in the DMPC/DMPG/DHPC mixture is consistent with previous results 39 and suggests that this peptide promotes lipid mixing. This possibility is supported by the observation that, the DMPC-d54 and DMPG-d54 spectra in panels (a) and (b) of Figure 6 are very similar suggesting that the two long-chain lipid components occupy nearly identical environments. Magainin 2, in contrast, seems to promote the coexistence of two distinct DMPG-d54 environments above 34◦ C, as seen from the apparent superposition of two different DMPG-d54 spectral components above this temperature in spectrum (c) of Figure 6. Spectra (c) and (d) of Figure 6 illustrate the distinct DMPC-d54 and DMPG-d54 environments in the presence of magainin 2. The spectra for DMPC-d54 /DMPG/DHPC (3:1:1) plus magainin 2 in Figure 5 (b) show that, once established at about 26◦ C, the magnetically oriented environment of DMPC-d54 , in this mixture, persists to at least 50◦ C. As noted above, spectrum (c) in Figure 6 appears to be a superposition of spectral components arising from DMPG-d54 molecules in two distinct environments. If bilayers were randomly oriented in either of the two environments, the quadrupole doublets arising from deuterons on chain segments in that environment would be Pake doublets which would include some spectral intensity at small splittings corresponding to reorientation about symmetry axes close to β = 54.7◦ from the applied magnetic field. The absence of intensity between the methyl doublets and the methylene doublet with the next smallest splitting thus suggests that

22


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Langmuir

both of the DMPG-d54 environments in the DMPC/DMPG-d54 /DHPC plus magainin 2 sample also remain magnetically oriented as the temperature is increased. One of the DMPG-d54 environments reflected by the spectrum of Figure 6 (c) gives rise to sharp doublet features that are at smaller splittings than those in the DMPC-d54 spectrum of Figure 6 (d) but that, otherwise, correspond qualitatively to those in the magnetically-orientable phase. This interpretation is reinforced by inspection of the series of spectra in Figure 5 (d) where it can be seen that the spectral component comprising the sharp features in the higher temperature DMPC/DMPG-54 /DHPC plus magainin 2 spectra corresponds closely to those of the magnetically orientable phase observed at 30◦ C in that series of spectra. The resolved doublets in Figure 6 (c) may thus reflect a fraction of DMPG-54 that remains associated with DMPC in the presence of magainin 2 as the sample temperature is increased. The other DMPG-d54 environment reflected by the spectrum in Figure 6 (c) gives rise to a larger plateau deuteron quadrupole splitting. Spectral doublets arising from DMPG-d54 deuterons on specific acyl chain segments in that environment are not clearly resolved suggesting that acyl chain reorientation is slower than in the coexisting, less ordered phase. This suggests an enviroment in which DMPG is orientationally and dynamically constrained. This may reflect preferential interaction of the anionic DMPG with the cationic magainin 2 peptide in this environment. Figure 6 (e) shows first spectral moments for the DMPC-d54 and DMPG-d54 spectra in DMPC/ DMPG/DHPC (3:1:1) mixtures containing either SP-B63−78 or magainin 2 for 32◦ C and above. The circles and triangles show the first spectral moments for both lipids in the mixtures containing SP-B63−78 and magainin 2 respectively. Spectral moments are larger, for both lipid components, in the presence of magainin 2 compared to those observed in the presence of SP-B63−78 indicating that, on average, magainin 2 has a stronger ordering effect on the bilayer lipids, than SP-B63−78 . Above 40◦ C, the DMPG-d54 component of the mixture is, on average, more ordered by magainin 2 than the DMPC-d54 component in corresponding mixtures. This is consistent with the comparison of spectra in Figures 6 (c) and (d). Peptide-induced sorting of anionic and zwitterionic lipids has been suggested to be one im-

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Page 24 of 36

portant mechanism to account for the antimicrobial activity of amphipathic cationic peptides. 75–79 In a study using DSC to study the effects of cationic amphipathic AMPs on model membranes comprising POPE and cardiolipin, 30 it was concluded that magainin 2 had little tendency to promote separation of the anionic and zwitterionic lipid components compared to synthetic AMPs including MSI-78, a synthetic AMP derived from magainin 2. This smaller lipid-sorting capacity of magainin 2, relative to the synthetic AMPs, was attributed to its relatively lower charge density. 30 These observations reported here do, however, indicate that magainin 2 promotes separation of lipids in the long-chain regions of the DMPC/DMPG/DHPC mixtures into DMPG-rich regions and DMPC/DMPG mixed regions. Detection of partial DMPC/DMPG separation in the current study is likely a result of the capacity of 2 H NMR to directly and sensitively detect DMPGd54 populations in different environments. Using 2 H and

31 P

NMR, a study of lipid aggregation

in the presence of the AMP cateslytin provided evidence that the peptide induced separation of the lipids into domains of different lipid chain orientational order and it was suggested that the boundaries between coexisting domains might be an important factor in peptide-induced membrane permeabilization. 75 Observations, by 31 P NMR, of bilayers containing the AMP maculatin also showed evidence of segregation of PC versus PG. 80 In the spectra of Figure 5 for temperatures above 36◦ C, the coexisting DMPG populations resulting from peptide-induced segregation, display different degrees of orientational order. The phase that is more enriched in DMPG is more ordered, an observation suggesting that magainin 2 may also be capable of inducing the formation of boundaries between domains of differing lipid chain orientational order.

Comments and Summary The comparisons drawn above suggest that SP-B63−78 promotes mixing of zwitterionic and anionic lipid components while magainin 2 appears to promote their segregation. These tendencies may reflect the differing functional properties of these peptides. SP-B is thought to facilitate respiration by helping to maintain contact between the active surfactant layer and surfactant reservoirs, and

24


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Langmuir

promote recruitment of surfactant material into the surface active layer, as lung volume cycles. SP-B63−78 has been found to retain some of that functional capacity. A tendency to promote interaction of lipid components in complex mixtures might be relevant to such activity. Magainin 2, in contrast, exhibits antimicrobial activity which is presumed to arise from the peptide’s capacity to disrupt bacterial membranes. 11 A tendency to induce separation of anionic and zwitterionic lipid components in such membranes might contribute to such activity. More generally, this work shows that the organization and behavior of a bicellar lipid mixture can be sensitive to specific details of peptide-lipid interaction. In particular, this work demonstrates that two cationic amphipathic peptides having the same surface charge but different residue sequences can interact with and modify the properties of both zwitterionic and mixed anionic/zwitterionic lipid assemblies in qualitatively different ways. This suggests that details of the peptide side-chain sequence and charge distribution are significant determinants of surface activity and function.

Acknowledgement This work was supported by a Discovery Grant (RGPIN 06345) from the Natural Sciences and Engineering Research Council of Canada. The authors thank Donna Jackman for valuable assistance with peptide synthesis.

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TOC Graphic SP-B63-78

Magainin 2:

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Effects of amphipathic polypeptides on membrane organization

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