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.
Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers
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.
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.
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.
Yujie Guo Yujie joined the lab in 2019 as a PhD student as part of the King’s-China Scholarship Council PhD Scholarship programme. Her project uses iSCAT microscopy to understand polymerisation.
Artificial Signal Transduction across Membranes. A key conundrum in the construction of an artificial cell is to simultaneously maintain a robust physical barrier to the external environment, while also providing efficient exchange of information across this barrier. Biomimicry provides a number of avenues by which such requirements might be met. Herein, we provide a brief introduction to the challenges facing this field and explore progress to date.
Modifying membrane morphology and interactions with DNA origami clathrin-mimic networks
We designed a three-arm DNA origami nanostructure whose shape resembles that of the clathrin triskelion. We describe the triggered assembly of a bioinspired DNA origami meshwork on a lipid membrane. DNA triskelia, three-armed DNA origami nanostructures inspired by the membrane-modifying protein clathrin, are bound to lipid mono- and bilayers using cholesterol anchors. Polymerization of triskelia, triggered by the addition of DNA staples, links triskelion arms to form a mesh. Using transmission electron microscopy, we observe nanoscale local deformation of a lipid monolayer induced by triskelion polymerization that is reminiscent of the formation of clathrin-coated pits. We also show that the polymerization of triskelia bound to lipid bilayers modifies interactions between them, inhibiting the formation of a synapse between giant unilamellar vesicles and a supported lipid bilayer.
Daisy Rogers-Simmonds Daisy joins us through the Institute of Chemical Biology CDT programme. She works between the Wallace Group and the Membrane Biophysics Group at Imperial College in collaboration with Oxford Nanopore Technologies. Her research interests lie in the development and study of droplet interface bilayers using block copolymers, and the subsequent incorporation of nanopores into these systems. Prior to joining the group, Daisy obtained a BSc (Hons) in Chemistry from Imperial College.
Daisy has a keen interest in and passion for public outreach and science communication, and recently began voluntary work as a STEM Ambassador alongside her studies.
Electroporation-Based Technologies and Treatments
Mark presented work on electroporation imaging at the 2018 EBTT workshop in Ljubljana organised by Damijan Miklavčič and Lluis M. Mir. This international workshop is intended both for novices and experts in electroporation, including PhD students, researchers and users.