d sc peptide1

Di¡erential scanning calorimetric study of the e¡ect of the antimicrobialpeptide gramicidin S on the thermotropic phase behavior of

phosphatidylcholine, phosphatidylethanolamine andphosphatidylglycerol lipid bilayer membranes

Elmar J. Prenner a, Ruthven N.A.H. Lewis a, Leslie H. Kondejewski a;b,Robert S. Hodges a;b, Ronald N. McElhaney a;*

a Department of Biochemistry, University of Alberta, Edmonton, Alta. T6G 2H7, Canadab Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alta. T6G 2H7, Canada

Received 22 September 1998; received in revised form 21 December 1998; accepted 7 January 1999

Abstract

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of largemultilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) anddimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GSon the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and chargeof their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phasetransition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles areexposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel toliquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in thetemperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition isabolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS onDMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionicDMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higherpeptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover,the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cyclingthrough the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protectsthe phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. Theseresults indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because

0005-2736 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 9 9 ) 0 0 0 0 4 - 8

Abbreviations: GS, gramicidin S; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol ; DPG, diphos-phatidylglycerol ; PS, phosphatidylserine; SpM, sphingomyelin; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatid-ylcholine; DMPE, dimyristoylphosphatidylethanolamine; DMPG, dimyristoylphosphatidylglycerol ; DSC, di¡erential scanning calorim-etry; ESR, electron spin resonance; NMR, nuclear magnetic resonance; IR, infrared; NBD-GS, N-4-nitrobenz-2-oxa-1,3-diazolegramicidin S; Lc or L

0c, lamellar crystalline phase with untilted or tilted hydrocarbon chains, respectively; LL or L

0L, lamellar gel phase

with untilted or tilted hydrocarbon chains, respectively; P0L, lamellar rippled gel phase with tilted hydrocarbon chains; LK, lamellar liquid-

crystalline phase* Corresponding author. Fax: +1-403-492-0095; E-mail : [email protected]

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Biochimica et Biophysica Acta 1417 (1999) 211^223

of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negativelycharged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact morestrongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of theformer system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionicphospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarilywith the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipidhydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove usefulin the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids. ß 1999Elsevier Science B.V. All rights reserved.

1. Introduction

Gramicidin S (GS)1 is a cyclic decapeptide[cyclo-(Val^Orn^Leu^D^Phe^Pro)2] ¢rst isolated from Ba-cillus brevis (see [1]) and is one of a series of anti-microbial peptides produced by this microorganism(see [2]). This peptide exhibits appreciable antibioticactivity against a broad spectrum of both Gram-neg-ative and Gram-positive bacteria as well as againstseveral pathogenic fungi [3,4]. In aqueous solutionGS forms an amphiphilic, two-stranded, antiparallelL-sheet structure in which the four hydrophobic Leuand Val residues project from one side of this disk-shaped peptide molecule and the two basic Orn res-idues project from the other [2,5^8]. In general, theantimicrobial and hemolytic activity of GS analogsincrease with the degree of hydrophobicity and am-phiphilicity of the peptide up to some optimal valueand the presence of two positively charged aminoacids are essential for maximal activity [3,9,10].Although the Orn residues of GS may be replacedby Lys or Arg residues, the presence of two posi-tively charged amino acid residues on the same faceof the peptide molecule are also known to be re-quired for maximal antimicrobial activity [2,4,6,11].

Considerable evidence exists that the primary tar-get of GS is the lipid bilayer of cell surface mem-branes and that this peptide kills cells by destroyingthe structural integrity of the lipid bilayer by induc-ing the formation of pores or other localized defects[12^17]. Unfortunately, GS is rather nonspeci¢c in itsactions and exhibits appreciable hemolytic as well asantimicrobial activity [18^20]. The therapeutic uti-lization of GS has therefore been limited to topicalapplications [19]. A major goal of our current studieson the interaction of GS with model and biologicalmembranes is to provide the fundamental knowledgeof the mechanism of action of this peptide on lipid

bilayers which is required to design GS analogs withenhanced activity for bacterial membranes and di-minished activity against the plasma membranes ofhuman and animal cells.

