AbstractThe amphibian antimicrobial peptide pseudin-2 is a peptide derived from the skin of the South-American frog Pseudis paradoxa (Olson et al., 2001). This peptide possesses tremendous potential as a therapeutic lead since it has been shown to possess both antimicrobial as well insulin-releasing properties (Olson et al., 2001; Abdel-Wahab et al., 2008). This study aimed to develop pseudin-2’s potential by understanding and improving its properties as an antimicrobial agent. The structure-function relationships of pseudin-2 were explored using a combination of in-vitro and in-silico techniques, with an aim to predict how the structure of the peptide may be altered in order to improve its efficacy. A library of pseudin-2 mutants was generated by randomizing codons at positions 10, 14 and 18 of a synthetic gene, using NNK saturation mutagenesis. Analysis of these novel peptides broadly confirmed, in line with literature precedent, that anti-microbial activity increases with increased positive charge. Specifically, 2 positively-charged residues at positions 10 and 14 and a hydrophobic at position 18 are preferred. However, substitution at position 14 with some polar, non-charged residues also created peptides with antimicrobial activity. Interestingly, the pseudin-2 analogue [10-E, 14-Q, 18-L] which is identical to pseudin-2, except that the residues at positions 10 and 14 are switched, showed no anti-microbial activity at all.
Molecular dynamics simulations of pseudin-2 showed that the peptide possesses two equilibrium structures in a membrane environment: a linear and a kinked a-helix which both embed into the membrane at an angle. Biophysical characterization using circular dichroism spectroscopy confirmed that the peptide is helical within the membrane environment whilst linear dichroism established that the peptide has no defined orientation within the membrane. Collectively, these data indicate that Pseudin-2 exerts its antimicrobial activity via the carpet model.
|Date of Award||16 Dec 2013|
|Supervisor||Anna V. Hine (Supervisor)|