Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane α-helical barrels with conductive channels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) low-conductance states that correlate with variations in the designed amino-acid sequences and modeled coiled-coil barrel geometries, indicating stable transmembrane α-helical barrels; and (ii) high-conductance states in which single channels change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic channels, as observed in natural barrel-stave peptide channels. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry.

Ion channels are ubiquitous in Nature, performing complex and essential tasks in our bodies. Synthetic chemists have begun to understand how to form artificial channels, which hold great promise as components in artificial cells, and in synthetic biology more widely. Future generations of these systems will be critical in the treatment of channelopathies; diseases where ion channels malfunction, and which are challenging to treat with current modalities. Despite advances in the design of synthetic ion transporters, the current generation cannot approach the selectivity and controllability of the biological ion channels they seek to emulate, and multimodal control over activity remains hard to achieve. Herein, we present a synthetic ion channel whose activity can be controlled by three orthogonal stimuli (light, pH, guest/ligand). The channel is formed from a pillar[5]arene functionalized with photoswitchable tetrafluoroazobenzene moieties. We demonstrate excellent control over E:Z switching across ten incorporated photoswitches (E to Z 87%, Z to E quant.). We show that the most active isomer – the Z-isomer – forms dimeric ion channels in membranes, with selectivity for M+/Cl− symport. Single molecule planar bilayer conductance studies show distinct high and low conductance states dependent on irradiation wavelength. Finally, we demonstrate that this activity can be modulated over 170-fold by controlling pH, irradiation, and guest addition, creating a powerful addition to the canon of synthetic ion channels.

Herein, we present a synthetic peptidomimetic, TBP2, which forms artificial membrane channels within droplet interface bilayers (DIBs). Real-time electrophysiology via TIRF microscopy, and single-channel recordings, we demonstrate the ability of TBP2 to mediate high-conductance transport of Na+ and K+ across the DIBs, highlighting its potential for nanobiotechnology applications in cell-free systems.

Living crystallization-driven self-assembly (CDSA) of semicrystalline block copolymers is a powerful method for the bottom-up construction of uniform polymer microstructures with complex hierarchies. Improving our ability to engineer such complex particles demands a better understanding of how to precisely control the self-assembly process. Here, we apply interferometric scattering (iSCAT) microscopy to observe the real-time growth of individual poly(ε-caprolactone)-based fibers and platelets. This label-free method enables us to map the role of key reaction parameters on platelet growth rate, size, and morphology. Furthermore, iSCAT provides a contrast mechanism for studying multi-annulus platelets formed via the sequential addition of different unimers, offering insights into the spatial distribution of polymer compositions within a single platelet.

Interferometric scattering (iSCAT) microscopy enables high-speed and label-free detection of individual molecules and small nanoparticles. Here we apply point spread function engineering to provide adaptive control of iSCAT images using spatial light modulation. With this approach, we demonstrate improved dynamic spatial filtering, real-time background subtraction, focus control, and signal modulation based on sample orientation.

Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane α-helical barrels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) a low-conductance states that correlate with variations in the modeled coiled-coil barrel geometries, indicating stable transmembrane α-helical barrels; and (ii) high-conductance states in which single pores change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic pores through the recruitment and expulsion of peptides, as observed in natural barrel-stave peptide pores. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry. ### Competing Interest Statement The authors have declared no competing interest.

Methods capable of controlling synthesis at the level of an individual nanoparticle are a key step towards improved reproducibility and scalability in engineering complex nanomaterials. To address this, we combine spatially-patterned activation of the photoreductant sodium pyruvate with interferometric scattering microscopy to achieve fast, label-free monitoring and control of hundreds of gold nanoparticles in real-time. Individual particle growth kinetics are well-described by two-step nucleation autocatalysis model, but with a distribution of individual rate constants that changes with reaction conditions.

Nanopore-based DNA sequencing offers high throughput and long reads by measuring blockades in current as a strand is translocated through the pore, providing a single-molecule readout with high time resolution. However, electrical measurements alone offer only information regarding the current block itself; to improve nanopore sensing, a molecular picture of the steps of analyte binding, capture and translocation is required. Optical single channel recording of calcium flux through the nanopore enables parallel single-molecule capture events to be distinguished.

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. While freely diffusing TOM-CC molecules are predominantly in a high permeability state, non-mobile molecules are mostly in an intermediate or low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus represent a β-barrel membrane protein complex to exhibit membrane state-dependent mechanosensitive properties, mediated by its two Tom22 subunits.

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore-formation of the archetypical cholesterol-dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 millisecond temporal resolution and 20 nanometre spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes.

