Real-Time Monitoring and Control of Nanoparticle Formation.

Methods capable of controlling synthesis at the level of an individual nanoparticle are a key step toward improved reproducibility and scalability in engineering complex nanomaterials. To address this, we combine the 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 a two-step nucleation-autocatalysis model but with a distribution of individual rate constants that change with reaction conditions.

Single-molecule tracking of perfringolysin O assembly and membrane insertion uncoupling.

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 ms temporal resolution and 20 nm 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.

Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity.

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 ß-barrel pores of TOM-CC. TOM-CC could thus represent a ß-barrel membrane protein complex to exhibit membrane state-dependent mechanosensitive properties, mediated by its two Tom22 subunits.

Fast slow folding of an outer membrane porin.

In comparison to globular proteins, the spontaneous folding and insertion of ß-barrel membrane proteins are surprisingly slow, typically occurring on the order of minutes. Using single-molecule Förster resonance energy transfer to report on the folding of fluorescently labeled outer membrane protein G we measured the real-time insertion of a ß-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 rapid, folding resolves this apparent dichotomy between slow ensemble kinetics and the typical timescales of biomolecular folding.

Constructing ion channels from water-soluble α-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. 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.

Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers.

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.

Modifying Membrane Morphology and Interactions with DNA Origami Clathrin-Mimic Networks.

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.

Controlling Anomalous Diffusion in Lipid Membranes.

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.

Dynamic tuneable G protein-coupled receptor monomer-dimer populations.

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.

Measuring the potential energy barrier to lipid bilayer electroporation.

Electroporation is a common tool for gene transfection, tumour ablation, sterilization and drug delivery. Using experimental methods, we explore the temperature dependence of electropore formation in a model membrane system (droplet-interface bilayers), using optical single-channel recording to image the real-time gating of individual electropores. We investigate the influence of the agarose substrate on electropores formed in this system. Furthermore, by examining the temperature-dependent kinetics of pore opening and closure we are able to estimate a barrier to pore opening in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes to be 25.0 ± 8.3 kBT, in agreement with previous predictions. Overall these measurements help support the toroidal model of membrane electroporation.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Correction: On demand modulation of lipid composition in an individual bilayer.

Correction for 'On demand modulation of lipid composition in an individual bilayer' by John S. H. Danial et al., Soft Matter, 2017, 13, 1788-1793.

On demand modulation of lipid composition in an individual bilayer.

Changes in local lipid composition are thought to play a key role in regulating many complex cellular processes. By studying lipid organization in artificial lipid bilayers the physical principles underlying these process can be studied in detail. However, such in vitro measurements are often hindered by heterogeneities in the lipid composition of individual bilayers prepared by current bulk methods. Here, the lipid composition of an individual droplet interface bilayer is varied by lipid titration into the bilayer from the oil phase in a microfluidic device. Control of lipid composition allows the reversible switching between single- and two-phase regions and sampling of specific lipid compositions in an individual bilayer. This method enables controlled modulation of composition-sensitive processes in a single lipid membrane.

Assembling the Tat protein translocase.

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

Imaging the dynamics of individual electropores.

Electroporation is a widely used technique to permeabilize cell membranes. Despite its prevalence, our understanding of the mechanism of voltage-mediated pore formation is incomplete; methods capable of visualizing the time-dependent behavior of individual electropores would help improve our understanding of this process. Here, using optical single-channel recording, we track multiple isolated electropores in real time in planar droplet interface bilayers. We observe individual, mobile defects that fluctuate in size, exhibiting a range of dynamic behaviors. We observe fast (25 s(-1)) and slow (2 s(-1)) components in the gating of small electropores, with no apparent dependence on the applied potential. Furthermore, we find that electropores form preferentially in the liquid disordered phase. Our observations are in general supportive of the hydrophilic toroidal pore model of electroporation, but also reveal additional complexity in the interactions, dynamics, and energetics of electropores.

Chemical amplification of magnetic field effects relevant to avian magnetoreception.

Magnetic fields as weak as the Earth's can change the yields of radical pair reactions even though the energies involved are orders of magnitude smaller than the thermal energy, kBT, at room temperature. Proposed as the source of the light-dependent magnetic compass in migratory birds, the radical pair mechanism is thought to operate in cryptochrome flavoproteins in the retina. Here we demonstrate that the primary magnetic field effect on flavin photoreactions can be amplified chemically by slow radical termination reactions under conditions of continuous photoexcitation. The nature and origin of the amplification are revealed by studies of the intermolecular flavin-tryptophan and flavin-ascorbic acid photocycles and the closely related intramolecular flavin-tryptophan radical pair in cryptochrome. Amplification factors of up to 5.6 were observed for magnetic fields weaker than 1 mT. Substantial chemical amplification could have a significant impact on the viability of a cryptochrome-based magnetic compass sensor.

Length-Dependent Formation of Transmembrane Pores by 310-Helical α-Aminoisobutyric Acid Foldamers.

The synthetic biology toolbox lacks extendable and conformationally controllable yet easy-to-synthesize building blocks that are long enough to span membranes. To meet this need, an iterative synthesis of α-aminoisobutyric acid (Aib) oligomers was used to create a library of homologous rigid-rod 310-helical foldamers, which have incrementally increasing lengths and functionalizable N- and C-termini. This library was used to probe the inter-relationship of foldamer length, self-association strength, and ionophoric ability, which is poorly understood. Although foldamer self-association in nonpolar chloroform increased with length, with a ∼ 14-fold increase in dimerization constant from Aib6 to Aib11, ionophoric activity in bilayers showed a stronger length dependence, with the observed rate constant for Aib11 ∼ 70-fold greater than that of Aib6. The strongest ionophoric activity was observed for foldamers with >10 Aib residues, which have end-to-end distances greater than the hydrophobic width of the bilayers used (∼ 2.8 nm); X-ray crystallography showed that Aib11 is 2.93 nm long. These studies suggest that being long enough to span the membrane is more important for good ionophoric activity than strong self-association in the bilayer. Planar bilayer conductance measurements showed that Aib11 and Aib13, but not Aib7, could form pores. This pore-forming behavior is strong evidence that Aibm (m ≥ 10) building blocks can span bilayers.

Imaging potassium-flux through individual electropores in droplet interface bilayers.

Using total internal reflection fluorescence microscopy of droplet interface bilayers containing the potassium-sensitive fluorophore APG-4, we imaged the ionic flux through individual electropores. We are able to monitor up to 30 individual pores in parallel and show voltage dependent responses in fluorescence that corresponds to the measured ionic current. These experiments help quantify the scope and current limitations of optical single channel recordings of potassium flux. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

High-throughput optical sensing of nucleic acids in a nanopore array.

Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼10(4) nanopores per mm(2) in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording.

Dynamic label-free imaging of lipid nanodomains.

Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220±60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.