School of Chemistry - Theses
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ItemMembrane interactions of the Alzheimer’s Aβ42 peptide and pore-forming protein equinatoxin II in atomistic detail( 2015)Cytolytic properties of membrane active peptides and proteins (MAPS), in particular the 42-residue isoform of the amyloid beta peptide (Aβ42) from Alzheimer’s disease and the potent cytolysin Equinatoxin II (EqtII) from the sea anemone Actinia equina, are fundamentally dependent on their interactions with membrane interfaces. Atomsitic details of these interactions, however, cannot be identified by current experimental approaches, but may be infrerred from additional molecular dynamics (MD) simulation methods. In Chapter 2, high-resolution NMR of Aβ42 is enabled by development of a high-yielding biosynthetic protocol for producing uniformly 15N and 13C15N labelled material in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods of spectral acquisition by solution state NMR, and further characterised by thioflavin T assays, circular dichroism, electrospray mass spectrometry and dynamic light scattering. Chapter 3 details solid-state NMR investigations, complemented by thioflavin T assays and circular dichrosim, to characterise perturbations to model phospholipid membranes comprising cholesterol, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, ganglioside GM1 or brain total lipid extract. Perturbations to headgroup and tail regions of model bilayers were modulated by lipid composition and by addition of Cu2+. The theory and applicability of MD simulation for studying protein-lipid interactions are discussed Chapter 4, in addition to analytical code developed to detect highly-specific cation-π complexes, hydrogen bonds and hydrophobic interactions. While MD applications for studying Aβ42-membrane interactions remain limited due to availability of data, structural and mechanistics aspects of EqtII are described by experimental research. In Chapter 5, the phosphocholine binding site of EqtII was mapped by solution NMR using uniformly 15N-labelled EqtII with dodecylphosphocholine (DPC). Subsequent docking of EqtII onto a DPC micelle, followed by all-atom MD simulation, identified several lipid-binding pockets stabilized by cation-π, hydrogen-bonding and hydrophobic interactions. Additional simulation with an N-acetyl sphingomyelin (SM) micelle suggested well-documented SM specificity might occur via hydrogen bonding to unique 3-OH and 2-NH functional groups by several conserved residues at the POC binding site and proximate to the hinge of the N-terminal helix that detaches upon pore formation. By combining NMR and MD results, atomistic details of membrane active peptides and proteins can be revealed.