Investigation into the structure function relationship of the membrane interaction of amphiphilic alpha helical antimicrobial peptides

Dennison, Sarah Rachel orcid iconORCID: 0000-0003-4863-9607 (2004) Investigation into the structure function relationship of the membrane interaction of amphiphilic alpha helical antimicrobial peptides. Doctoral thesis, University of Central Lancashire.

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Many eukaryotic organisms produce membrane interactive, a-helical antimicrobial peptides (a-AMPs) and a database of such peptides, together with selected physiochemical parameters was established. This database was divided into four
groups according to the a-AMPs target organism(s) (active against Gram-positive bacteria {G+}; active against Gram-negative bacteria {G-}; active against Grampositive and Gram-negative bacteria {G+, G-}; or active against Gram-positive
bacteria, Gram-negative bacteria and fungi {G+, G-, F}). Analysis of the database showed that there was no statistically significant correlation between specificity and p1 (range 4.2 to 12.7) or net charge (range -5 to +16). The peptides exhibited variable hydrophobicity, <H> (range -0.8 to +0.7) whilst amphiphilicity (measured by the hydrophobic moment, <jAH>) ranged from 0.2 to 1.1. A statistically significant negative correlation between <pH> and <H> was noted for each group of a-AMPs and this may relate to the amphiphilic balance required for antimicrobial activity, a-AMPs showed some differences in amino acid composition compared to the McCaldon and Argos dataset of unrelated oligopeptides, suggesting functional relevance of some amino acid residues. The groups {G+, G-} and {G+, G-, F}, for example are characterised by being rich in weakly hydrophobic or hydrophilic amino acids.
Minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) can provide a measure of AMP potency. A statistical analysis of MIC's for peptides from {G+, G-} group, showed no significant differences in the potency of these
peptides when directed against either Gram-positive or Gram-negative bacteria. In contrast, a statistical analysis of MIC's for peptides from the {G+, 0-, F} group showed that peptides from this group were effective at lower concentrations against bacterial targets as compared to fungal targets. Increases in hydrophobic arc size were generally accompanied by increases in peptide antimicrobial potency and in addition, a negative correlation between MIC and net charge was observed.
Regression analyses indicated that an appropriate amphiphilicity/hydrophobicity balance was required for the antimicrobial action of a-AMPs and this may indicate a general structure/function relationship underlying both the efficacy and specificity of these peptides. Oblique orientated a-helices are highly specialised protein structural elements that penetrate membranes at a shallow angle and are used to promote membrane destabilisation by a number of protein classes. Here, the use of extended <pH> methodology showed that over 50% of the a-AMPs are candidate oblique ahelices providing some insight into possible modes of action.
Peptides VP I and BYDV-MP were identified here as candidate AMPs based on amino acid composition and the potential to form oblique orientation. The biological activity of VPI and BYDV-MP was confirmed in vivo when an MLC of 3 mM was
demonstrated on both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Monolayer studies using lipid extract from these target organisms and parallel studies using mimetic monolayers confirmed a high level of peptide membrane interaction. The single lipid monolayer results suggested that VPI and BYDV-MP have a lower affinity for zwitterionic lipid (DMPE surface pressure increase of 4 mNm') but a high affinity for anionic lipid (DMPS surface pressure increase of 7 to 9 mN m') and may have a requirement for this specific lipid or anionic lipids in general to achieve higher levels of membrane penetration.
To test the ability of VP! to penetrate membranes protonated and deuterated homologues were analysed by neutron diffraction, in the presence of POPC: POPS (10:1 molar ratio). The data analysed from these studies showed the protonated
homologue to penetrate the membrane core but the deuterated homologue showed no significant levels of membrane interaction. Monolayer studies confirmed that the protonated homologue interacted strongly with anionic and zwitterionic membranes (surface pressure increase 4 mNm'), however, the deuterated homologue did not have the ability to interact with POPC:POPS monolayers. FTIR conformational analysis showed the protonated homologue to adopt high levels of a-helical stmcture (65 %) and in contrast the deuterated homologue exhibited low levels of a-helical structure (C 20 %). These results support the original predictions and also appear to show that deuteration has directly or indirectly, affected the ability of the VP! peptide to interact with membranes, possibly by inhibiting a-helix formation by the peptide or decreasing structural stability.

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