The influence of proteins and peptides on the phase properties of lipids

Chemistry and Physics of Lipids, 40 (1986) 259-284 Elsevier Scientific Publishers Ireland Ltd.

259

THE INFLUENCE OF PROTEINS AND PEPTIDES ON THE PHASE PROPERTIES OF LIPIDS

J.A. KILLIAN and B. de KRUIJFF Department of Biochemistry and Institute of Molecular Biology and Medical Biotechnology, State University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands) Received March 20th, 1986 This paper reviews model membrane studies on the modulation of the macroscopic structure of lipids by lipid-protein interactions, with particular emphasis on the gramicidin molecule. This hydrophobic peptide has three main effects on lipid polymorphism: (1) in lysophosphatidylcholine it triggers a miceUar to bilayer transition, (2) in phosphatidylethanolamine it lowers the bilayer to hexagonal HII phase transition temperature and (3) in phosphatidy!choline and other bilayer preferring lipids it is able to induce the formation of an HII phase. From experiments in which the gramicidin molecule was chemically modified it can be concluded that the tryptophan residues play a determining role in the peptide-induced changes in polymorphism. The experimental data lead to the proposal that gramicidin molecules have a tendency to self-associate, possibly mediated by tryptophan-tryptophan interactions and organize into tubular structures such as found in the HII phase. Keywords: lipid dynamics; protein/lipid interaction; lipid polymorphism; nuclear magnetic resonance; gramicidin.

Introduction The functioning o f biological membranes is determined to a large extent by the chemical, structural and dynamical properties of its main components, lipids and proteins, and by the interactions among and between these molecules. Concerning the lipid backbone of the membrane it is now evident that the bilayer is the basic structure which is ideally suited for the main general function of a membrane, namely to act as a semi-permeable barrier. Proteins are supposed to provide the membrane with its specific and selective functions such as signal transmission, pumping and other transport activities and energy conservation. In the last decade it has become clear that the above structural view of the lipid part of a membrane is too simple. In particular the realization that each membrane contains substantial amounts of lipids which upon isolation and dispersion in aqueous buffers do not organize themselves in a bilayer but in a nonbilayer configuration has suggested that lipids might play additional structural and functional roles (for review see Refs. 1 and 2). The most commonly encountered non-bilayer configurations adopted by lipids in model membrane systems are the hexagonal HII phase and the related lipidic particles. These non-bilayer structures can greatly affect the functional behavior of the model membranes. They 000%3084/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

260 might be intermediate in vesicle fusion, they appear to be involved in lipid flipflop, protein insertion and translocation and might act as carriers for polar compounds. Although unambiguous proof for the existence of non-bilayer lipid structures in biological membrance is still lacking, there are now several morphological and functional indications which strongly suggest that these structures can be formed in a variety of membranes [1-5]. Moreover, non-bilayer lipid structure preferring lipids (non-bilayer lipids) appear to be crucial for particular membrane functions, such as regulation of enzymatic or transport properties of membrane-bound proteins [6-9]. From the functional point of view it is clear that mechanisms should exist by which the structure of lipids can be locally and/or transiently modulated to allow specific processes to occur at discrete sites. Lipid-protein interactions are among the most likely candidates for such regulatory roles. Also in this area of membrane research the model membrane approach has been successful. The chemical and structural complexicity of the biomembrane and the dynamics of its components, makes it virtually impossible to study the in vivo or in vitro modulation of such local and transient lipid or lipid-protein organizations. The model membrane offers the opportunity not only to systematically approach the structural aspects of lipid-protein interactions, by which a molecular framework of protein induced changes in lipid polymorphism can be constructed, but also to test the consequences of structural transitions for selected membrane functions. We will illustrate in this article first the wide range of structural possibilities lipid-protein interactions can give rise to and secondly, we will examplify in detail the molecular aspects of peptide-induced changes in lipid polymorphism using gramicidin as a model peptide.

General Aspects of the Modulation of Lipid Polymorphism by Lipid-Protein Interactions The first suggestions that proteins can exert a strong effect on macroscopic lipid organization came from studies in which the overall lipid structure in biomembranes was compared with that of the isolated total lipids dispersed in buffer under physiological conditions of ionic composition, hydration and temperature. In a number of cases most notably for the rod outer segment [10] and mitochondrial inner membrane [11], the total lipids adopted (in part) non-bilayer structures, whereas the overall majority of the lipids in the biomembrane were organized in a bilayer configuration. These studies indicated that membrane proteins could stabilize bilayer structure. Subsequently, it was unambiguously proven in model membranes that particular membrane proteins could indeed exert a pronounced bilayer stabilization. For the best studied example, the intrinsic red cell membrane glycoprotein glycophorine, the bilayer stabilization is so strong that incorporation of only one protein molecule per 400 18:1cj18:1c phosphatidylethanolamine (DOPE) molecules results in a bilayer structure for all the lipids

