Advanced biophysical approaches to interrogate the structure and function of membrane proteins and antimicrobial peptides in droplet interface bilayers

Baird, Hannah 2025. Advanced biophysical approaches to interrogate the structure and function of membrane proteins and antimicrobial peptides in droplet interface bilayers. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Membranes are fundamental structures in cells, allowing compartmentalisation of cellular contents, regulating entry and exit of substances, and acting as hubs for chemical and electrical signalling. Understanding membranes is fundamental in identifying targets for drug development, optimising the passage of substances across this barrier, and identifying how to target and disrupt it. Artificial membrane models provide a simplified system for probing membrane structure, function, and interactions with substances, affording insights not possible in complex biological systems. Droplet interface bilayers (DIBs), which form when monolayers arising from two aqueous components incubated in a lipid-oil environment are brought together to form a bilayer, have been adopted widely as artificial membrane models for the study of molecular transport across membranes, creation of artificial cellular systems, measurements of lipid bilayer properties, and characterisation of membrane-interacting compounds. This work explored peptides, Magainin 2 (Mag2) and Peptidyl Glycine Leucine carboxyamide (PGLa), and membrane proteins, human ryanodine receptor (hRyR2) and bacterial pesticidal proteins (BPPs), and in DIBs. Assessing the ion flux through the pores formed by these compounds with physical techniques, electrophysiology and Ca2+ flux imaging with Total Internal Reflection Fluorescence (TIFM) microscopy, insight into their function was gained. Furthermore, single molecule (SM) fluorescence imaging and Förster Resonance Energy Transfer (FRET) provided additional observations into the dynamics of and peptide-peptide interactions between Mag2 and PGLa, providing some additional evidence of the mechanism behind the synergistic antimicrobial effect known to exist between peptides. Electrophysiology and Ca2+ flux imaging found that the ion flux through Mag2, PGLa, and Mag2-PGLa pores varied. The dynamics and kinetics of the Mag2-PGLa interaction was assessed by SM FRET, which revealed the presence of homo- and heterodimers, and evaluated their association with peptide pores. Imaging of mutated hRyR2 suggested a change in clustering as the cause of some cardiomyopathies. The activity of BPPs were assessed, and the results of mutational iii studies showed that the mechanism of interaction between a BPP and the membrane may be via a folded coil rather than a straight coil, as previously assumed. The work presented in this thesis demonstrates the value of Ca2+ flux imaging and SM FRET in DIBs to quantify membrane protein function and assess dynamics in real-time, providing crucial spatiotemporal information about membrane proteins and peptides that is typically inaccessible through ensemble-averaged techniques at short experimental timescales.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Pharmacy
Subjects:	Q Science > Q Science (General)
Funders:	EPSRC
Date of First Compliant Deposit:	14 May 2026
Last Modified:	14 May 2026 11:34
URI:	https://orca.cardiff.ac.uk/id/eprint/186998

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