Although considerable evidence exists that theprinciple target of GS is the lipid bilayer of cell sur-face membranes, only a limited number of studies ofthe interaction of this peptide with lipid bilayer mod-el membranes have been published to date. More-over, the conclusions reached from these studies donot always agree with one another. Pache et al. [21]reported that even at a low lipid/peptide ratio (10:1),GS had almost no e¡ect on the gel to liquid-crystal-line phase transition temperature, enthalpy or coop-erativity of DPPC multilamellar vesicles, nor was theorganization the DPPC bilayer signi¢cantly per-turbed by the presence of the peptide, as monitoredby NMR or ESR spectroscopy. The only signi¢cante¡ect of GS addition observed was a decrease in theDPPC P^O stretching frequency as monitored by IRspectroscopy. Pache and coworkers thus concludedthat GS is bound to the surface of the DPPC bilayerand interacts only with the lipid polar headgroups. Asimilar conclusion was reached by Datema et al. [22],based on their observations of the very small reduc-tion of the temperature and cooperativity of themain phase transition of DPPC observed by DSCeven at low lipid/peptide ratios (6:1), the insensitivityof the motional characteristics of the GS molecule tothe DPPC gel to liquid-crystalline phase transition,and the ability of GS to induce an isotropic compo-nent in the 31P-NMR spectrum at higher tempera-tures. However, based on the generally appreciablehydrophobicity of the GS molecule, the sensitivity ofthe lateral di¡usion coe¤cient of this peptide to thehost bilayer chain-melting phase transition, and theability of cholesterol to induce lateral aggregation ofGS in DPPC and particularly in DMPC bilayers, Wu

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et al. [23] suggested that GS must penetrate at leastpartially into the hydrophobic core of the host lipidbilayer. This conclusion was later supported by Zi-dovetzki et al. [24], who demonstrated by 2H-NMRspectroscopy that GS alters the orientational orderparameter pro¢le of the hydrocarbon chains ofDMPC, at least at low lipid/protein ratios (5.5:1),and by 31P-NMR spectroscopy, which suggestedthat the structure of the bilayer is abolished at evenlower lipid/peptide ratios (2.7:1). Thus these earlystudies, which were done exclusively on zwitterionicPC systems, failed to resolve either the issue of thelocation of GS in lipid model membranes or the na-ture of its interactions with the host lipid bilayer.

We have recently studied the e¡ect of GS on thethermotropic phase behavior of a variety of syntheticphospholipids using primarily 31P-NMR spectros-copy supplemented by DSC and X-ray di¡raction.We found that at physiologically relevant concentra-tions of GS (lipid/protein ratios of 25:1), GS doesnot a¡ect the lamellar phase preference of the zwit-terionic lipids PC and SpM nor of the anionic lipidsDPG and PS, even at high temperatures. However,GS was found to potentiate inverted cubic phaseformation in zwitterionic PE and, to a lesser extent,in anionic PG dispersions, as well as in total polarlipid extracts from the glycolipid-based membranesof Acholeplasma laidlawii and from the phospholip-id-based membranes of Escherichia coli. Moreover,the ability of GS to induce nonlamellar phase for-mation was found to increase with the intrinsic non-lamellar phase-forming propensity of the PE molec-ular species studied. We suggested that the ability ofGS to induce localized regions of high curvaturestress in the lipid bilayers of biological membranesmay be relevant to the mechanism by which thispeptide disrupts cell membranes.

It thus seems likely that interactions between GSand biological membranes will vary markedly withthe physical properties, and thus with the lipid com-position, of the target membrane. Also, because lipidpolar headgroups essentially determine the surfaceproperties of biological membranes, interactions be-tween GS and biological membranes should be par-ticularly sensitive to membrane lipid polar head-group composition. We are thus studying thenature of the interactions of GS with lipid bilayermodel membranes which contain a wide range of

polar headgroups. This research is intended to pro-vide basic information on the sensitivity of variousmembrane lipid classes to the action of GS and onhow these are a¡ected by variations in the structureof the peptide molecule. Because the lipid composi-tions of bacterial and eucaryotic cell membranes arequite di¡erent and because antimicrobial and hemo-lytic activities can be at least partially dissociated inGS analogs [4], such information could potentially beused in the design of more therapeutically useful GSderivatives. In fact, our recent work has shown forthe ¢rst time that GS does exhibit an appreciablelipid polar headgroup speci¢city, at least as regardsthe lamellar/nonlamellar phase behavior of variousglycerophospho- and sphingophospholipids [25].The present high-sensitivity DSC study of the e¡ectof GS on the thermotropic phase behavior ofDMPC, DMPE and DMPG bilayer membranes rep-resents a continuation of this research program.Since PCs are virtually absent in bacterial mem-branes but are generally the most abundant phos-pholipid in eucaryotic plasma membranes, DMPCcan serve as a model for the surface membrane ofthe cells of higher animals, particularly so since PCsare typically found primarily in the outer monolayerof the lipid bilayer of such membranes. Similarly,PGs are absent in eukaryotic plasma membranesbut are ubiquitous and often abundant in bacterialmembranes, so that DMPG can serve as a goodmodel for the bacterial membrane. Finally, PEs arealso major components of virtually all eukaryoticplasma membranes but are also abundant in mostGram-negative bacteria and in members of theGram-positive Bacillus genus of bacteria as well.However, in eukaryotic cell membranes PEs are usu-ally enriched in the inner monolayer of the lipid bi-layer. Thus information gained from a study ofDMPE may be applicable to both bacterial and ani-mal plasma membranes, but especially to the former.

