Measuring the potential energy barrier to lipid bilayer electroporation.
Sengel JY, Wallace MI.
Phil. Trans. Royal Soc. B 2017
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 kT, in agreement with previous predictions. Overall these measurements help support the toroidal model of membrane electroporation.
On demand modulation of lipid composition in an individual bilayer.
Danial JSH, Cronin B, Mallik C, Wallace MI.
Soft Matter 2017
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.
Alcock F, Stansfeld PJ, Basit H, Habersetzer J, Baker MAB, Palmer T, Wallace MI, Berks BC.
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.
Sengel JT, Wallace MI.
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.
Kattnig DR, Evans EW, Déjean V, Dodson CA, Wallace MI, Mackenzie SR, Timmel CR, Hore PJ.
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.
Dissecting the Self-Assembly Kinetics of Multimeric Pore-forming Toxins.
Lee AA, Senior M, Wallace MI, Woolley TE Griffiths IM.
J.Royal Soc. Interfaces 2016
TPore-forming toxins are ubiquitous cytotoxins that are exploited by both bacteria and the immune response of eukaryotes. These toxins kill cells by assembling large multimeric pores on the cell membrane. However, a quantitative understanding of the mechanism and kinetics of this self-assembly process is lacking. We propose an analytically solvable kinetic model for stepwise, reversible oligomerization. In biologically relevant limits, we obtain simple algebraic expressions for the rate of pore formation, as well as for the concentration of pores as a function of time. Quantitative agreement is obtained between our model and time-resolved kinetic experiments of Bacillus thuringiensis Cry1Ac (tetrameric pore), aerolysin, Staphylococcus aureus α-haemolysin (heptameric pores) and Escherichia coli cytolysin A (dodecameric pore). Furthermore, our model explains how rapid self-assembly can take place with low concentrations of oligomeric intermediates, as observed in recent single-molecule fluorescence experiments of α-haemolysin self-assembly. We propose that suppressing the concentration of oligomeric intermediates may be the key to reliable, error-free, self-assembly of pores.
Length-Dependent Formation of Transmembrane Pores by 310-Helical α-Aminoisobutyric Acid Foldamers
Jones JE, Diemer V, Adam C, Raftery J, Ruscoe RE, Sengel JT, Wallace MI, Bader A, Cockroft SL, Clayden J, Webb SJ.
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.
The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.
Cleon F, Habersetzer J, Alcock F, Kneuper H, Stansfeld PJ, Basit H, Wallace MI, Berks BC, Palmer T.
Mol. Microbiol. 2015
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 comprised 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 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 five of TatC. Our findings show that TatC functions as an obligate oligomer.
Imaging potassium-flux through individual electropores in droplet interface bilayers.
Szabo M, Wallace MI.
Biochim Biophys Acta. 2015
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.
High-throughput optical sensing of nucleic acids in a nanopore array.
Huang S, Romero-Ruiz M, Castell OK, Bayley H, Wallace MI.
Nature Nano. 2015
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 ∼104 nanopores per mm2 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
de Wit G, Danial JS, Kukura P, Wallace MI.
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.
Probing channel, pump, and transporter function using single-molecule fluorescence.
Weatherill EE, Danial JSH, Wallace MI.
in Pumps, Channels and Transporters, Methods of Functional Analysis. Wiley 2015
The ability of patch clamping to monitor individual ion channels revolutionized our approach to understanding the discrete functional states of these membrane proteins, including how ligands, point mutations, and the electrochemical potential affect channel function
Fluorescence-detected magnetic field effects on radical pair reactions from femtolitre volumes.
Dodson CA, Wedge CJ, Murakami M, Maeda K, Wallace MI, Hore PJ.
Chem Comm 2015
We show that the effects of applied magnetic fields on radical pair reactions can be sensitively measured from sample volumes as low as ∼100 femtolitres using total internal reflection fluorescence microscopy. Development of a fluorescence-based microscope method is likely to be a key step in further miniaturisation that will allow detection of magnetic field effects on single molecules.
Membrane pore formation at protein-lipid interfaces.
Gilbert RJ, Dalla Serra M, Froelich CJ, Wallace MI, Anderluh G.
Trends Biochem Sci. 2014
Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate ‘proteolipidic’ pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer’s Aβ1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further.
Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers.
Baker MA, Rojko N, Cronin B, Anderluh G, Wallace MI.