261 [12]. In the absence of the protein DOPE prefers to organize in the hexagonal HH phase. From experiments with proteolytic enzymes and lee[ins it could be concluded that for the bilayer stabilization, both the large hydrophilic carbohydrate-rich headgroup of glycophorin and the hydrophobic membrane spanning part of the protein are essential [12,13]. From the receptor point of view an intriguing outcome of these experiments was that the multivalent lectin wheatgerm agglutinin was able via clustering of the glycophorin molecules in the model membranes to trigger a bilayer ~ hexagonal HII transition for part of the lipids [13]. Other examples of intrinsic membrane proteins which stabilize bilayer structure in model membranes are cytochrome c oxidase [14] and chlorophyllase [15]. That pure electrostatic interactions have a drastic influence on the phase behavior of lipids is well known and is described in detail elsewhere in this issue [16]. Poly-L-lysine, which is commonly used as a model for extrinsic membrane proteins, has a pronounced bilayer stabilizing effect in model membranes of cardiolipin due to its electrostatic interactions with the lipid headgroups [17,18]. It effectively blocks the bilayer hexagonal HII transition induced by Ca2÷, when the number of lysine residues per molecule exceeds 5. A major class of membrane proteins reacts both with the polar and apolar part of the lipids. Although the amount of investigated polypeptides is still limited, it appears that this class of amphipathic proteins and peptides destabilizes bilayer structure. Formation of inverted non-bilayer lipid structures was demonstrated in appropriate lipid systems for cytochrome c [19], possibly for its biosynthetic precursor apocytochrome c [20], A1 basic protein from myelin [21], cardiotoxin from snake venom [22] and mellitin from bee venom (Batenburg and de Kruijff, unpublished). It appeared that the tendency to form non-bilayer lipid structures is related to the insertion of the protein or peptide in the membrane suggesting that such lipid organizations might play a role in protein transport through membranes. That protein translocation can occur through the membrane of a pure lipid vesicle was recently demonstrated for apocytochrome c [23] and the M13 major coat protein [241. To get a more systematic view on the way in which a polypeptide affects lipid structure requires detailed analysis by a variety of physical techniques of simple peptide molecules in appropriate model membrane systems. In the next sections we will summarize our recent studies on gramicidin which peptide turned out to be an excellent model for such an approach. General Features of Gramicidin

Gramicidins are polypeptide antibiotics, produced by Bacillus brevis strain ATCC 8185. They are linear pentadecapeptides, consisting of alternating D- and Lamino acids. Their N-terminal and C-terminal parts are blocked by a formyl group and ethanolamine group, respectively. Due to the absence of any charged or polar

262 amino acids, these molecules are extremely hydrophobic. The natural mixture consists for 85% of grarnicidin A, the structure of which is: 1

5

HCO- L-Val-GIy-L-Ala-D-Leu-L-Ala-D-VaI-L-Val-D-Val10

15

L-Trp-D-Leu.L-Trp-D-I_au-L-Trp-D-Leu-L-Trp-NHCH2 CH~OH The less abundant gramicidin species B and C differ from gramicidin A in the substitution of Trp n by a phenylalanine or tyrosine residue, respectively. In 5-20% of the molecules, the Val ~ is substituted by an isoleucine residue [25]. The gramicidins are synthesized by multi-enzyme complexes [26] and most likely are, together with the tyrocidine type of peptide antibiotics produced by B. brevis, involved in gene regulation during the shift from the vegetative phase to the sporulation phase of growth of the bacteria [27]. Gramicidin can interact in vivo with the DNA-tyrocidine complex, which results in activation of overall RNA synthesis [28]. Furthermore, gramicidin is able to inhibit transcription by binding to the o-subunit of the RNA polymerase [29,30]. The main interest in gramicidin has arisen from the observation that it can dramatically influence the barrier properties of membranes [31,32]. The addition of very small amounts of the peptide to either model or biological membranes results in a loss of barrier function due to the formation of transmembrane channels through which water and small cations can pass the membrane (recent review: Ref. 33). In the channel conformation the molecule is completely hydrophobic at the outside and it spans, as a dimer, the membrane. Since this conformation resembles that of the membrane-spanning part of intrinsic membrane proteins, gramicidin has been a popular model for such proteins. In particular the molecule has been used to study various aspects of lipid-protein interactions in model membrane systems. Studies with lamellar lipid systems formed by saturated phosphatidylcholines (PCs) showed that gramicidin can perturb acyl chain packing of the lipid molecules [34-37]. Below the transition temperature it decreased chain order. In the liquid-crystalline state for gramicidin-lipid ratios less than about 1 : 15 (molar) the chains are more ordered, at higher concentrations the polypeptide causes chain disordering. Recently it was found, that gramicidin has a dramatic influence on lipid polymorphism. Three types of lipids were used to investigate the molecular details of the lipid structure modulating activity of the polypeptide: (1) lysophosphatidylcholine, which in excess water and under physiological conditions of pH, temperature and ionic strength organizes in micellar structures, (2) the hexagonal HU phase preferring lipid phosphatidylethanolamine and (3) the typical bilayer forming lipid phosphatidylcholine. The chemical structure of these lipids is shown in Fig. 1. We will first describe the effect of gramicidin incorporation on the structure of these different types of lipids. Then we will discuss and illustrate how specific features in the chemical structure of the polypeptide can determine the peptide-lipid interaction. Finally, we will present our ideas on the molecular basis of the lipid structure modulating activity of gramicidin.

263 CH3 I

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1 PALMITOYL GLYCERO-3PHOSPHOCHOLINE (LPC) PHOSPHATIDYL ETHANOLAMINE (PE) PHOSPHATIDYL CHOLINE (PC) F i g . 1.

Chemical structures of lipids.

G~micidin-Lysophosphatidylcholine Interactions As is described by Tilcock [16] the phase preference of lipids is often related to the geometrical shape of the lipid molecules. In this concept LPC, with its relatively large polar headgroup has a cone shape and prefers organization in miceUes. Space-filling models of gramicidin show, that also the peptide can be considered to be cone shaped, due to the presence of four bulky tryptophan GR/LPC

GR/LPC

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Fig. 2. 81.0 MHz 31P-NMR spectra of aqueous dispersions of gramicidin (GR) and LPC at various molar ratios. For details see Ref. 39.