2. Materials and methods

DMPC, DMPE and DMPG were purchased fromAvanti Polar Lipids (Alabaster, AL) and were usedwithout further puri¢cation. Gramicidin S was ob-tained from Sigma (St. Louis, MO) and dissolvedin pure ethanol and stored at 320³C. The lipid stock

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solutions were dissolved in redistilled methanol. Lip-id/peptide mixtures were prepared by mixing appro-priate amounts of the lipid and peptide stock solu-tions, drying under N2, and exposure of the lipid/peptide ¢lms to high vacuum overnight. The multi-lamellar GS-containing vesicles were then preparedby vortexing in excess bu¡er (10 mM Tris^HCl,100 mM NaCl and 2 mM EDTA) normally at tem-peratures above the main phase transition tempera-ture of the phospholipid. All chemicals were pur-chased from BDH (Toronto, Ont.).

We found that the e¡ects of GS on the thermo-tropic phase behavior of the phospholipid vesiclesstudied depended signi¢cantly on the method ofpreparation of these peptide^phospholipid complexesand on their thermal history. In order to mimic thein vivo e¡ects of GS as closely as possible, we ini-tially added peptide to preformed large unilamellarvesicles at temperatures above the phospholipid gelto liquid-crystalline phase transition temperature.However, the chain-melting phase transition of theselarge unilamellar vesicles was so broad, even in theabsence of peptide, that the e¡ects of the addition ofsmaller quantities of GS on phospholipid thermo-tropic phase behavior could not be accurately deter-mined. Moreover, the e¡ects of the addition of largeramounts of GS to these vesicles varied considerablywith the number of heating and cooling scans per-formed, presumably because GS can cause vesicularlysis and fusion at higher concentrations, allowingthe peptide access to both surfaces of the lipid bi-layer. Alternatively, when small amounts of GS areadded to preformed large multilamellar vesicles,which exhibit much more cooperative phase transi-tions in the absence of peptide, only very small ef-fects on phospholipid thermotropic phase behaviorwere observed, probably because GS was interactingonly with the outer monolayer of the single bilayeron the surface of these multilamellar vesicles. Again,when larger amounts of peptide were added to thesesystems, the characteristic e¡ects of GS on the ther-motropic phase behavior of the multilamellar phos-pholipid vesicles became increasingly more markedwith each DSC heating and cooling cycle, presum-ably because the peptide was progressively gainingaccess to the surfaces of bilayers which were initiallyinaccessible. Thus, in order to maximize the observ-able e¡ects of GS on phospholipid thermotropic

phase behavior in a system exhibiting highly cooper-ative lipid phase transitions and to minimize the ther-mal history dependence of the system, lipid^peptidecomplexes were prepared as described above andthen these complexes were hydrated to produce largemultilamellar vesicles in which the GS was presum-ably uniformly distributed among the various bi-layers. In this way we could accurately measure thee¡ects of very small amounts of GS on phospholipidthermotropic phase behavior while minimizing,although not entirely eliminating, any thermal his-tory dependence of the sample. This latter e¡ectwas obviated by performing three heating and cool-ing cycles in the DSC instrument before collectingdata.

The calorimetry was performed on a MicrocalMC-2 high-sensitivity Di¡erential Scanning Calorim-eter (Microcal, Northampton, MA). For all samplesa scan rate of 10³C/h was used. Sample runs wererepeated at least three times to ensure reproducibil-ity. Data acquisition and analysis was done usingMicrocal's DA-2 and Origin software (Microcal).The total lipid concentrations used for the DSC anal-yses were about 0.5 mg/ml, providing for full hydra-tion for the GS^phospholipid mixtures. Samples con-taining GS alone, dissolved in bu¡er at peptideconcentrations corresponding to those of the highestpeptide/lipid molar ratios studied (1:10), exhibitedno thermal events over the temperature range 0^90³C. This indicates that GS does not denatureover this temperature range and that the endother-mic events observed in this study arise exclusivelyfrom phase transitions of the phospholipid vesicles.

3. Results

3.1. Thermotropic phase behavior of GS/DMPCmixtures

DSC heating endotherms illustrating the e¡ects ofGS on the thermotropic phase behavior of large,multilamellar vesicles of DMPC alone are presentedin Fig. 1. Aqueous dispersions of DMPC alone,when not extensively annealed at low temperatures,exhibit only two endothermic events, a less energeticpretransition near 14³C and a more energetic maintransition near 24³C. Under these conditions a sub-

BBAMEM 77564 24-2-99


transition centered at 18³C is not observed, sincethere is insu¤cient time for the formation of an L

0c

phase and this phase was not nucleated by exposureto low temperatures (320³C). The pretransitionarises from the conversion of the L

0L to the P

0L phase

and the main or chain-melting phase transition fromthe conversion of the P

0L to the LK phase. For a more

detailed discussion of the thermotropic phase behav-ior of DMPC and other members of the homologousseries of linear saturated PCs, see Lewis et al. [26].