Equinatoxin II (EqtII), a sea anemone cytolysin, is known to oligomerize to form pores that spontaneously insert into membranes. Crystallographic and cryo-EM studies of structurally similar cytolysins offer contradictory evidence for pore stoichiometry. Here we used single-molecule photobleaching of fluorescently labeled EqtII to determine the stoichiometry of EqtII oligomers in supported lipid bilayers. A frequency analysis of photobleaching steps revealed a log-normal distribution of stoichiometries with a mean of 3.4±2.3 standard deviations. Comparison of our experimental data with simulations of fixed stoichiometries supports our observation of a heterogeneous distribution of EqtII oligomerization. These data are consistent with a model of EqtII stoichiometry where pores are on average tetrameric, but with large variation in the number of subunits in individual pores.
High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning.
Spillane KM, Ortega-Arroyo J, de Wit G, Eggeling C, Ewers H, Wallace MI, Kukura P.
Nano Lett. 2014
The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane “pickets” or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20-40 nm scattering gold beads with simultaneous 2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.
Combining single-molecule imaging and single-channel electrophysiology.
Weatherill EE, Wallace MI.
J. Mol. Biol. 2014
Combining simultaneous single-molecule fluorescence measurements of ion channel conformational change with single-channel electrophysiology would enable a direct link between structure and function. Such methods would help us to create a truly molecular “movie” of how these important biomolecules work. Here we review past and recent progress toward this goal.
Fluorescence imaging of MACPF/CDC proteins. New techniques and their application.
Senior MJ, Wallace MI.
Subcell Biochem. 2014
Structural and biochemical investigations have helped illuminate many of the important details of MACPF/CDC pore formation. However, conventional techniques are limited in their ability to tackle many of the remaining key questions, and new biophysical techniques might provide the means to improve our understanding. Here we attempt to identify the properties of MACPF/CDC proteins that warrant further study, and explore how new developments in fluorescence imaging are able to probe these properties.
Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA.
Marcoux J, Politis A, Rinehart D, Marshall DP, Wallace MI, Tamm LK, Robinson CV.
The transmembrane domain of the outer membrane protein A (OmpA) from Escherichia coli is an excellent model for structural and folding studies of β-barrel membrane proteins. However, full-length OmpA resists crystallographic efforts, and the link between its function and tertiary structure remains controversial. Here we use site-directed mutagenesis and mass spectrometry of different constructs of OmpA, released in the gas phase from detergent micelles, to define the minimal region encompassing the C-terminal dimer interface. Combining knowledge of the location of the dimeric interface with molecular modeling and ion mobility data allows us to propose a low-resolution model for the full-length OmpA dimer. Our model of the dimer is in remarkable agreement with experimental ion mobility data, with none of the unfolding or collapse observed for full-length monomeric OmpA, implying that dimer formation stabilizes the overall structure and prevents collapse of the flexible linker that connects the two domains.
Imaging the lipid-phase-dependent pore formation of equinatoxin II in droplet interface bilayers.
Rojko N, Cronin B, Danial JS, Baker MA, Anderluh G, Wallace MI.
Biophys J. 2014
Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.
Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
Alcock F, Baker MA, Greene NP, Palmer T, Wallace MI, Berks BC.
The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.
A radical sense of direction. signalling and mechanism in cryptochrome magnetoreception.
Dodson CA, Hore PJ, Wallace MI.
Trends Biochem. Sci. 2013
The remarkable phenomenon of magnetoreception in migratory birds and other organisms has fascinated biologists for decades. Much evidence has accumulated to suggest that birds sense the magnetic field of the Earth using photochemical transformations in cryptochrome flavoproteins. In the last 5 years this highly interdisciplinary field has seen advances in structural biology, biophysics, spin chemistry, and genetic studies in model organisms. We review these developments and consider how this chemical signal can be integrated into the cellular response.
Constructing droplet interface bilayers from the contact of aqueous droplets in oil.
Leptihn S, Castell OK, Cronin B, Lee EH, Gross LC, Marshall DP, Thompson JR, Holden M, Wallace MI.
Nat. Protocols 2013
We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the α-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes < 30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.
One-step synthesis of fluorescein modified nano-carbon for Pd(II) detection via fluorescence quenching.
Panchompoo J, Aldous L, Baker M, Wallace MI, Compton RG.
