STUDIES ON THE PORE FORMATION INDUCED BY THE ANTIMICROBIAL PEPTIDE Ctx(Ile21)-Ha BY ELECTRON PARAMAGNETIC RESONANCE: ORIENTATION, CONFORMATION AND OLIGOMERIZATION

• Grant number: 13/18211-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2013
• End date: June 30, 2014
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Antonio José da Costa Filho
• Grantee: Eduardo Festozo Vicente
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

During the past decades, a great number of antimicrobial peptides (AMPs) have been identified from several species of vertebrates and invertebrates, such as magainin, aurein, cecropin, ceratotoxin, among others. Furthermore, several antimicrobial and hemolytic peptides have also been isolated particularly from frog species. Due to the property of permeabilizing and destroying bacterial membranes, thus leading the pathogen to death, these peptides are an interesting target for the development of new antibiotics. In particular, the antimicrobial peptide Ctx(Ile21)-Ha, extracted and isolated from the skin secretion of an arboreal South American frog, Hypsiboas albopunctatus, has been studied and demonstrated to be a very promising molecule for this objective due to its biological activities against fungi and bacteria and a moderate toxicity against human cells. In this context, this project aims to perform a thorough investigation of the peptide structure, conformation and topology, since this information is still not known and can tremendously contribute to unravelling basic aspects related to oligomerization during interaction with the membrane and pore formation. A strategy to address such issues consists of attaching spin probes to the peptide backbone, allowing the evaluation of the relationship between conformational properties and biological activity. Thus, by the insertion of spin labels such as TOAC and MTSSL in the peptide backbone and using the techniques of continuous wave EPR, power saturation, pulsed EPR (DEER) and complementary studies, such as circular dichroism, we intend to produce important data about the structure and function of the peptide when interacting with membranes as well as data on the pore formation mechanisms by measuring the distances between the paramagnetic centers. This crucial information to improve activity modulation and to achieve a better understanding of the mode of action of antimicrobial peptides. Nowadays, in the pharmaceutical market, antibiotics based on peptides as active ingredient are not yet available and our results have the potential to contribute to that. Hence, this project aims, by aggregating the knowledge at a molecular level of the peptide/membrane interactions, at producing relevant information needed for the construction of new active molecules that can be used as an alternative therapy to the conventional methods.