The interaction of GS with DMPC vesicles clearlyalters the thermotropic phase behavior of the latter,as illustrated in Fig. 1. Small amounts of GS de-crease the temperature (Fig. 2) and enthalpy (Fig.3) of the pretransition and the pretransition is abol-ished entirely in DMPC vesicles having lipid/peptideratios of 100:1 or less (Fig. 1). Moreover, the pres-ence of increasing quantities of GS also results in theinduction of a two-component main phase transition,with a more cooperative, lower temperature endo-therm superimposed over a less cooperative, higher

temperature endotherm. The temperature (Fig. 2),enthalpy (Fig. 3) and cooperativity (Fig. 1) of bothcomponents of the chain-melting phase transitionalso decrease with increases in peptide concentrationrelative to DMPC alone. However, the GS-induceddecreases in the phase transition temperature of the

Fig. 1. High-sensitivity DSC heating scans illustrating the e¡ectof the addition of increasing quantities of GS on the thermo-tropic phase behavior of DMPC multilamellar vesicles. The topscan is of DMPC alone, and the lipid/peptide molar ratios ofthe lower scans are indicated on the ¢gure itself.

Fig. 2. A plot of variations in the midpoint temperatures forthe pretransition and main transition of DMPC multilamellarvesicles as a function of GS concentration. E, pretransition tem-perature; b, overall temperature for main phase transition; k,temperature of the sharp component of the main phase transi-tion; a, temperature of the broad component of the mainphase transition.

Fig. 3. A plot of the transition enthalpies for the pretransitionand main transition of DMPC multilamellar vesicles as a func-tion of GS concentration. The symbols used are the same asfor Fig. 2.

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two main phase transition components are modest(2^3³C) and the total enthalpy of chain melting isreduced by only 10^15%, even at a phospholipid/pep-tide ratio of 10:1. These results indicate that thepresence of GS produces only a modest destabiliza-tion of the gel state of DMPC bilayers, even aftermaximizing GS^DMPC interactions by multiple cy-cling through the gel to liquid-crystalline phase tran-sition. Generally similar although less detailed resultswere reported previously for GS/DMPC and GS/DPPC mixtures studied by low-sensitivity DSC[22,23].

3.2. Thermotropic phase behavior of GS/DMPEmixtures

DSC heating scans illustrating the e¡ects of GSaddition on the thermotropic phase behavior oflarge, multilamellar vesicles of DMPE are presentedin Figs. 4 and 5. Aqueous dispersions of DMPEalone, in the absence of extensive incubation at lowtemperature, exhibit a single, relatively energetic LL/LK phase transition near 50³C (see [27], and referen-ces therein for a more complete description of thethermotropic phase behavior of DMPE and othermembers of the homologous series of linear saturatedPEs). If GS is added to DMPE vesicles at temper-

atures above but near to the gel to liquid-crystallinephase transition temperature of DMPE, essentiallyno e¡ect on the thermotropic phase behavior is ob-served upon heating (Fig. 4), even at high GS con-centrations. However, if GS-containing DMPEvesicles are exposed to temperatures well above thegel to liquid-crystalline phase transition temperature,increasing quantities of GS lower the temperature,enthalpy and cooperativity of the chain-meltingphase transition of DMPE slightly when DSC cool-ing curves are run (Fig. 5). Moreover, upon subse-quent reheating, the characteristic e¡ects of the pres-ence of the peptide on the phase behavior of DMPEvesicles are retained (Fig. 5). However, the e¡ects ofGS addition on the temperature, enthalpy and coop-erativity of the main phase transition observed uponcooling and subsequent reheating are very small,even at the highest peptide^phospholipid ratio tested.These ¢ndings indicate that the presence of GS pro-duces only a very slight destabilization of the gelstate of DMPE bilayers and then only if the GS-containing vesicles are ¢rst exposed to high temper-atures. However, the interaction of GS with DMPE

Fig. 5. High-sensitivity DSC heating and cooling scans illustrat-ing the e¡ect of the exposure to high temperature on the ther-motropic phase behavior of DMPE multilamellar vesicles hav-ing a lipid/peptide molar ratio of 25:1. Heating and coolingthermograms of DMPE alone are illustrated in A and B, re-spectively, for comparison. The heating scan of GS-containingDMPE vesicles not previously exposed to high temperature isshown in C, while heating and cooling scans of the samevesicles exposed brie£y to a temperature of 75³C are shown inD and E, respectively.