Carbon black (CB) nanoparticles modified with fluorescein, a highly fluorescent molecule, were prepared using a facile and efficient methodology. Simply stirring CB in aqueous solution containing fluorescein resulted in the strong physisorption of fluorescein onto the CB surface. The resulting Fluorescein/CB was then characterised by means of X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), fluorescence microscopy and fluorescence spectroscopy. The optimum experimental conditions for fluorescence of Fluorescein/CB viz. fluorescence excitation and emission wavelengths, O(2) removal and the amount of Fluorescein/CB used, were investigated. The Fluorescein/CB was used as a fluorescent probe for the sensitive detection of Pd(II) in water, based on fluorescence quenching. The results demonstrated that the fluorescence intensity of Fluorescein/CB decreased with increasing Pd(II) concentration, and the fluorescence quenching process could be described by the Stern-Volmer equation. The limit of detection (LOD) for the fluorescence quenching of Fluorescein/CB by Pd(II) in aqueous solution was found to be 1.07 μM (based on 3σ). Last, approaches were studied for the removal of Fe(III) which interferes with the fluorescence quenching of Fluorescein/CB. Complexation of Fe(III) with salicylic acid was used to enhance and control the selectivity of Fluorescein/CB sensor towards Pd(II) in the presence of Fe(III).
Quantification of membrane protein inhibition by optical ion flux in a droplet interface bilayer array.
Castell OK, Berridge J, Wallace MI.
Angew Chem Int Ed. 2012
Optical platforms for assaying membrane protein function offer a promising route to scalable high-throughput screening (see picture). For the first time quantitative measurements of membrane protein inhibition are reported in an optically addressable lipid bilayer array. Wide-field total internal reflection fluorescence (TIRF) imaging of Ca2+ flux enables the quantification of α-hemolysin inhibition by γ-cyclodextrin.
Rapid assembly of a multimeric membrane protein pore.
Thompson JR, Cronin B, Bayley H, Wallace MI.
Biophys J. 2011
We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in less than 5 ms. No lower order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen, and the cholesterol-dependent cytolysins, and their eukaryotic analogs, such as the complement pore membrane attack complex and perforin domain. The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore formers.
Determining membrane capacitance by dynamic control of droplet interface bilayer area.
Gross LC, Heron AJ, Baca SC, Wallace MI.
By making dynamic changes to the area of a droplet interface bilayer (DIB), we are able to measure the specific capacitance of lipid bilayers with improved accuracy and precision over existing methods. The dependence of membrane specific capacitance on the chain-length of the alkane oil present in the bilayer is similar to that observed in black lipid membranes. In contrast to conventional artificial bilayers, DIBs are not confined by an aperture, which enables us to determine that the dependence of whole bilayer capacitance on applied potential is predominantly a result of a spontaneous increase in bilayer area. This area change arises from the creation of new bilayer at the three phase interface and is driven by changes in surface tension with applied potential that can be described by the Young-Lippmann equation. By accounting for this area change, we are able to determine the proportion of the capacitance dependence that arises from a change in specific capacitance with applied potential. This method provides a new tool with which to investigate the vertical compression of the bilayer and understand the changes in bilayer thickness with applied potential. We find that, for 1,2-diphytanoyl-sn-glycero-3-phosphocholine membranes in hexadecane, specific bilayer capacitance varies by 0.6-1.5% over an applied potential of ±100 mV.
Imaging multiple conductance states in an alamethicin pore.
Harriss LM, Cronin B, Thompson JR, Wallace MI.
Alamethicin is the archetypal antimicrobial pore-forming peptide. Although the peptide has long been known to form pores of characteristic conductances in lipid membranes, the precise nature of these pores is not known. Simultaneous calcium-flux imaging and single-channel recording in a droplet interface bilayer allowed us to directly attribute multiple conductance states to a single point diffusing in the bilayer.
Dynamic and reversible control of 2D membrane protein concentration in a droplet interface bilayer.
Gross LC, Castell OK, Wallace MI.
We form an artificial lipid bilayer between a nanolitre aqueous droplet and a supporting hydrogel immersed in an oil/lipid solution. Manipulation of the axial position of the droplet relative to the hydrogel controls the size of the bilayer formed at the interface; this enables the surface density of integral membrane proteins to be controlled. We are able to modulate the surface density of the β-barrel pore-forming toxin α-hemolysin over a range of 4 orders of magnitude within a time frame of a few seconds. The concentration changes are fully reversible. Membrane protein function and diffusion are unaltered, as measured by single molecule microscopy and single channel electrical recording.
In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers.
Leptihn S, Thompson JR, Ellory JC, Tucker SJ, Wallace MI.