264

residues, all located at the carboxyterrninal part of the molecule. Recent energy calculations indicate that as a result of the hydrophobicity of these trp-residues an orientation of the grarnicidin molecule with its N.terminal part at the lipid/ water interface is energetically more favourable, thus predicting complementary shapes for LPC and the gramicidin molecule [38]. In addition these theoretical studies showed that positioning of the gramicidin molecules at an air-water interface allows the peptide to be surrounded by 4 LPC moleules, to form an almost cylindrical complex, which according to the shape-structure relationship, should result in the formation of bilayers. The results of these calculations are in good agreement with earlier experimental evidence for the formation of lamellar structures of LPC and gramicidin [39]. Phospholipid structure can be conveniently monitored by aIP-NMR. The. 31p-NMR spectrum of a micellar solution of LPC is characterized by an isotropic signal (Fig. 2). This is the result of rapid reorientation of the lipids by micelle tumbling and I

; I '

A

/!1 I

: /',

c / I

D _

/--J /,'

-20

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Fig. 3. 81.0 MHz 31P-NMR spectra of aqueous dispersions of gramicidin and LPC (1:5, m/m) recorded after 0 (A), 15 (B) and 60 h (C) exposure to the magnetic field in the spectrometer and after storage at -20°C for several days outside the magnet (D). Spectra were recorded as described elsewhere [39].

265 lateral diffusion by which the chemical shift anisotropy of the phosphorus atom is almost completely averaged [40,41]. Incorporation of gramicidin by hydration of a mixed lipid-gramicidin film results in the gradual appearance of a broad spectral component with an asymmetrical line shape [39]. This line shape is typical for a lamellar organization of the phospholipids in which the chemical shift anisotropy is only partially averaged by fast axial rotation of the lipid molecules [40,41]. The growth of this bilayer component goes at the expense of the isotropic NMR signal, such that at a molar ratio of 1 gramicidin to 4 LPC molecules, the isotropic signal has almost completely vanished (Fig. 2). Quantification of the spectral component reveals that as a result of the gramicidin-lipid interaction, a lamellar gramicidin-LPC (1:4, molar) complex is formed [39]. The bilayer stabilization exerted by gramicidin on LPC is a surprising observation, since detergents, such as LPC normally destabilize bilayer structures. During alP-NMR investigations on the LPC/gramicidin system another interesting phenomenon was encountered. A LPC/gramicidin (5 : 1, m/m) sample was positioned in the magnet for a prolonged time and at different time intervals 31P-NMR spectra were recorded. As is shown in Fig. 3, a sharp low field peak occurs which increases with time. The appearance of such a low field peak was observed before but to a smaller extent in gramicidin containing samples of soya-PC [42]. In line with previous suggestions [42] we attribute this as a magnetic field induced aUignment of the gramicidin molecules resulting in a concomittant preferential orientation of the phospholipid molecules with their rotational axis parallel to the magnetic field. After storing the sample for several days outside the magnet this low field peak had completely disappeared (Fig. 3D), which indicates that a reversible macroscopic ordering of the gramicidin/LPC complex had taken place in the magnetic field. A similar protein-induced orientation of lipids in a magnetic field has been reported for rod outer segments under the influence of rhodopsin [43]. These phenomena are especially of interest in the light of the observed behavior of pure phospholipids, which in contrast show a tendency to orient themselves perpendicular to the magnetic field [44]. Within the bilayer gramicidin and LPC can form ordered structures as was earlier suggested on the basis of tryptophan fluorescence measurements and negative staining electron microscopy [45]. A model was proposed in which linear arrays of hexamers of gramicidin are present with a two-dimensional periodicity and in which the space between the hexamers is ftlled with LPC molecules. Also in our studies freeze-fracture electron microscopy suggested the presence of ordered aggregates. Although the gramicidin-LPC, 1 : 4, molar complex is characterized by smooth fracture faces, in case of a molar ratio of 1 : 2.5 regularly structured fracture faces are observed (Fig. 4). Finally the lamellar LPC/gramicidin complex was characterized by 2H-NMR. With this technique it is possible to gain information on acyl chain order by using specifically deuterated compounds [46]. The quadrupolar splitting (Avq) is related

266

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4 6 8 10 12 ACYL CHAIN POSITION

I

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16

Fig. 5. Quadrupolar splitting at 45°C of the labelled carbon atom in the acyl chain, in aqueous

dispersions of DPPC (= ~.), LPC/DPPC (1:4, m/m) (o c), LPC/gramicidin (4 : 1, m/m) (~ ~) and LPC/cholesterol (1:1, m/m) (× X). Reproduced with permission from Killian et al. [47]. The data on DPPC are obtained from Ref. 58.

