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
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.
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