The ability to routinely study eukaryotic ion channels in a synthetic lipid environment would have a major impact on our understanding of how different lipids influence ion channel function. Here, we describe a straightforward, detergent-free method for the in vitro reconstitution of eukaryotic ion channels and ionotropic receptors into droplet interface bilayers and measure their electrical activity at both the macroscopic and single-channel level. We explore the general applicability of this method by reconstitution of channels from a wide range of sources including recombinant cell lines and native tissues, as well as preparations that are difficult to study by conventional methods including erythrocytes and mitochondria.
Visualizing helicases unwinding DNA at the single molecule level.
Fili N, Mashanov GI, Toseland CP, Batters C, Wallace MI, Yeeles JT, Dillingham MS, Webb MR, Molloy JE.
Nuc. Acids Res. 2010
DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.
Lucky imaging. improved localization accuracy for single molecule imaging.
Cronin B, de Wet B, Wallace MI.
Biophys. J. 2009
We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells.
Simultaneous measurement of ionic current and fluorescence from single protein pores.
Heron AJ, Thompson JR, Cronin B, Bayley H, Wallace MI.
The ability to simultaneously monitor both the ionic current and fluorescence from membrane channels and pores has the potential to link structural changes with function in such proteins. We present a new method for simultaneously measuring single-channel electrical currents and fluorescence from membrane proteins by using water-in-oil droplet bilayers. We demonstrate the simultaneous fluorescence and electrical detection of stochastic blocking by cyclodextrin in multiple staphylococcal alpha-hemolysin pores. The combined fluorescence signal from individual pores exhibits the same sequence of blocking events as the total current recording, showing that the two signals from each pore are correlated.
Protein modification for single molecule fluorescence microscopy.
Dillingham MS, Wallace MI.
Org. Biomol. Chem. 2008
Single molecule methods have emerged as a powerful new tool for exploring biological phenomena. We provide a brief overview of the scope of current experiments and assess the limitations of both fluorescent labels and the means to achieve protein modification for single molecule microscopy.
Droplet interface bilayers.
Bayley H, Cronin B, Heron A, Holden MA, Hwang W, Syeda R, Thompson JR, Wallace MI.
Our efforts on droplet interface bilayers in Oxford began in the summer of 2005 following a suggestion made by David Needham at “Lipids, liposomes and biomembranes”, a conference in Vancouver. He proposed that a bilayer would form between two droplets brought together in an oil (squalene) containing a lipid (glycerol monooleate) and that this might be a means to miniaturize planar bilayer recording.
Direct detection of membrane channels from gels using water-in-oil droplet bilayers.
Heron AJ, Thompson JR, Mason AE, Wallace MI.
We form planar lipid bilayers between an aqueous droplet and a hydrogel support immersed in a lipid-oil solution. By scanning the bilayer over the surface of an SDS-PAGE gel, we are able to directly detect membrane proteins from gels using single-channel recording. Using this technique, we are able to examine low levels of endogenous protein from cell extracts without the need for over-expression. We also use droplet bilayers to detect small molecules from hydrogels. The bilayers show enhanced stability compared to conventional planar lipid bilayers, and both bilayer size and position can be controlled during an experiment. Hydrogel scanning with droplet bilayers provides a new method for the discovery and characterization of ion channels with the potential for high-throughput screening.
Enhanced stability and fluidity in droplet on hydrogel bilayers for measuring membrane protein diffusion.
Thompson JR, Heron AJ, Santoso Y, Wallace MI.
Nano Lett. 2007
We form artificial lipid bilayers suitable for single-molecule fluorescence microscopy by contacting an aqueous droplet with a hydrogel support immersed in a solution of lipid in oil. Our results show that droplet on hydrogel bilayers (DHBs) have high lipid mobilities, similar to those observed in unsupported lipid bilayers. DHBs are also stable over a period of several weeks. We examine membrane protein diffusion in these bilayers and report a decreased lateral mobility of the heptameric beta-barrel pore-forming toxin alpha-hemolysin versus that of its monomeric precursor. These results corroborate previous models of the alpha-hemolysin insertion mechanism where the monomer binds to the lipid bilayer without insertion.
Membrane protein stoichiometry determined from the step-wise photobleaching of dye-labelled subunits.
Das SK, Darshi M, Cheley S, Wallace MI, Bayley H.