to the order parameter of the C-D segment and in case of dipalmitoyl-PC (DPPC) is constant for the first 9 methylene groups and decreases towards the terminal part of the acyl chain as is shown in Fig. 5. A very similar order profile is observed for LPC in lameUar structures with either gramicidin or as present in dispersions of LPC with DPPC (molar ratio, 1:4) [47]. That the quadrupolar sphtting of chain labelled lyso-PC is very sensitive towards changes in lipid packing can be observed in Fig, 5 for the lameUar complex formed with cholesterol. The condensing effect of cholesterol [48] is manifested as an overall increase of the quadrupolar splitting which translates into an increase in acyl chain order. The lack of any significant effect of gramicidin on AVq despite the: very high peptide content, i.e. 4 acyl chains per gramicidin monomer, suggests that either the acyl chains are unperturbed by the presence of the gramicidin molecule, or indicates that indeed, in agreement with previous suggestions, self-association of gramicidin occurs whereby the contact area between the peptide and the lipid decreases. Gramicidin-Phosphatidylethanolamine Interactions Aqueous dispersions of PEs can undergo in the lameUar phase a gel to liquidcrystalline phase transition and in addition a lamellar liquid-crystaUine to hexagonal H I transition [1]. This is shown in the thermogram of dielaidoylphosphatidylethanolamine (DEPE) dispersed in buffer (Fig. 6). The gel to liquidcrystalline phase transition occurs around 356C. The bilayer to hexagonal transition occurs around 60°C and has a much smaller transition enthalpy due to the fluid character of both the lamellar and hexagonal HI/phase.

268

GR/DEPE 0 f

k I:::)

i

i

i

2o 30 4'0 do do ' TEMPERATURE

(~C)

Fig. 6. DSCiheating curves of aqueous dispersions of mixtures of DEPE and gramicidin (GR) with the indicated molar composition. For details see Ref. 49.

From Figs. 6 and 7 can be seen that incorporation of even 1 gramicidin per 5 DEPE molecules has virtually no effect on the gel to liquid-crystalline phase transition. Since apparently no gramicidin-lipid interactions occur in the gel state, these measurements suggest that also in PE extensive segregation takes place between the peptide and the lipid. The effect of gramicidin incorporation on the lamellar to hexagonal transition in the DEPE is shown in Fig. 8. The presence of only one gramicidin molecule per 100 DEPE molecules causes already a significant downward shift in transition temperature. The presence of higher concentrations of gramicidin even lowers the bilayer to hexagonal transition temperature till about that of the gel to liquidcrystalline phase transition [49,50].

-,a

0

0

0.10 0.20 G R / L I P I D (m/m) - -

7. Effect of gramicidin (GR) incorporation on the enthalpy of the gel ~ liquid-crystalline phase transition in aqueous dispersions of DEPE. For details see Ref. 49.

Fig.

269 100

I

1

I

I

o----

I

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I

0

1 t~

1 0

' ~ o I00 10203O4O5O6070 TEMPERATURE (*C) Fig. 8. Percentages of bilayer and HII component in the 31P-NMR spectra of dispersions of DEPE (o o) and gramicidin/DEPE mixtures of molar ratios of 1 : 100 (~ ~) and 1 : 10 (× × ) as a function of temperature. For details see Ref. 49.

alP-chemical shift anisotropy m e a s u r e m e n t s showed that in case o f co-existing liquid-crystaUine and hexagonal HI] phase the gramicidin molecules are preferentiaUy located in the hexagonal HI/ phase and cause a decrease in headgroup order [49]. The possible m e c h a n i s m o f the gramicidin-induced H I / p h a s e f o r m a t i o n in PE systems is shown in Fig. 9.

-,TEMPERATURE T

TO

T

TC

Fig. 9. Schematic representation of the possible mechanism of gramicidin-induced HII phase formation in PE systems. In the lamellar gel state, gramicidin forms aggregates at all concentrations of the peptide. Upon raising the temperature till the gel liquid-crystalline phase transition temperature Tc, phase separation occurs between a fluid bilayer of pure DEPE (route 1) and a hexagonal HII phase which is very rich in gramicidin (route 2). Upon further increasing the temperature, more PE will organize in this gramicidin-containing HI1 phase. The remaining part of the lipids, the extent of which is dependent on the gramicidin concentration, will undergo a normal bilayer to hexagonal HII phase transition (route 3). Reproduced with permission from Ref. 49.

270

Gramicidin-Phosphatidylcholine Interactions Naturally occurring PCs are typically bilayer-forming lipids. As compared to PE, the presence of the quatemary ammonium group increases headgroup hydration and decreases intermolecular interactions, both resulting in an increase of effective size of the headgroup. The gramicidin-induced bilayer destabilization observed in studies with PEs is so strong that even in case of PCs an HI1 phase can be induced under the appropriate conditions [42,50]. The 31p NMR spectra of a series of dispersions of unsaturated liquid-crystalline PCs containing gramicidin in a 10:1 molar ratio is shown in Fig. 10. For acyl chain lengths exceeding 16 carbon atoms, incorporation of gramicidin results in the appearance of a new spectral component with a 31p. NMR line shape typical of the hexagonal HII phase [42]. Due to the fast lateral diffusion of the lipid molecules around the tubes in the hexagonal HII phase, additional averaging of the chemical shift anisotropy occurs, resulting in a reversal in anisotropy of the spectrum and a reduction in line width [40,41]. As a result, the dominant spectral component for lipids in the hexagonal HII phase occurs at around - 7 p p m . HI] phase formation is maximal for the longest chain tested (22 : lc-PC). Similar behavior is observed for a series of saturated PCs, in which also incorporation of

16 : 1c

.

18:1tr

18:1 c

22:1 c I

- 50

I

1

1

I

0

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50

H

Fig. 10. 81.0 MHz 31P-NMR spectra of aqueous dispersions of mixtures or: gramicidin with di-unsaturated PC species in a 1 : 10 molar ratio. Reproduced with permission from van Echteld et al. [42].