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of β-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

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.

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. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

Combining single-molecule fluorescence measurements of ion channels with single-channel recordings of ionic current would allow for the construction of a molecular movie of the working of an ion channel, thereby directly linking structural changes to functional responses. Such measurements would provide an important bridge between high-resolution, but static, protein structures, and the rich kinetic information provided by patch-clamp recording. However, significant incompatibilities between the experimental conditions required to measure these two signals makes progress in this area challenging. Optical single-channel recording shifts the measurement of ionic current from an electrical to an optical readout, allowing parallel interrogation of multiple channels. This shift provides a straightforward route to combine single-molecule Förster Resonance Energy Transfer (smFRET) microscopy of channel conformational dynamics with optical single-channel recording (oSCR) of channel function. Here we report on our recent work to establish parallelized smFRET and oSCR recordings, using stochastic sensing of fluorescently labelled DNA in protein nanopores to provide well-controlled simultaneous events.

Diffusion in cell membranes is not just simple two-dimensional Brownian motion but typically depends on the timescale of the observation. The physical origins of this anomalous subdiffusion are unresolved, and model systems capable of quantitative and reproducible control of membrane diffusion have been recognized as a key experimental bottleneck. Here, we control anomalous diffusion using supported lipid bilayers containing lipids derivatized with polyethylene glycol (PEG) headgroups. Bilayers with specific excluded area fractions are formed by control of PEG lipid mole fraction. These bilayers exhibit a switch in diffusive behavior, becoming anomalous as bilayer continuity is disrupted. Using a combination of single-molecule fluorescence and interferometric imaging, we measure the anomalous behavior in this model over four orders of magnitude in time. Diffusion in these bilayers is well described by a power-law dependence of the mean-square displacement with observation time. Anomaleity in this system can be tailored by simply controlling the mole fraction of PEG lipid, producing bilayers with diffusion parameters similar to those observed for anomalous diffusion in biological membranes.

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.

Diffusion in biological membranes is seldom simply Brownian motion; instead, the rate of diffusion is dependent on the time scale of observation and so is often described as anomalous. In order to help better understand this phenomenon, model systems are needed where the anomalous diffusion of the lipid bilayer can be tuned and quantified. We recently demonstrated one such model by controlling the excluded area fraction in supported lipid bilayers (SLBs) through the incorporation of lipids derivatized with polyethylene glycol. Here, we extend this work, using urea to induce anomalous diffusion in SLBs. By tuning incubation time and urea concentration, we produce bilayers that exhibit anomalous behaviour on the same scale as that observed in biological membranes.

Characterizing the lateral organization of lipid membranes is important for understanding the function of cell membranes. The most common way to determine miscibility phase behavior is using fluorescence microscopy, where the presence of separate phases is imaged by the preferential partitioning of a labeled lipid. However, fluorescence microscopy requires adding an additional lipid component, and can also lead to photooxidation. Interference scattering microscopy (iSCAT) is a label-free imaging technique that images the differences in light scattering from different regions of a sample. iSCAT is capable of imaging coexisting domains. We quantify the sensitivity of iSCAT to physical parameters including thickness mismatch, topography, and the presence of defects.

G protein-coupled receptors (GPCRs) are the largest class of membrane receptors, playing a key role in the regulation of processes as varied as neurotransmission and immune response. Evidence for GPCR oligomerisation has been accumulating that challenges the idea that GPCRs function solely as monomeric receptors; however, GPCR oligomerisation remains controversial primarily due to the difficulties in comparing evidence from very different types of structural and dynamic data. Using a combination of single-molecule and ensemble FRET, double electron–electron resonance spectroscopy, and simulations, we show that dimerisation of the GPCR neurotensin receptor 1 is regulated by receptor density and is dynamically tuneable over the physiological range. We propose a “rolling dimer” interface model in which multiple dimer conformations co-exist and interconvert. These findings unite previous seemingly conflicting observations, provide a compelling mechanism for regulating receptor signalling, and act as a guide for future physiological studies.

Characterizing the lateral organization of lipid membranes is important for understanding the function of cell membranes. Several groups have reported submicron heterogeneity in model membranes, using techniques such as neutron scattering, x-ray scattering, FRET, or AFM. Here, we study these systems using iSCAT microscopy, a recently developed technique that images the differences in light scattering from different regions of a sample. This technique is capable of label-free detection of nanometer-scale lipid domains, and is compatible with fully hydrated, micron-scale membranes. This allows us to directly measure the membrane organization and dynamics, and to compare data from the same technique across widely different length scales.