Chembiochem. 2007 Jun 18;8(9):994-9</i></b><p>Body Count. In a generally applicable approach, the number of subunits in fluorescently-labelled protein complexes has been determined by counting photobleaching steps from individual molecules (see figure). The distribution of steps in the pore-forming toxins α-haemolysin and leukocidin indicate seven subunits for α-hemolysin, and four LukF and four LukS subunits for leukocidin.
Prepore for a breakthrough.
Bayley H, Jayasinghe L, Wallace MI.
Nat. Struct. Mol. Biol 2005
A key to understanding bacterial pathogenicity is the mechanism by which water-soluble protein toxins assemble on cell membranes to form oligomeric bilayer-spanning pores. The recent reconstructions from cryo-electron micrographs of three-dimensional pore and prepore structures of the cholesterol-dependent toxin pneumolysin shed new light on the later steps of the assembly of large toxin pores.
A model of stereocilia adaptation based on single molecule mechanical studies of myosin I.
Batters C, Wallace MI, Coluccio LM, Molloy JE.
Phil. Trans. RSB. 2004
We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features that have not been seen using fast skeletal muscle myosin II. (i) The working stroke occurs in two, distinct phases, producing an initial 3 nm and then a further 1.5 nm of movement. (ii) Two types of binding interaction were observed: short-lived ATP-independent binding events that produced no movement and longer-lived, ATP-dependent events that produced a full working stroke. The stiffness of both types of interaction was similar. (iii) In a new type of experiment, using feedback to apply controlled displacements to a single acto-myosin cross-bridge, we found abrupt changes in force during attachment of the acto-Myo1b cross-bridge, a result that is consistent with the classical ‘T2’ behaviour of single muscle fibres. Given that these myosins might exhibit the classical T2 behaviour, we propose a new model to explain the slow phase of sensory adaptation of the hair cells of the inner ear.
Nanometre resolution tracking of myosin-1b motility.
Wallace MI, Batters C, Coluccio LM, Molloy JE.
IEE Proc Nanobiotechnol. 2003
The movement produced by a small number of myosin molecular motors was measured with nanometre precision using single-molecule fluorescence localisation methods. The positional precision of the measurements was sufficient to reveal fluctuations in sliding velocity due to stochastic interactions between individual myosin motors and the actin filament. Dependence of sliding velocity upon filament length was measured and fluctuations in velocity were quantified by autocorrelation analysis. Optical tweezers-based nanometry was used to measure the myosin-1b step-size directly. The 10 nm power-stroke and its duty cycle ratio were consistent with values derived from in vitro sliding assays.
Photon counting histogram for one-photon excitation.
Perroud TD, Huang B, Wallace MI, Zare RN.
An advance that counts! A method is presented for analyzing single-molecule behavior using one-photon excitation of fluorescence. It is based on the photon counting histogram (PCH) procedure that has enjoyed much success for two-photon excitation, but has not previously been applied to one-photon excitation. This corrected PCH model is able to resolve fluorescent species with different degrees of brightness, for example, a mixture of tetramethylrhodamine-5′-maleimide and Cy3-maleimide (see graph). Because one-photon excitation is often much easier to do than two-photon excitation, this method has the potential to be widely used and represents an important advance in the field of single-molecule spectroscopy.
Combined single-molecule force and fluorescence measurements for biology.
Wallace MI, Molloy JE, Trentham DR.
J. Biology 2002
Recent advances in single-molecule techniques allow the application of force to an individual biomolecule whilst simultaneously monitoring its response using fluorescent probes. The effects of applied mechanical load on single-enzyme turnovers, biomolecular interactions and conformational changes can now be studied with nanometer precision and millisecond time resolution.
Coupled electrorotation of polymer microspheres for microfluidic sensing and mixing.
Wilson CF, Wallace MI, Morishima K, Simpson GJ, Zare RN.
Anal Chem. 2002
We show that coupled electrorotation (CER) of microscopic particles using microfabricated electrodes can be used for localized sensing and mixing. The effective use of microelectromechanical systems and micro total analysis systems requires many types of control. These include the abilityto (1) manipulate objects within microchannels by noncontact means, (2) mix fluids, and (3) sense local chemical parameters. Coupled electrorotation, in which the interactions between induced electric dipoles of adjacent particles lead to particle rotation, addresses aspects of all three challenges simultaneously. CER is a simple means of controlling the rotation of dielectric objects using homogeneous external radio frequency electric fields. CER is sensitive to several chemical and physical parameters such as the solution conductivity, pH, and viscosity. As a step toward integrating CER devices into microfluidic systems, a simple chip was designed to induce local mixing and to detect local changes in salt concentration, pH, and viscosity.
