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Title: Biophysical investigations into membrane-active peptides and proteins
Author: Yin, Daniel
ISNI:       0000 0004 6497 4429
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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The inexorable spread of antibiotic or antimicrobial resistance is a global problem, described by the UK Chief Medical Officer to be "as big a threat as terrorism". Due to uncontrolled, unnecessary overuse of antibiotics in medicine and agriculture, bacterial resistance has evolved to even the antibiotics of last resort. Antimicrobial peptides (AMPs) are a promising class of organic molecule that have been proposed to exert a potent antimicrobial effect, which, directly or indirectly, involve complex interactions with cell membranes. Three broad mechanisms have been proposed for AMPs: carpet, barrel-stave and toroidal pore. However, the molecular basis for the mode of action of AMPs, and the relationship between primary structure and antimicrobial activity, remains poorly understood. In this thesis, interactions of membrane-active peptides and proteins with model lipid membranes are studied, to understand better the peptide-lipid interactions of two de novo AMPs and a functionally related protein puroindoline-b (pinB), which is implicated in antimicrobial plant defence. Quartz crystal microbalance (QCM), solid-state nuclear magnetic resonance (ssNMR), electron paramagnetic resonance (EPR) and neutron reflectivity (NR) are used to achieve this. The two AMPs were designed rationally with their primary structure predicted to display specific peptide-lipid interactions. Tilamin (tilted antimicrobial insert) was designed by modifying amhelin (antimicrobial insert), a pore-forming AMP. The modified peptide was predicted to disrupt model membranes mimicking bacterial membranes via a different mode of action to transmembrane barrel stave pore formation. Chom (chopped cecropin mutant) was designed by shortening the length of a natural AMP, cecropin-B, and was predicted to operate via a carpet mechanism. To model the biophysical properties such as morphology, thickness and charge of native membranes, simplified phospholipid liposomes were used to better understand the membrane-perturbing influence of the AMPs, and whether this was correlated with antimicrobial activity. In the presence of anionic model membranes (mimicking Gram-negative inner membranes and Gram-positive membranes), tilamin and chom adopt amphipathic alpha helix conformations as determined by circular dichroism, while remaining unstructured in solution and in the presence of zwitterionic model membranes mimicking mammalian model membranes. Adoption of a folded conformation appears to be important for the lytic effect of the AMPs. Calcein leakage experiments performed show that the AMPs induce leakage of calcein from the interior of anionic liposomes, consistent with the proposal that membrane permeabilisation is important for antimicrobial activity. The peptide-lipid interactions of the AMPs were then probed using QCM and ssNMR, giving mechanistic evidence that chom operates via the carpet mechanism as predicted. The nature of the mode of action of tilamin remained uncertain. From order parameters of lipids in bilayers, obtained using ssNMR upon interaction with tilamin, a toroidal pore mechanism was proposed, along with a new mode of action that caused monoleaflet poration, though it was not possible to resolve the two mechanism based on data obtained from symmetrical vesicles alone. Adapting a newly established protocol to control the leaflet distribution of lipids in model membranes, an asymmetrically distributed nitroxide probe reveals for the first time leaflet-specific peptide-lipid interactions using cw-EPR. Tilamin shows changes in bilayer lipid order parameters that do not match those seen for either an all-surface or transmembrane control peptide, indicating more complex interactions. Unique QCM data, heterogeneous changes in order parameter profiles observed with acylchain 2H ssNMR, as well as a lack of interaction with the inner leaflets of anionic model membranes seen by cw-EPR taken in combination suggest tilamin operates via a more complicated mechanism. Supported by tilt angles obtained by geometric analysis of labelled alanines (GALA) of deuterium-labelled tilamin and atomic force microscopy (AFM) imaging performed with collaborators, the results are consistent with a new mechanism; monolayer poration. The puroindolines are also studied, due to their potential role as antimicrobial proteins in food safety, and in controlling wheat endosperm texture. The mode of insertion of wild-type puroindoline-B (pinB+) and a single-point mutant (pinBs) into bacterial model bilayers was probed for the first time with ssNMR, EPR and NR. In contrast to previous work on monolayers, pinBs does not cause changes in bilayer lipid order in the gel phase, while pinB+ forms a protein layer on the surface of a membrane. The results suggest that in more native-like model membranes, the tryptophan-rich domain (TRD) of pinBs and pinB+ greatly affects the membrane binding properties, with implications for the role of the proteins in vivo.
Supervisor: Judge, Peter ; Watts, Anthony ; Ryadnov, Max Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available