Fig. 4. High-sensitivity DSC heating scans illustrating the e¡ectof the addition of increasing quantities of GS on the thermo-tropic phase behavior of DMPE multilamellar vesicles not ex-posed to high temperatures (i.e., temperatures above 65^70³C).The top scan is of DMPE alone, and the lipid/peptide molarratios of the lower scans are indicated on the ¢gure itself.

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Fig. 6. High-sensitivity DSC heating scans illustrating the e¡ect of the addition of relatively large or relatively small quantities of GSon the main phase transition of DMPG multilamellar vesicles are presented in panels A and B, respectively. In each panel the topscan is of DMPG alone, and the lipid/peptide molar ratios of the lower scans are indicated on the ¢gure panels. The insets of panelB are blow-ups of the DSC scan in the temperature range just above the sharp component of the main phase transition, illustratingthe behavior of the minor broad endothermic peak present in these samples. High-sensitivity DSC scans illustrating the e¡ect of theaddition of small quantities of GS on the pretransition of DMPG are presented in panel C. The DMPG concentration is threefoldthat of the samples utilized in panels A and B, and only the pretransition regions of the DSC thermograms are illustrated.

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bilayers at high temperatures can also induce the LK

phase of this phospholipid to form an inverted cubicphase [25], indicating that structurally signi¢cant in-teractions between GS and a su¤ciently £uid phaseof DMPE can occur, and that these interactions dopersist after cooling to temperatures below the mainphase transition temperature.

3.3. Thermotropic phase behavior of GS/DMPGmixtures

DSC heating scans illustrating the e¡ect of GSaddition on the thermotropic phase behavior oflarge, multilamellar vesicles of DMPG are presentedin Fig. 6A,B. Aqueous dispersions of DMPG, notextensively annealed at low temperatures, also exhib-it two endothermic events upon heating, a less ener-getic pretransition near 14³C and a more energeticmain near 24³C. Again, under these conditions asubtransition is not observed. The pretransitionarises from the conversion of the L

0L gel to the P

0L

gel phase and the main transition or chain-meltingphase transition from the conversion of the P

0L to the

LK phase. For a more detailed discussion of the ther-motropic phase behavior of DMPG and other mem-bers of the homologous series of linear saturatedPGs, see Zhang et al. [28].

The interaction of GS with DMPG has a majore¡ect on the thermotropic phase behavior of this

anionic phospholipid. Speci¢cally, the presence ofincreasing quantities of GS reduces the temperatureand enthalpy of the pretransition, which appears tobe abolished entirely at lipid/peptide ratios of 500:1or less. Moreover, at low concentrations of peptide,two endotherms are clearly present in the DSC heat-ing thermograms, a sharp, relatively energetic endo-therm centered at 22³C and a broad, less energeticendotherm centered near 27³C (Fig. 6B). As the GSconcentration increases, the initially higher temper-ature endotherm decreases in temperature and coop-erativity but becomes relatively more energetic ascompared to the lower temperature, more coopera-tive endotherm (Fig. 6A). However, at higher peptideconcentrations, both endotherms decrease in temper-ature (Fig. 7) but the less cooperative transition,which becomes increasingly more prominent, isnow centered at a lower temperature than the morecooperative transition. Moreover, the total enthalpyassociated with both transitions decreases with in-creasing peptide concentration (Fig. 8), particularlyat higher peptide concentrations. If one assumes thatthe sharp endotherm is due to the chain-melting ofDMPG domains relatively poor in peptide and thebroad endotherm is due to the presence of domainsof DMPG enriched in peptide, then it appears that atlower concentrations GS stabilizes the gel state ofDMPG bilayers, as might be expected for a posi-tively charged peptide. However, at higher GS con-

Fig. 7. A plot of variations in the midpoint temperatures of themain phase transition of DMPG multilamellar vesicles as afunction of GS concentration. The symbols used are the sameas for Fig. 2.

Fig. 8. A plot of the transition enthalpies of the main phasetransition of DMPG multilamellar vesicles as a function of GSconcentration. The symbols used are the same as for Fig. 2.

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centrations, the presence of GS appears to destabilizeboth the relatively peptide-rich and peptide-poor do-mains of DMPG vesicles. Note that the e¡ects of GSon the temperature, enthalpy and cooperativity ofthe gel to liquid-crystalline phase transition ofDMPG vesicles are much larger than those observedin DMPE or DMPC vesicles at comparable peptideconcentrations.