Non-Arrhenius kinetics for the loop closure of a DNA hairpin.
Wallace MI, Ying LM, Balasubramanian S, Klenerman D.
Intramolecular chain diffusion is an elementary process in the conformational fluctuations of the DNA hairpin-loop. We have studied the temperature and viscosity dependence of a model DNA hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained by analyzing the correlation amplitude through a two-state model and are consistent with steady-state fluorescence measurements. The kinetics of closing the loop show non-Arrhenius behavior, in agreement with theoretical prediction and other experimental measurements on peptide folding. The fluctuation rates show a fractional power dependence (beta = 0.83) on the solution viscosity. A much slower intrachain diffusion coefficient in comparison to that of polypeptides was derived based on the first passage time theory of SSS [Szabo, A., Schulten, K. and Schulten, Z. (1980) J. Chem. Phys. 72, 4350-4357], suggesting that intrachain interactions, especially stacking interaction in the loop, might increase the roughness of the free energy surface of the DNA hairpin-loop.
Two-state model of conformational fuctuation in a DNA hairpin-loop.
Ying L, Wallace MI, Klenerman D.
Chem. Phys. Lett. 2001
Stretched exponential kinetics have been observed in the conformational ̄uctuation of a DNA hairpin-loop under equilibrium conditions. In this paper, we employ a simple multiple-pathway two-state jump model to calculate single- molecule proximity ratio distributions. The simulation can reasonably reproduce the experimental single-molecule data of the conformational ̄uctuations in water, indicating that static disorder is dominant. In contrast, there exists sig- ni®cant discrepancy between the two-state simulation and experiment in buer (2.5 mM Tris-HCl, 250 lM EDTA, 100 mM NaCl), suggesting that both static and dynamic disorder may contribute to the non-exponential kinetics.
Diamond deposition in a DC-arc Jet CVD system. investigations of the effects of nitrogen addition.
Smith JA, Rosser KN, Yagi H, Wallace MI, May PW, Ashfold MNR.
Diamond and Rel. Mat. 2001
Studies of the chemical vapour deposition of diamond films at growth rates 100 m h1 with a 10-kW DC-arc jet system are described. Additions of small amounts of N2 to the standard CH4H2Ar feedstock gas results in strong CN(B->X). emission, and quenches C2(d->a). and H-alpha emissions from the plasma. Species selective, spatially resolved optical emission measurements have enabled derivation of the longitudinal and lateral variation of emitting C2, CN radicals and H (n=3). atoms within the plasma jet. Scanning electron microscopy and laser Raman analyses indicate that N2 additions also degrade both the growth rate and quality of the deposited diamond film; the latter technique also provides some evidence for nitrogen inclusion within the films.
FRET fluctuation spectroscopy. Exploring the conformational dynamics of a DNA hairpin-loop.
Wallace MI, Ying LM, Balasubramanian S, Klenerman D.
J. Phys. Chem. B 2000
The motions of a dye-labeled DNA hairpin loop (Cy5-5′-GGGTT-(A)30-AACCC-3′-TMR) have been investigated through the fluctuations in proximity ratio from fluorescence resonance energy transfer (FRET). We examine three solution conditions: (1) MilliQ water, (2) Tris-EDTA buffer, and (3) Tris-EDTA buffer plus an excess of DNA complementary to the loop sequence, (T)30. Correlations in proximity ratio show submillisecond dynamics. Static heterogeneity is revealed from the distribution of proximity ratio amplitudes. The observed stretched exponential kinetics are consistent with a model based on the transition between two states over a complex energy landscape.
Ratiometric analysis of single-molecule Fluorescence Resonance Energy Transfer using logical combinations of threshold criteria. A study of 12-mer DNA.
Ying LM, Wallace MI, Balasubramanian S, Klenerman D.
J. Phys. Chem. B 2000
Single-molecule fluorescence resonance energy transfer (FRET) combined with bulk fluorescence lifetimes, anisotropy, and spectra have been used to study a donor-acceptor labeled model DNA system (Cy5-5′- ACCTGCCGACGC-3′-TMR). A general ratiometric analysis method using independent donor and acceptor thresholding has been developed. Use of two logical combinations of thresholding criteria provides more information than either method alone, revealing heterogeneity within this system. Conditions yielding similar bulk fluorescence spectra can be readily distinguished by this single-molecule method. Fluorescence lifetimes and anisotropy measurements also suggest nonnegligible fluorophore-DNA interaction.