271

1 gramicidin per 10 PC molecules results in hexagonal HI/phase formation when the chain length exceeds 16 carbon atoms [42]. The difference in amount of hexagonal HI/ phase induced by gramicidin in DEPC (18: It) and dioleoyl-PC (DOPC 18: lc) suggests that in addition to the chain length, also the nature of unsaturation plays an important role in the gramicidin-induced HI] phase formarion. Although in these experiments, samples were prepared by hydration of a mixed PC-gramicidin f'dm, it was shown that also the addition of gramicidin as an ethanolic solution to preformed DOPC liposomes results in HII phase formation, demonstrafing that the spontaneous insertion of the peptide in a bilayer can trigger a bilayer + HII transition [51 ]. DOPC was chosen to investigate in more detail the molecular aspects of the gramicidin-induced HI] phase formation [52]. Since HII phase-preferring lipids typically have a low headgroup hydration [53], it can be expected that hydration might play an important role in the gramicidin-induced HII phase formation. Investigations by 31P-NMR, 2H-NMR and small-angle X-ray diffraction of mixed DOPC-gramicidin films as a function of the 2H20 content led to a number of interesting observations [52]. Figure 11 illustrates this with the 3~P-NMR spectra of DOPC-gramicidin films in a molar ratio of 10: 1, containing increasing amounts of 2I-I20. Whereas in the absence of the peptide, DOPC at all stages of hydration gives rise to the typical 31P-NMR spectrum indicative of a bilayer organization, the

DOPC

15.7

5.6 7.4

15.6 27.0 EXCESS I

0

L

I

I

25

0 -25 (pprn)

- 50

Fig. 11. 81.0 MHz 31P-NMR spectra of aqueous dispersions of DOPC and DOPC/gramicidin (10 : 1, m/m) samples containing different amounts of 3H20. N = ~H20/PC (m/m). Reproduced with permission from Killian and de Kruijff [52].

272

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x 50 \

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0.3 N-1

Fig. 12. Nil phase formation in DOPC/gramicidin systems of varying 2H20 content. The insert shows the relation between the amount of HII phase and the gramicidin concentration (tool%) in excess 2H20. Molar ratios gramicidin/DOPC 0 (; ~), 1:50 (c o), 1:25 (, ~) and 1 : 10 (t~ t~). Reproduced with permission from Killian and de Kruijff [52]. presence of gramicidin has a number of different effects. For values of N (which is the number of 2H20 molecules per PC) lower than 5, typical bilayer 3~p-NMR spectra are observed. For intermediate 2H20 contents (N = 5 - 1 5 ) a second bilayer spectral component appears, which has a marked reduced chemical shift anisotropy. The hexagonal HII phase is induced for N > 15. Thus, the presence of water is essential for gramicidin-induced hexagonal Hn phase formation in PC systems. This is opposite to what one would expect from the phase behavior of pure lipid systems and suggests that a specific hydrated conformation of gramicidin is essential for HII phase formation. That gramicidin has a different structure in hydrated lipid systems than in dry films was reported earlier on basis of Raman spectroscopy [57]. Very similar behavior was also observed for other gramicidin concentrations and the data are summarized in Fig. 12. The fraction of DOPC in the hexagonal phase decreases with decreasing gramicidin and 2H20 content; The molecular efficiency of the HII phase formation by gramicidin is rather low: 2.5 mol DOPC per gramicidin monomer in excess water for DOPC-gramicidin ratios up to 25 : 1 (insert Fig. 12).. The new lamellar phase observed at intermediate 2H20 content was also detected by X-ray diffraction and was characterized by a smaller repeat distance. In this intermediate phase headgroup order appears to be greatly decreased, as indicated by the large reduction in chemical shift anisotropy [52]. Information on the behavior of water in these systems was obtained by 2H-NMR. A set of representative spectra is shown in Fig. 13. For pure DOPC from the lowest amount of 2H20 tested up to N = 21, a single quadrupolar splitting is observed with a value which decreases in a biphasic way with increasing 2H20 content (Fig. 8, Ref. 52). Above N = 21 an isotropic component becomes visible of which the intensity increases with further increase of N. This is due to phase separation

273

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Fig. 13. 30.7 MHz ~H-NMR spectra of DOPC/gramicidin/~H20 samples. The numbers given with the spectra are the molar ratio's of 2H20 to PC. Reproduced with permission from Killian and de Kruijff [52].

between the maximally swollen DOPC/2H20 phase and excess 2H20. Apparently, these two types of molecules cannot exchange with each other at least not on the time scale of the 2H-NMR measurement. Gramicidin incorporation has a dramatic influence on the properties of the 2H20 molecules as revealed by 2H-NMR. Two effects are most noticeable. At the lowest stage of hydration and for N = 3-5, the spectra are broadened, but a single quadrupolar splitting is observed with a value which decreases linearly with increasing gramicidin concentration up to 4 mol% of the peptide. Above N = 5, multi-component spectra are observed demonstrating phase separation [52]. One component has the quadrupolar splitting very similar to that of the gramicidin-free bilayer. The other component has a broad isotropic line shape or has a very small value of the quadrupolar splitting. The intensity of these components increases with increasing gramicidin concentration at any given stage of hydration. Analysis of these data revealed that for N = 5, gramicidin becomes prefer-

