Trichogin topology and activity in model membranes as determined by fluorescence spectroscopy.

Antibiotic peptides, exhibiting activity against different types of microorganisms, are part of the innate immune system of most organisms. Their mechanism of action is basically that of altering the permeability of cell membranes, bringing about the cell death by collapse of transmembrane electrochemical gradients and osmolysis, though, despite the large number of studies, the molecular details of the mechanism are still uncertain. In view of the fact that bioactivity relies on both peptide affinity for membranes and ability to self-associate, it is easily understandable why cationic and hydrophobic peptides behave differently. Cationic peptides can bind to the charged surface of membranes, but their insertion in the hydrophobic core of the phospholipid bilayer or their aggregation is hindered by electrostatic effects. Therefore, their activity is best described by the Shai-Matsuzaki-Huang model, also known as the “carpet” or “toroidal pore” model, in which peptides bind to the membrane surface in a carpet-like fashion, by insertion into the polar headgroups region only. As a result, an unfavorable elastic tension arises, leading to the formation of transient defects or pores. On the other hand, because hydrophobic peptides include in their sequence only a few or no charged amino acids, they tend to bring together, so that their mechanism of action is best described by the so called “barrel stave” model, in which several peptide chains assemble in a transmembrane orientation, forming well defined channels. These two classes of peptides also differ in activity, in the sense that anionic lipids, such as those in bacteria, but not the zwitterionic bilayers, present in mammal or fungal cells, favor the binding of cationic peptides only. Therefore, these latter peptides are more selective than the hydrophobic ones. A similar difference holds in aqueous solution, in that the entropy-driven hydrophobic interactions definitely favor gathering, while the opposite occurs for cationic peptides, owing to electrostatic effects. Lipopeptaibols are members of a unique family of membrane active peptides, comprising a linear sequence of 6-10 aminoacids, a large amount of the helix-promoting Aib (α-aminoisobutyric acid), a N-terminal fatty acyl group and a 1,2-amino alcohol at the C-terminus. One of the main components of this family is Trichogin GA IV (TR), a natural peptide having the following primary structure, where Oct is n-octanoyl, and Lol leucinol. Oct-Aib1-Gly2-Leu3-Aib4-Gly5-Gly6-Leu7-Aib8-Gly9-Ile10-Lol (TR) This peptide can modify membrane permeability, displaying both antibacterial and hemolytic effects. However, the full details of bioactivity are still unknown, and different models, including bilayer destabilization,3 channel formation or diffusion through the membrane as ion carrier,11 have been put forward. We report here the results of a thorough investigation on the behavior of this peptide in both aqueous solution and model membranes. Because peptide-membrane interactions are best studied by fluorescence experiments, two fluorescent trichogin analogs, both having a leucine methyl ester at the C-terminus, replacing Lol, were employed. One, denoted A3, is labeled with azulene [Aal: β-(1-azulenyl)-L-alanine], replacing Leu 3, and the other, labeled with a fluorene moiety (Fmoc: fluorenyl-9-methylcarbonyl group) linked to the side-chain of 2 -diamino-L-butyric acid (Dab), replacing Ile10, is denoted F10. The primary structure of the analogs is as follows, where TR-OMe is the reference peptide, and Boc a protective group (t-butyloxycarbonyl). Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe (TR-OMe) Oct-Aib-Gly-Aal-Aib-Gly-Gly-Leu-Aib-Gly-Dab(Boc)-Leu-OMe (A3) Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Dab(Fmoc)-Leu-OMe (F10) The fluorophores, chosen because they can act as a donor-acceptor pair in Förster energy transfer,12 were incorporated in such positions as to substitute