The interaction of a peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with DPPC model membranes: a DSC study

https://doi.org/10.1016/S0040-6031(02)00074-6Get rights and content

Abstract

Depending on their hydrophobicity, peptides can interact differently with lipid membranes inducing dramatic modifications into their host systems. In the present paper, the interaction of a synthetic peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes has been investigated by differential scanning calorimetry (DSC), adopting three different experimental approaches. In the first, the peptide is forced to be included into the hydrocarbon region of the lipid bilayer, by codissolving it with the lipid giving rise to mixed multilamellar vesicles–peptide systems; in the second, this system is passed through an extruder, thus producing large unilamellar vesicles–peptide systems; in the third, it is allowed to interact with the external surface of the membrane.

The whole of the DSC results obtained have shown that the incorporation of the peptide into the lipid bilayer by means of the first method induces a decrease in the enthalpy of the gel–liquid crystal transition of the membrane and a shift of the transition to the lower temperatures, thus resembling, in spite of its prevalently hydrophilic nature, the behavior of transbilayer hydrophobic peptides. The extrusion of these systems creates unilamellar vesicles free of peptides but of smaller size as evidenced by the decreased cooperativity of the transition. The peptide, added externally to the DPPC model membrane, has no effect on the phase behavior of the bilayer.

These findings suggest that the effect of the interaction of scrambled hydrophobic/hydrophilic peptides into lipid bilayers strongly affects the thermotropic behavior of the host membrane depending on the preparation method of the lipid/peptide systems. The whole of the results obtained in the present paper can be useful in approaching studies of bioactive peptides/lipids systems.

Introduction

The lipid component of prokaryotic and eukaryotic cell membranes is formed by a complex mixture of phospholipids, giving rise to different phases [1], [2], depending on the different individual lipid classes present in such membranes, pH and ionic strength. A considerable body of evidence has now accumulated indicating that the various phase-preferring lipid components play important structural and functional roles in eukaryotic membranes [2]. The phase that a fully hydrated membrane lipid prefers under a given set of conditions can be rationalized by considering the geometric packing [3] and, on turn, the curvature stress induced in the bilayer [4]. The modulation of both these two factors by inclusions, such as proteins and peptides, can markedly affect the phase behavior of lipid membranes [5]. The α-helical conformation and transbilayer orientation of synthetic peptides [6], [7], [8], [9], [10], [11] within lipid bilayers have been proven by a combination of different spectroscopic and X-ray diffraction measurements [12]. DSC [13] and 2H NMR spectroscopic studies [14] have shown that the incorporation of the peptides into 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and DPPE bilayers broadens the gel–liquid crystalline phase transitions and reduces its enthalpy. The effect of the hydrophobic length of the peptide is usually invoked to explain the promotion of the preferred phase [9].

One of the common structural features in biologically active peptides and proteins, such as polypeptide hormones, polypeptide antibiotics and venoms is an α-helical structure in which the aminoacidic sequence has both hydrophobic and hydrophilic character [15]. The arrangement of hydrophobic/hydrophilic residues in the sequence has been shown to modulate the cell-lytic properties of the α-helical transbilayer peptide [16], [17], [18]. For these reasons, studies on peptides/membrane association phenomena have been recently concentrated on the modifications of the physical parameters of the lipidic bilayer as a consequence of peptide incorporation. However, in spite of their potential importance, the effects of different methods of peptide/membrane incorporation on the thermotropic behaviour of the bilayer, are poorly investigated.

In this light, here we report the DSC measurements of a DPPC/peptide system where the peptide chosen as a model was a fragment (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with a scrambled hydrophobic/hydrophilic sequence. This peptide has been shown to possess a considerable propensity to form stable α-helices and therefore is a good candidate for this kind of studies [19]. The DSC results have been discussed in terms of different preparation protocols of the lipid/peptide systems, thus pointing out the not negligible effect of the sample preparation in avoiding artifacts in the interpretation of results.

Section snippets

Chemicals

The peptide (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) was synthesized on a Milligen 9050 peptide synthesizer as previously reported [19].

DPPC was obtained from FLUKA.

All inorganic salts for phosphate buffer preparation were purchased from Sigma Chemical co.

Preparation of pure DPPC multilamellar vesicles (MLV)

Pure membranes were prepared drying DPPC/CHCl3 solutions by evaporating them under high vacuum to dryness in round-bottomed flasks and by removing all residual solvent by a gentle nitrogen flow. The resulting lipid

Results and discussion

In the upper panel of Fig. 2, the Cpexc profile of pure (curve a) MLVs-DPPC and peptide/MLVs-DPPC (curve b) bilayer, prepared according to method A described in the experimental section, are reported. The corresponding thermodynamic parameters are reported in Table 1. It can be noted that the DSC peak relating to the peptide/DPPC system is broadened and shifted to lower temperatures with respect to the pure lipid curve. In particular, the Tm is lowered by about 1.5 °C and the enthalpy is

Conclusions

The primary structure has been shown to play an important role in cell-lytic and antimicrobial peptides that act by perturbing the barrier function of membranes [16], [17], [18], [23]. Depending on their hydrophobic/hydrophilic balance, the peptides either stabilize or lyse the membrane [17], [24]

Some authors have recently analyzed the relationship between the relative magnitude of the hydrophobic–hydrophilic moiety and membrane-binding properties [25], [26], [27]. It turned out that the

Acknowledgements

This work was partially supported by Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) (Grants: MM03194891 and 9903032282) and Università degli Studi di Catania. Thanks are due to Salvatore Petrantoni for his assistance in sample preparation.

References (27)

  • T. Heimburg

    Biochim. Biophys. Acta

    (1998)
  • J.A. Killian

    Biochim. Biophys. Acta

    (1998)
  • S. Morein et al.

    Biophys. J.

    (2000)
  • P.H. Axelsen et al.

    Biophys. J.

    (1995)
  • J.C. Huschilt et al.

    Biochim. Biophys. Acta

    (1989)
  • A. Kitamura et al.

    Biophys. J.

    (1999)
  • G. Saberwal et al.

    Biochim. Biophys. Acta

    (1994)
  • R.C. MacDonald et al.

    Biochim. Biophys. Acta

    (1991)
  • O.G. Mouritsen et al.

    Biophys. J.

    (1984)
  • A.J. Verkleij et al.

    Biochim. Biophys. Acta

    (1973)
  • R. Brasseur

    J. Biol. Chem.

    (1991)
  • R. Brasseur et al.

    Trends Biochem.

    (1997)
  • P. Yeagle, The Structure of Biological Membranes, CRC Press, Boca Raton, FL,...
  • Cited by (0)

    View full text