274

N<6

6
N>12

Hit Fig. 14. Schematic representation of the effect of hydration on the phase properties of DOPC/ gramicidin mixtures. The scheme depicts the situation for a DOPC/gramicidin ratio of 25 : 1. For N < 6, one lamellar phase exists with similar structural properties as the pure DOPC system. The gramicidin molecules, represented by the zig-zags, are thought to be randomly oriented in the hydrophobic part of the bilayer in a condensed and anhydrous conformation. Increasing water concentration will cause preferential hydration of the gramicidin molecule. Above N = 6, phase separation occurs resulting in a lamellar phase which is highly enriched in the hydrated gramicidin molecule, shown here in the head-to-head dimer channel conformation. This bilayer is relatively thin and the headgroups of the lipid molecules have obtained increased motional freedom. The other lameUar phase behaves like the pure DOPC lamellar system but contains some hydrated gramicidin. Above N = 12, the gramicidin-rich lameUar phase converts to a gramicidin-rich hexagonal HII phase possibly as the result of an interbilayer fusion event, involving linear arrays of gramicidin molecules. The gramicidin molecules in the HII phase most likely are oriented with their axis perpendicular to the aqueous tubes present in the lipid cylinders as there is rapid exchange between the gramicidin-associated water and the water within these tubes. entiaUy h y d r a t e d over DOPC and takes up approx. 140 2H20 molecules per gramicidin m o n o m e r . Thus, the grarnicidin molecules dehydrate the PC headgroup. These results are summarized in the h y d r a t i o n m o d e l shown in Fig. 14. Preliminary sucrose density centrifugation e x p e r i m e n t s with DOPC/gramicidin (10/1,

275

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I

O.10 0.20 GR/LIPID (m/m)

Fig. 15. Effect of gramicidin (GR) incorporation on the enthalpy of the gel ~ liquid-crystalline phase transition in aqueous d~spersionsof DEPC. For details see Ref. 49. molar ratio) samples in excess water unambiguously demonstrated that indeed a macroscopic phase separation occurs between a bilayer and an HII phase. Pure fractions of both phases could be isolated. In the bilayer phase a peptide to lipid ratio of about 1/16 was found, whereas the HII phase appeared to be 2-3-fold enriched in gramicidin (K,J.N. Burger, J.A. Killian and B. de Kruijff, in prep.). Thus, similar to the gramicidin-induced HII phase in PE systems, also in DOPC the HII phase has an extremely high peptide content. However, in PC it appears that gramicidin has a limited solubility in the bilayer phase. More insight into the nature of the gramicidin/PC interaction can be obtained by investigating the thermodynamic details of this interaction and comparing the results to those of the previously described gramicidin/PE system. The effect of gramicidin on the energy content of the gel-to-liquid-crystalline phase transition was measured for DEPC (Fig. 15). In contrast to PE systems and similar to the behavior in DPPC [49,54] incorporation of gramicidin in DEPC results in a decrease in transition enthalpy up to a molar ratio of gramicidin to lipid of about 1:15. At a higher peptide concentration no further decrease is observed. These results indicate that also in the gel state of PCs gramicidin has a limited solubility and can dissolve in the bilayer up to molar ratio of 1 gramicidin per about 15-20 PC molecules. From both the thermodynamical and structural data a molecular picture of gramicidin-induced HII phase formation in PC model membranes emerges which is schematically represented in Fig. 16.

Gramicidin-Negatively Charged Lipid Interactions The induction of the HII phase by gramicidin in bilayer-forming lipid systems is not restricted to PC [55]. Under conditions that the negative charge on the anionic phospholipids cardiolipin, phosphatidylserine (PS) and phosphatidylglycerol (PG) is expressed (100 mM NaCI, pH 7.4, for instance), these lipids are typically organized in bilayers [2], which often are widely separated due to strong

276

,,*TEMPERATURE T < TC

T > TC

I

PC

3

Fig. 16. Schematic representation of the gramicidin PC interaction in relation to HII phase formation in PC systems in excess water. In the lameUar gel state aggregation of the peptides occurs only at high gramicidin concentrations (peptide to lipid molar ratio > -+1/20), whereby probably smaller aggregates are formed as in PE systems. Upon increasing the temperature at low peptide concentrations gramicidin will be present in the fluid bilayer as mono- or oligomers (route 1). At higher concentrations the aggregate size increases (route 2). These large aggregates are able to induce HII formation (route 3) when the chain length exceeds 16 carbon atoms.

electrostatic interbilayer repulsive forces [56]. Incorporation of gramicidin in these lipid systems also results in Ha phase formation as is evidenced for DOPG in Fig. 17. The freeze-fracture micrograph shows the characteristic striated fracture patterns observed for lipids organized in the HI1 phase. Quantification by 3~P-NMR revealed a similar extent o f HII phase formation in these anionic phospholipids as in the zwitterianic DOPC, indicating that the presence o f a negative charge on the headgroup virtually has no effect on HII phase formation [55]. Since a location o f charged headgroups around the narrow aqueous cylinders present in the H a phase must be energetically unfavorable, due to strong intermolecular repulsive forces, these observations suggest that the HII phase-promoting

277

Fig. 17. Freeze-fracture micrograph of an aqueous dispersion (100 mM NaC1, 10 mM Tris, pH 7.4) of gramicidin and DOPG in a 1 : 10 molar ratio. Final mag. × 100 000. For details see Ref. 55. ability of gramicidin must be very strong. Recently, gramicidin-induced HII phase formation was also observed in total lipid extracts of rat liver microsomes and mitochondria (G. van Duijn, A. Rietveld and B. de Kruijff, unpublished observations), demonstrating that this effect is not restricted to single component synthetic lipid systems.

