Biophysical investigation of biomolecules in bio-membrane models

Abstract

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Therefore there remains an urgent need for a new generation of antifungal agents. The present work concerns the synthesis, the antifungal activity and the biophysical characterization of a set of linear and cyclic peptides (AMT1, cyclo-AMT1, AMT2, cyclo-AMT2, AMT3, cyclo-AMT3) including aminoacids characteristic of membrane-active antimicrobial peptides (AMP). The peptides were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans. To shed light on the role played by the membrane cell in the antifungal activity an extensive biophysical study was carried out using different spectroscopic techniques. Our structural investigation provides data to exclude the ability of the peptides to penetrate the membrane of the fungal cell, highlighting their attitude to interact with the external surface of the bilayer. Taken together our data support the hypothesis that the membrane cell of the fungi may be an important platform for specific interactions of the synthesized peptides with more specific targets involved in the cell wall synthesis. Viral fusion glycoproteins present a membrane-proximal external region (MPER) which is usually rich in aromatic residues and presents a marked tendency to stably reside at the membrane interfaces, leading, through unknown mechanisms, to a destabilization of the bilayer structure. This step has been proposed to be fundamental for the fusion process between target membrane and viral envelope. In present work, we investigate the interaction between an octapeptide (C8) deriving from the MPER domain of gp36 of Feline Immunodeficiency Virus and different membrane models by combining experimental results obtained by Nuclear Magnetic Resonance, Electron Spin Resonance, Circular Dichroism and Fluorescence Spectroscopy with Molecular Dynamics simulations. Our data indicate that C8 binds to the lipid bilayer adsorbing onto the membrane surface without deep penetration. As a consequence of this interaction, the bilayer thickness decreases. The association of the peptide with the lipid membrane is driven by hydrogen bonds as well as hydrophobic interactions that the Trp side chains form with the lipid headgroups. Notably these interactions may be the key to interpret at molecular level the function played by Trp residues in all the fragments of viral envelope involved in fusion mechanism with target membrane. [edited by author]