The amphipathic helix concept: length effects on ideally amphipathic LiKj(i=2j) peptides to acquire optimal hemolytic activity.

In a minimalist approach to modeling lytic toxins, amphipathic peptides of LiKj with i=2j composition and whose length varies from 5 to 22 residues were studied for their ability to induce hemolysis and lipid vesicle leakage. Their sequences were designed to generate ideally amphipathic alpha helices with a single K residue per putative turn. All the peptides were lytic, their activities varying by more than a factor of 103 from the shortest 5-residue-long peptide (5-mer) to the longest 22-mer. However, there was no monotonous increase versus length. The 15-mer was as active as the 22-mer and even more than melittin which is used as standard. Partition coefficients from the buffer to the membrane increased in relation to length up to 12 residues, then weakly decreased to reach a plateau, while they were expected to increase monotonously with peptide length and hydrophobicity as revealed from HPLC retention times. Fluorescence labeling by a dansyl group at the N-terminus, or by a W near the CO-terminus, show that up to 12 residues, the peptides were essentially monomeric while longer peptides strongly aggregated in the solution. Lipid affinity was then controlled by peptide length and was found to be limited by folding and self-association in buffer. The lytic activity resulted both from lipid affinity, which varied by a factor of 20-fold, and from efficiency in disturbing the membrane when bound, the latter steeply and monotonously increasing with length. The 15-residue-long peptide, KLLKLLLKLLLKLLK, had the optimal size for highest lytic activity. The shallow location of the fluorescent labels in the lipids is further evidence for a model of peptides remaining flat at the interface.

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.

The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin.

An original series of 12- to 22-residue-long peptides was developed, they are only constituted by apolar Leu and charged Lys residues periodically located in the sequence in order to general ideal highly amphipathic alpha-helices. By circular dichroism, the peptides are proven to be mainly alpha-helical in organic and aqueous solvents and in the presence of lipids. The peptides are highly hemolytic, their activity varies according to the peptide length. The 15-, 20-, and 22-residue-long-peptides have LD50 approximately 5 x 10(-8) M for 10(7) erythrocytes, i.e. they are 5-10 times more active than melittin, and are indeed several orders of magnitude more active than magainin or mastoparan.