Relationship between Chemical Structure of Gramieidin and its Lipid StructureModulating Activity The lipid structure-modulating activity of gramicidin, most likely is the result of the specific chemical and spatial structure of the molecule. This can be inferred from the hydration experiments, which indicated that a hydrated conformation of gramicidin is essential, but also is suggested from the observation that other hydrophobic polypeptides such as the 23 amino acid long membrane-spanning segment of the red cell membrane glycoprotein glycophorin, in contrast to gramicidin, acts as a bilayer stabilizer in HII phase-preferring lipid systems [12,13]. In an attempt to unravel the structural requirements in the gramicidin molecule for its potency to induce an H I phase in DOPC systems, the derivatives of gramicidin shown in Fig. 18 were synthesized [55]. By removing the formyl group

278 GRAMICIDIN 1 15 HCO-L-Vol. ,, ( -L - Trp-D-Leu)3-L - Trp-NHCH2CH20H

GR DESI:-6JI (+)

1 L-Vol . . . . . . . . . . . . . . . . . . .

15 -k - Trp-NHCH2CH20H

N-Suc-GIi ( - )

1 HOOCCH2CH2CO-L-Vo] . . . . . . . . . . . . . . . . . . .

15 -L - TrP-NHCH2CH20H

O-Suc-GR ( - )

1 HCO-L-Vol . . . . . . . . . . . . . . . . . . .

15 -L - TrP-NHCH2CH2OOCCH2CH2COOH

1 15 HCO-L-Vol , . , ( - L - r . rp-u-Leu)3-L-FTrp-NHCH2CH2uH

FTRP-6R

~

~

. . ^

Fig. 18. Chemical structures of gramicidin A and some derivatives.

(yielding desformyl-gramicidin, DESF-GR) and by subsequent coupling o f a succinate group (yielding N-succinyl-gramicidin, N-Suc-GR), N-terminal modified gramicidin analogs were prepared which, when incorporated in model membrane systems, can be expected to behave like positively and negatively charged molecules, respectively. The coupling o f succinate to the ethanolamine group at the C-terminal end o f the molecule (yielding O-succinyl-gramicidin: (O-Suc-GR), also will result in a negatively charged molecule but now with an opposite direction o f polarity. Incorporation o f the N- and C-terminal modified gramicidin analogs in DOPC systems in a 1:10 molar ratio in all cases results in HII phase formation to an extent which is of the same magnitude as that observed for the unmodified molecules (Table I). Moreover, also the two other lipid structure-modulating activities o f the peptide, i.e. bilayer formation in mixtures with LPC and a downward shift in bilayer to hexagonal transition temperature in DEPE were observed for these TABLE 1 EFFECT OF GRAMICIDIN AND C- AND N-TERMINAL MODIFIED ANALOGUES ON LIPID STRUCTURE IN AQUEOUS DISPERSIONS

No peptide Gramicidin DESF-GR N-S uc-GR O-Suc-GR

% HII in DOPCa

Structure b with lyso-PC

Decrease (°C) in bilayer --*HII transition temperature in DEPEc

0 44 33 40 31

Micelle Bilayer Bilayer Bilayer Bilayer

0 10 13 13 17

DOPC/peptide, 10 : 1 (molar). yso-PC/peptide, 4 : 1 (molar). eDEPE/peptide, 50 : 1 (molar).

279 9

I0

|1

12

13

14

15

GRAM.A -L-TrI~-D-Leu'-L-TrI:~,-D-Leu-L-Tq>-D-Leu-L-TrI~31p NMR GRAMICIDINA

~ i

GR/DOPC(1/10) Fig. 19. Effect of formylation of the tryptophans of gramicidin on the structure of peptide/ DOPC (1 : 10) model membranes as detected by 81.0 MHz 3~P-NMR spectroscopy. For details

see Ref. 59.

analogs [55]. These data clearly demonstrate that the presence of ionizable groups at the C- and N-terminal part of the gramicidin molecule, which can be expected to be charged under the experimental conditions used, do not interfere with the specific interactions of the peptide with the lipid. Most interestingly, the tryptophan residues in the gramicidin molecule appear to be very important for the gramicidin-lipid interaction [59]. Replacement of the indol proton of all 4 tryptophans by a formyl group (FTRP-GR), results in a complete loss of HII phase formation in DOPC model membranes as evidenced for instance in Fig. 19 by 31P-NM1L Deformylation of the tryptophans results in the reappearance of the HU phase-inducing ability of gramicidin [59]. Summarizing Discussion on the Molecular Details of Gramicidin-Lipid Interactions

Besides for the long-known, well-studied channel-forming properties of gramicidin [33] the more recent observations on gramicidin-RNA polymerase interaction [29,30] and the lipid structure-modulating activity of the peptide reviewed in this paper, make gramicidin an even more intriguing multi-functional compound. Before we will try to relate these various properties of gramicidin, we will first discuss the molecular basis of the lipid structure-modulating effect of the peptide. We consider this effect to be a very specific property of the gramicidin molecule. This is because of: (1) the sharply defined stoichiometry in the interaction with lyso-PC, (2) the very strong HII promoting ability in bitayer structure-preferring lipids, the extent of which is almost independent of temperature [42] and the nature of the headgroup and (3) the requirement for a specific hydrated conformation to induce this H a phase in DOPC model systems.

