The Role of Peptide Self Association in Solution andor in Membranes

Self-association of AMPs is an important parameter which affects their selectivity toward different cells. Self-association is driven either by the peptidic chain, or by the attachment of extrinsic hydrophobic groups such as fatty acids, resulting in the formation of a-helical bundles that could initiate strong hydrophobic binding to zwitterionic membranes (Oren et al. 1999; Strahilevitz et al. 1994; Ghosh et al. 1997). The role of self-association of AMPs in their selective binding to target cells is elaborated in the following paragraphs.

(1) AMP self-association driven by amino-acid interaction. The effect of peptide self-association on the function of AMPs has been demonstrated in several studied.

(a) Dermaseptin and its native mutants, isolated from the skin of the Phyl-lomedusa frog (Mor et al. 1994), were shown to have different cell specificities. Mode-of-action studies demonstrated that those which are non-cell-selective oligomerize in the membrane and bind and permeate both zwitterionic and negatively charged membranes (Strahilevitz et al. 1994; Kustanovich et al. 2002). Assembly was driven either by the hydrophobic N-terminal of one mutant or by the additional negative charge (Asp at the fifth position) in another mutant. In another study, dermaseptin S3 (DS3) was shown to be toxic only toward the intraerythrocytic parasite, while another mutant, dermaseptin S4 (DS4), was toxic to both the parasite and the host erythrocyte (Ghosh et al. 1997). Studies with fluorescentlylabeled peptides revealed that DS4 forms larger aggregates in aqueous solution compared with DS3.

(b) The role of peptide oligomerization in selective lytic activity was addressed also with the human cecropin-like LL-37, which is cytotoxic to both bacteria and normal eukaryotic cells. In contrast, its N-terminal truncated form, FF-33, preserved the antimicrobial activity of the parental LL-37 but was devoid of hemolytic activity. Using fluorescently labeled peptides, it was found that LL-37, but not FF-33 exists in equilibrium between monomers and oligomers in solution atverylowconcentrations (Oren et al. 1999).

(c) A series of amphipatic all-L-amino acid peptides and their diastereo-mers were de novo synthesized. The template for the sequence was KX3KWX2KX2K where X = Gly, Ala, Val, He, or Leu (Avrahami et al. 2001). The data revealed that most of the L-amino acid peptides oligomerized and adopted distinct structures (random coil, a-helix or p sheet) in solution and in a membrane mimetic environment. Among this group, only the Leu-containing peptide was active on both bacteria and human erythrocytes, while its diastereomer, which did not oligomerize in solution, was active only on bacteria. The large size of these oligomers probably prevented them from penetration into the bacterial phospholipid membrane, and therefore, their all L-amino acid parental peptides were practically inactive (Avrahami et al. 2001).

(2) AMP self-association driven by the conjugation of fatty acids. The attachment of fatty acids has been shown to control hydrophobicity and self-assembly of monomeric AMPs, without altering the properties of the peptidic chain. In a first study magainin was used (Avrahami and Shai 2002). When aliphatic acids with different lengths were attached to its N-terminal they affected its organization in solution. The attachment of heptanoic, undecanoic, and palmitic acids resulted in lipopeptides with three distinct structures and oligomeric states in solution, at their minimal inhibitory concentration (MIC): (a) the attachment of heptanoic acid resulted in a monomeric, unordered structure; (b) the attachment of undecanoic acid yielded concentration-dependent oligomers of a-helices; and (c) the attachment of palmitic acid yielded concentration-independent a-helical monomers, a novel lipopeptide structure, which is resistant to proteolytic digestion. A cartoon illustrating possible organizations of these three lipopeptides is shown in Fig. 7.2. As expected, the increase in hydrophobicity and the oligomeric state of magainin analogs increased its activity toward mammalian cells. Similar results were obtained with fatty acid-conjugated de-novo synthesized 12-mer diastereomeric AMPs (Avrahami and Shai 2003).

In another study conjugation of fatty acids to non-membrane active peptides endowed them with antimicrobial activities (Avrahami and Shai 2003, 2004). A new group of lipopeptides with potent antifungal or both antibacterial and antifungal activities was developed by conjugation of palmitic acid to short positively charged peptides, which are devoid of biological activity. The parental peptides also did not have the threshold hydrophobicity required for

Antibacterial Peptide Membrane

Fig. 7.2. A cartoon illustrating possible organizations of three lipophilic magainin analogs in solution. Thick lines and helices represent the peptides and thin lines represent the fatty acid moieties (modified from Avrahami and Shai 2002)

C7-magainin C11-magainin C16-magainin

Fig. 7.2. A cartoon illustrating possible organizations of three lipophilic magainin analogs in solution. Thick lines and helices represent the peptides and thin lines represent the fatty acid moieties (modified from Avrahami and Shai 2002)

membrane binding and permeation. Specifically, a group of diastereomeric peptides with a general sequence K4X7W (X designates one of the following aliphatic amino acids: Gly, Ala, Val, or Leu) were palmitoylated at their N-terminus. Most importantly, palmitoylated K4G7W and K4A7W gained potent antibacterial and antifungal activity with low hemolytic activity, despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, palmitoylated K4L7W lost the potent antibacterial activity of the parental peptide but preserved antifungal activity albeit with different selectivities. Interestingly, both K4V7W and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptides was highly potent toward yeast. Both the Leu and Val derived lipopeptides were also endowed with hemolytic activity. Mode-of-action studies suggested that this group of lipopeptides act by increasing the permeability of the cell membrane, and that differences in their potencies and target specificities are the result of differences in their oligo-meric state and ability to dissociate and insert through the cell wall into the cytoplasmic membrane.

(3) Cyclization oflinear lytic peptides decreases assembly and increases selectivity towards bacteria. Cyclization oflinear amphipatic a-helical peptides reduced the extent of their a-helical structure, and as a consequence reduced their oli-gomeric state, which in turn affected their biological function. Examples include the following. Cyclic forms of magainin 2 and melittin were synthesized by incorporation of cysteins at both the N- and C-termini of the peptides (Av-rahami and Shai 2002). Cyclization of magainin markedly reduced its cytolytic activity toward both erythrocytes and bacteria. In contrast with magainin, cyclization of melittin analog significantly reduced its hemolytic activity but preserved or increased activity toward bacteria. The reduction in hemolytic activity of both peptides upon cyclization was correlated with a reduction in their binding and the ability to increase the permeability of PC/cholesterol membranes, the major component of the outer leaflet of red blood cells. Most importantly, at similar molar ratios of bound peptide:lipid, both linear and cyclic magainin analogs showed similar membrane permeation activity with both zwitterionic and negatively charged phospholipid membranes, indicating that the linearity of this peptides is not required for membrane binding and permeation. However, the finding that cyclic magainin is much less active than linear magainin in the killing of bacteria, points to the role of linearity in reaching the bacterial inner phospholipid membrane.

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