3.4. E¡ect of GS on the thermal stability of DMPGbilayers

The e¡ect of exposure to high temperature on thethermotropic phase behavior of DMPG vesicles, re-constituted with and without GS, is illustrated in Fig.9. In the absence of peptide, the DMPG vesicles ex-hibit complex DSC endotherms after repeated heat-ing to 95³C and subsequent cooling. We have shownby TLC chromatographic analysis (data not pre-sented) that the complex thermotropic phase behav-ior of the DMPG vesicles observed after repeatedheating to high temperatures is due to the progres-sive chemical hydrolysis of these phospholipids andthat such hydrolysis is not observed, or at least is

much less pronounced, with the DMPE andDMPC vesicles. However, GS-containing DMPGvesicles exhibit thermotropic phase behavior whichis much more similar to GS-containing DMPGvesicles not exposed to high temperatures, and infact the presence of increasing quantities of GS re-sults in a progressively greater inhibition of heat-cat-alyzed chemical hydrolysis of DMPG vesicles (Fig.9). These results suggest that the binding of GS toDMPG bilayers at high temperatures is relativelystrong and that the phospholipid^peptide complexformed provides protection from heat-catalyzedchemical degradation. Unfortunately, the intrinsi-cally strong resistance of DMPC and DMPE bilayersto hydrolysis upon exposure to high temperaturesprecluded an analysis of the e¡ect of the presenceof GS on the chemically stability of these phospho-lipids.

4. Discussion

The major new ¢nding of this work is that thee¡ect of GS on the thermotropic phase behavior of

Fig. 9. DSC heating scans illustrating the e¡ect of repeated heating to high temperature on the thermotropic phase behavior ofDMPG multilamellar vesicles alone (A), or containing smaller (lipid/peptide molar ratio 250:1) (B) or larger (lipid/peptide molar ratioof 25:1) (C) quantities of GS. The ¢rst heating scan terminating at 95³C is shown as the solid line and the fourth such scan is shownas the dotted line.

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phospholipid bilayers varies markedly with both thestructure and charge of the lipid polar headgroup.Speci¢cally, the addition of GS has essentially nodetectable e¡ect on the thermotropic phase behaviorof zwitterionic DMPE bilayers employed in thisstudy, even at very high peptide levels and even aftermultiple cycling through the gel to liquid-crystallinephase transition temperature. Only after exposure ofGS-containing DMPE vesicles to high temperaturesis a slight decrease in the phase transition temper-ature, enthalpy and cooperativity of this phospholip-id observed (see also [25]). Similarly, GS has only asmall e¡ect on the thermotropic phase behavior ofzwitterionic DMPC bilayers, inducing a small de-crease in the temperature and enthalpy, and a mod-erate decrease in the cooperativity, of the main phasetransition of this phospholipid. Moreover, underthese conditions the pretransition of DMPC is abol-ished at moderate GS concentrations. However, GShas a much more pronounced e¡ect on the thermo-tropic phase behavior of DMPG, substantially reduc-ing the temperature, enthalpy and cooperativity ofthe main phase transition at higher peptide concen-trations and abolishing the pretransition at lower GSconcentrations than are required for DMPC. Theseresults indicate that GS interacts much more stronglywith anionic rather than zwitterionic lipids, and, inthe latter category, more strongly with DMPC thanwith DMPE at comparable reduced temperatures inthe liquid-crystalline state.

The stronger interaction of GS with anionicDMPG bilayers in comparison with zwitterionicDMPC and DMPE bilayers is almost certainly duein part to the more favorable electrostatic interac-tions between the peptide and lipid, and speci¢callyto the strong attractive interactions between the twopositively charged ornithine residues of the peptideand the negatively charged phosphate moieties of thephosphorylglycerol headgroups of the former lipid.Although attractive electrostatic interactions betweenthe positively charged ornithine residues of GS andthe negatively charged phosphate moieties of thephosphorylcholine and phosphorylethanolamine po-lar headgroups probably also exist, the overallstrength of the electrostatic interactions between pep-tide and phospholipid will be markedly attenuated bythe presence of the positive charges on the cholineand ethanolamine moieties of the DMPC and DMPE

bilayers, respectively. However, the shielding of thepositively charged quaternary nitrogen of the cholinemoiety in DMPC by the three methyl groups at-tached to it may result in a somewhat stronger (butstill rather weak) net electrostatic attractive interac-tion between GS and DMPC as compared to DMPEbilayers, perhaps accounting in part for the slightlystronger interactions of GS with the former lipidobserved in the present study. The fact that the pres-ence of two ornithine or other positively chargedamino acid residues at speci¢c positions in the GSmolecule are required for antimicrobial activity[2,4,11] suggests that electrostatic interactions be-tween this peptide and anionic phospholipids, ofthe type observed here between GS and DMPG,are of functional signi¢cance in vivo.

