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Lucian Heeler Lucian joins the group as part of the KCL BiPAS CDT programme. In collaboration with Jeremy Carlton we will use new single-molecule tools and artificial membrane mimics to understand the mechanism of membrane fission by the Endosomal Sorting Complex Required for Transport (ESCRT).

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Guanzong Zhai Supported by the King’s-China Scholarship Council PhD Scholarship programme (K-CSC), Guanzhong Zhai joins the Wallace group working on on new methods for artificial cell fabrication.

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Zhongdao Li After a KURF summer studentship failed to dissuade Zhongdao from working in the lab he joins us for a PhD working on new methods to link artificial cells and real cells.

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Designer alpha-helical barrels.

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance.

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Our chapter in Methods 619. Single-channel recording from pore-forming toxins (PFTs) provides a clear and direct molecular readout of toxin action. However to complete any mechanistic understanding of PFT behavior, this functional kinetic readout must be linked to the underlying changes in toxin structure, binding, conformation, or stoichiometry. Here we review how single-molecule imaging methods might be used to further our understanding of PFTs, and provide detailed practical guidance on the use of droplet interface bilayers as a method capable of examining both single-molecule fluorescence and single-channel electrical signals from PFTs.

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Kharina Fenton Kharina joins the lab from Cardiff University after a placement in Australia studying 3D printable living electrodes. Her project will work to engineer a new class of artificial ion channels.

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Single-molecule FRET imaging of beta-barrel folding. In comparison to globular proteins, the spontaneous folding and insertion of β-barrel membrane proteins is surprisingly slow, typically occurring on the order of minutes. Using single-molecule Förster Resonance Energy Transfer to report on the folding of fluorescently-labelled Outer Membrane Protein G we measured the real-time insertion of a β-barrel membrane protein from an unfolded state. Folding events were rare, and fast (<20 ms); occurring immediately upon arrival at the membrane. This combination of infrequent, but rare, folding resolves this apparent dichotomy between slow ensemble kinetics, and the typical timescales of biomolecular folding.

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Tracking Perfingolysin O assembly and pore formation. We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway of the Cholesterol-Dependent Cytolysin, Perfringolysin O. This enables quantification of the stoichiometry of PFO complexes during assembly with millisecond temporal resolution and 20 nanometre spatial precision. Our results support a model of overall stepwise irreversible assembly, dominated by monomer addition, but with infrequent assembly from larger partial complexes. Furthermore, our results suggest a dominant proportion of inserted, but non-conductive intermediates in assembly.