280 Since the mode of organization of lipids in aqueous dispersions is determined by the balance of several attractive and repulsive forces, the strength of which is dependent on many different conditions, it can be expected that also in case of the gramicidin-induced changes in lipid polymorphism, various factors will be involved. We will subsequently consider: (a) the shape of the molecule, (b) its chain and headgroup disordering ability, (c) its dehydrating capacity and (d) its tendency to self-associate into different structures. It should be realized at forehand that the relative importance of any particular factor could be different for the various lipid structure-modulating activities of the peptide. In the /3-helical conformation space-filling models reveal a pronounced cone shape of the molecule, which is due to the location of the 4 bulky tryptophan residues at the C-terminal part of the molecule. The shape-structure concept of lipid polymorphism as applied to gramicidin in this conformation can explain most of the lipid structure-modulating effects of gramicidin, but two aspects deserve special attention. Firstly, it is in contradiction with the channel conformation, in which gramicidin is believed to be present as N- to N-terminal dimers, joined together by hydrogen-bonding. This conformation has been postulated for the structure of the gramicidin dimer in mixtures with LPC, prepared via heat-incorporation [60] and with DMPC [61]. Secondly, and more severely, is the notion that the /g and Osuccinyl monomers, which most likely are charged and therefore will have an opposite orientation at the membrane-water interface, affect lipid structure in a similar way as the native gramicidin molecule. It should be realized however that these discrepancies might be only apparent, because the three-dimensional structure of gramicidin and these analogues in the different lipid systems described in this paper is virtually unknown. Gramicidin causes a disordering of the acyl chains when incorporated in high amounts in bilayers of saturated PC's [34-37]. Furthermore, the peptide causes a decrease in headgroup order, especially in the lamellar phase of DOPC at low water content [52], and in the HII phase of PE systems [50], which is possibly caused by a location of the headgroups in part over the gramicidin molecules. Both observations are consistent with the suggestion that the shape of the complex of gramicidin and its surrounding lipids will be more cone-like than the sum of their individual shapes, thus favoring HII phase formation. The dependency of the extent of HII phase formation on acyl chain length could be explained by a mismatch in length between the g(amicidin dimer (30 h in the lipid-associated form [62] ) and the thickness of the hydrophobic part of the bilayer (30 A;DPPC [63], thus longer for those PC species in which the HII phase is induced) which could result in dimple formation and thus contribute to HII phase formation. However, it should be realized that due to geometrical reasons longer chained lipids by themselves are better packed into an HII phase than shorter ones. The lipid headgroup dehydrating capacity of gramicidin observed at low water contents also most likely will contribute to HII phase formation. However, it is not clear whether such a dehydrating effect also occurs in the presence of excess

281 water. Consistent with this dehydration idea is the visual observation that dispersion of mixed gramicidin-lipid films (including those of negatively charged lipids) is more difficult and swelling less extensively as in case of peptide-free systems. In several lipid systems it has now been observed that gramicidin has a pronounced tendency to self-associate. In case of mixtures with LPC, a two-dimensional array-wise organization of gramicidin hexamers was postulated [45]. Such an organization could well be involved, also in HII phase formation. Close approximation of bilayers at the site of parallel organized long arrays of such structures could result in line fusion and subsequent HII phase formation. Alternatively, it could be that gramicidin molecules in lateral self-association have themselves a strong tendency to be organized in tubular structures such as those found in the HII phase. The notion that the tube diameter of the HII phase induced by gramicidin is virtually independent on acyl chain length and nature of the headgroup supports this suggestion. The idea that peptides can form the backbone of a tubular structure such as found in the HII phase is biologically intriguing in that such stable tubes are found for instance in case of the tight junction [3,64]. It will be clear that information on the structure of gramicidin in the various lipid systems is essential to answer some of the questions raised above. The 4 tryptophan residues in gramicidin play an important role in the lipid structure-modulating effect of the peptide. In case of the LPC-gramicidin lamellar system, it was suggested that by stacking interactions the tryptophan indol rings are involved in the formation of ordered gramicidin aggregates [45]. We would like to propose that also for HI1 phase formation intermolecular indol ring stacking interactions are essential. If we now return to the two other weU-studied properties of gramicidin: channel formation and inhibition of RNA polymerase, then it is intriguing to note that also for these effects the tryptophans have been shown to be absolutely essential [65,66]. We hypothesize that tryptophan-tryptophan interactions occur which may determine the conformation of the peptide and are functionally essential in mediating gramicidin-gramicidin or gramicidin-protein interactions. Recently, the importance of aromatic-aromatic interactions for the structure of proteins was emphasized [67]. To what extent other structural characteristics of the molecule, such as the alternation of D- and L-amino acids in the sequence which gives the molecule the ability to organize in ~-type helices, is important for the various effects of the peptide, remains to be seen.

Concluding Remarks and Future Prospects Our studies on gramicidin suggest that due to the relative simplicity of the molecule, the modern methods of chemistry and purification of hydrophobic peptides and the powerful biophysical and computational methods to monitor both lipid and peptide structure dynamics it will become possible to obtain for the first time a detailed molecular picture of the lipid structure-modulating activity of a peptide.

282

With the available variety of model membrane systems then also the functional consequences of peptide-induced changes in lipid structure can be investigated. We foresee this will lead to a functional knowledge of peptide-lipid interactions which will serve as a basis to understand the structural and functional aspects of lipidprotein interactions in biomembranes.

Acknowledgements We would like to thank A.J. Verkleij, J. Leunissen-Bijvelt, C.J.A. van Echteld, R. van Stigt, J. de Gier, F. Borle, J. Seelig, R. Brasseur, V. Cabiaux, J.M. Ruysschaert, C.W. van den Berg, H. Tournois, S. Keur, A.J. Slotboom, G.J.M. van Scharrenburg, W.J. Timmermans and K.N.J. Burger for their expert contributions to parts of the research described in this paper.

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