However, other di¡erences between the physicalproperties of DMPC and DMPE bilayers may alsoaccount for the apparently stronger interactions ofGS with DMPC and compared to DMPE vesicles.For example, GS may interact preferentially withliquid-crystalline DMPC rather than DMPE bilayersbecause of the greater £uidity and decreased packingdensity of the former phospholipid as compared tothe latter (see [27]). This suggestion is supported byour present ¢ndings that GS interacts less stronglywith gel as compared to liquid-crystalline bilayers ofboth of these zwitterionic phospholipids and morestrongly with DMPC than with DMPE at compara-ble reduced temperatures in the liquid-crystallinestate, and by our previous observation that signi¢-cant GS^DMPE interactions are only observed aftersuch exposure to high temperatures [25]. This sugges-tion is also supported by the fact that the incorpo-ration of cholesterol into DMPC vesicles attenuatesthe e¡ect of GS addition on both their thermotropicphase behavior and permeabilization (unpublishedobservations from this laboratory). It is also possiblethat the capacity for hydrogen bond formation be-tween GS and the polar headgroup and interfacialregions of the host phospholipid bilayer may alsobe important in determining the strength and natureof peptide^phospholipid interactions. Additionalstudies on a wider variety of lipids employing variousspectroscopic techniques will clearly be required toelucidate the nature of GS^phospholipid interactionsin greater detail.

The comparative e¡ects of the incorporation of

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GS on the thermotropic phase behavior of zwitter-ionic and anionic phospholipid bilayers can also beused to deduce both the general location of this pep-tide relative to the lipid bilayer and the nature of thelipid^peptide interactions involved. For example, Pa-pahadjopoulos et al. [29] and McElhaney [30] haveproposed that many membrane-associated proteinscan be classi¢ed into one of three groups with regardto their interactions with phospholipid bilayers.Group I proteins are typically positively charged,water soluble, peripheral membrane proteins that in-teract much more strongly with anionic than withzwitterionic lipids. The interactions of such proteinswith anionic phospholipid bilayers typically increasesboth the temperature and enthalpy of the main phasetransition temperature while decreasing its coopera-tivity only slightly. Group I proteins are localized onthe bilayer surface where they interact only with thephospholipid polar headgroups primarily by electro-static interactions. Group II proteins are also typi-cally positively charged at neutral pH but are some-what less water soluble and also interact morestrongly with anionic than with zwitterionic phos-pholipid bilayers. The interactions of these proteinswith anionic phospholipid bilayers usually decreasesthe temperature, enthalpy and cooperativity of themain phase transition moderately, at least at rela-tively high protein concentrations. Group II proteinsare localized at the bilayer interface where they in-teract primarily with the polar headgroups and glyc-erol backbone region of the phospholipid moleculesby both electrostatic and hydrogen bonding interac-tions, although some hydrophobic interactions withthe region of the hydrocarbon chains near the bilayerinterface also occurs. Finally, Group III proteinshave a range of charges but are water-insoluble, in-tegral membrane proteins that interact equally wellwith anionic and zwitterionic phospholipid bilayers.The e¡ect of the incorporation of such proteins intophospholipid bilayers is usually to reduce the temper-ature only slightly but to decrease the enthalpy andcooperativity of the main phase transition markedly.Group III proteins penetrate into or through phos-pholipid bilayers and interact with the phospholipidhydrocarbon chains by hydrophobic and van derWaal's interactions, as well as with the phospholipidpolar headgroups. The incorporation of Group Iproteins also usually has little e¡ect on the perme-

ability of the anionic phospholipid vesicles withwhich they interact, while the incorporation ofGroup II and Group III proteins increases vesiclepermeability. The results of the present study indi-cate that overall GS behaves most like a Group IIprotein. Thus it is probably located in the interfacialregion of phospholipid bilayers, where it interactsprimarily with the polar headgroup and glycerol-backbone region of the phospholipid molecules byelectrostatic and hydrogen-bonding interactions, itsmodest perturbation of hydrocarbon chain packingin the gel state being a secondary e¡ect of its inter-actions primarily with the polar region of the phos-pholipid molecules. We also suggest, however, thatthe hydrophobic leucine and valine residues of theGS molecule may have limited interactions with thephospholipid hydrocarbon chains near the interfacialregion of the bilayer, and the somewhat amphiphilicphenylalanine residues may interact with both theglycerol backbone and hydrocarbon chains at thebilayer polar/nonpolar interface. However, the exactlocation of GS molecules in liquid-crystalline lipidbilayers may vary somewhat with the charge andstructure of the lipid polar headgroup (unpublisheddata from this laboratory). An interfacial location ofthe GS molecules would also explain the protectiona¡orded to DMPG bilayers from the thermally in-duced hydrolysis of the fatty acyl ester linkages ofthese phospholipid molecules by this peptide.

