Analytical studies on channel forming proteins

Abstract

Abstract. Antimicrobial peptides have shown great potential as pharmaceutical agents, they are being considered for their ability to fight bacterial and fungal infections and even to destroy cancerous cells by disrupting the cell membranes of their targets. A clear understanding of the mechanism behind their activity and how this is related to their structure is therefore essential if these peptides are to be considered as precursors for the next generation of a new range of drags. This research project has been involved in the development of a series of analogous model amphiphilic cyclic peptides, which were designed to adopt a ß- sheet conformation on inserting into lipid membranes. The model peptides were examined using a wide range of analytical techniques; these studies have enabled both the propensity of these peptides to adopt transmembrane ß sheet structures to be established and to gain some understanding of their behaviour under different environments. Circular dichroism and its sister technique linear dichroism have shown both the structure and the orientation of the peptide backbone on insertion into lipid membranes. Calorimetrie studies have demonstrated the extent of the cyclic peptide disruption on the phase transition of saturated and unsaturated phospholipid membranes and electron microscopy has revealed the ability of one of the model peptides to form fibrous structures on precipitation from a solution of the fluorinated alcohol, hexafluoroisopropanol (HFIP) in water. A natural ß -sheet forming protein, the C-terminal domain of the autotransporter protein BrKA from Bordetella pertussis was overexpressed to provide a comparison for the model )ff-sheet peptides. Attempts were made to grow highly ordered 2D arrays of the protein in phospholipid membranes for structural analysis by both transmission electron microscopy and atomic force microscopy. The effect of the fluorinated alcohol HFIP, which was used to solubilise the amphiphilic peptides, was examined on phospholipid systems. The study was considered to afford some understanding towards the integration of small molecules into membranes.