We believe that our suggestion for the generallyinterfacial localization of the GS molecule in lipidbilayers is actually compatible with most of thedata on GS^PC interactions previously published.For example, the failure of Pache et al. [21] to detectany e¡ect of GS on the thermotropic phase behaviorof DPPC bilayers was almost certainly due to theinsensitivity of the calorimeter employed, since sub-sequent workers [22,23] including ourselves ([25], andthe present work) have clearly shown a modest butsigni¢cant e¡ect of GS incorporation on the pretran-sition and main transition of DPPC and DMPC bi-layers. Thus, this piece of calorimetric evidence foran exclusively surface location is not valid. More-over, a careful inspection of the ESR spectrum pub-lished by Pache et al. [21] reveals a small but signi¢-cant degree of immobilization of the 12-nitroxylstearate spin-labeled PC molecule in the presence ofadmittedly high levels of GS, indicating some e¡ect

BBAMEM 77564 24-2-99


on the lipid hydrocarbon chains. Moreover, the DSCand lateral di¡usion and lateral aggregation meas-urements of Wu et al. [23], which suggested penetra-tion of the peptide into the bilayer, utilized NBD-GS, a £uorescent derivative in which one or bothof the ornithine residues in the GS molecules werechemically modi¢ed. Such a modi¢cation will reducethe positive charge distribution on the GS moleculeas well as altering its amphiphilicity, hydrophilicityand size. Thus the interactions of NBD-GS with PCbilayer may well not accurately re£ect the behaviorof the underivatized peptide. Moreover, it seems tous that the sensitivity of the lateral di¡usion coe¤-cient of NBD-GS to the phase transition of the PCbilayer, and the lateral aggregation of this derivat-ized peptide induced by the addition of cholesterol,would both be expected from a peptide moleculelocalized at the bilayer interface as well as onepenetrating partially or fully into the lipid bilayerhydrocarbon core. Moreover, the 2H-NMR resultsof Zidovetzki et al. [24], which show that GS incor-poration slightly reduces overall hydrocarbon chaindisorder in £uid DMPC bilayers and decreases signalintensity primarily in the methylene segments closestto the glycerol backbone, are fully compatible withthe postulated interfacial location of this peptide.The only information which does not seem compat-ible with a generally interfacial location of the GSmolecule in liquid-crystalline PC bilayers is the re-port of Datema et al. [22], which suggested that GSmolecular motion is insensitive to the phase state ofDPPC bilayers. However, this result is at any rate atvariance with the report of Wu et al. [23], who foundthat the lateral di¡usion coe¤cient of at least NBD-GS is markedly altered by the phase state of DMPCbilayers. Clearly, additional work will be required toresolve this latter discrepancy, but in our view thepreponderance of evidence supports a location ofGS in £uid lipid bilayers in which the primary inter-actions are between the peptide molecule and thepolar headgroups and the interfacial regions of thephospholipid molecule.

In closing, we note the importance of applying anumber of physical techniques to studying variousaspects of GS^phospholipid interactions in order toarrive at a more complete understanding of thesesystems. Thus, in our previous 31P-NMR spectro-scopic and X-ray di¡raction studies of the e¡ect of

GS on phospholipid phase preference at tempera-tures above the main phase transition temperature[25], we found that the major e¡ect of GS on lamel-lar/nonlamellar phase preference was on DMPE bi-layers, with a weaker e¡ect on DMPG and no e¡ectat all on DMPC bilayers. Moreover, in general GSinduced inverted cubic phases more readily in zwit-terionic as opposed to anionic phospholipids. In con-trast, our present DSC studies on the e¡ect of GS onthe main phase transition of these phospholipids in-dicate that GS interacts more strongly with theanionic DMPG vesicles than with the zwitterionicDMPC and DMPE vesicles, and more stronglywith the former than the latter. Moreover, both ofthese types of e¡ects of GS on phospholipid thermo-tropic phase behavior may be relevant to the actionof GS on natural membranes. For example, anioniclipids such as PG may increase the rate or extent ofGS binding to the bilayer surface, thus facilitatingthe interaction of this peptide with zwitterionic lipidssuch as PE or nonionic lipids such as monoglycosyldiacylglycerols, which can in turn form localized re-gions of nonlamellar structure which disrupt the lipidbilayer of the target membrane. We are thus continu-ing to study the e¡ects of GS on a variety of lipidsystems using additional physical techniques.

Acknowledgements

This work was supported by operating and majorequipment grants from the Medical Research Coun-cil of Canada (R.N.M. and R.S.H.), by major equip-ment grants from the Alberta Heritage Foundationfor Medical Research (R.N.M. and R.S.H.), and bythe Protein Engineering Network of Centres ofExcellence (R.S.H.). E.J.P. and L.H.K. were sup-ported by postdoctoral fellowships from the AlbertaHeritage Foundation for Medical Research and theProtein Engineering Network of Centres of Excel-lence, respectively.

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d sc peptide1

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