Mark I. Wallace

Droplet interface bilayers

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

Non-Arrhenius kinetics for the loop closure of a DNA hairpin

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 (β = 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. & 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.

Simultaneous measurement of ionic current and fluorescence from single protein pores.

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.

Membrane protein stoichiometry determined from the step-wise photobleaching of dye-labelled subunits.

Many important proteins function as multimeric complexes, which often contain different types and numbers of subunits. Such proteins play a key role in a wide variety of cellular events, including signal transduction, cell regulation, transport and energy generation. Protein oligomers are associated with many human diseases including early onset Parkinson’s, Alzheimer’s and Huntington’s disease. Such complexes also play key roles in the pathogenic action of many bacterial toxins. If we are to understand the widespread roles of protein complexes, additional methods must be developed that are capable of measuring the temporal variations in subunit stoichiometry that must occur during complex assembly. Several techniques provide information on protein stoichiometry. The mapping of high resolution X-ray crystal structures onto lower resolution cryoelectron microscopy images provides one method for determining high-resolution structures of large complexes. 6] The difficulty of this sophisticated approach, however, is reflected in the limited number of publications successfully combining both techniques. Atomic force microscopy (AFM) can also be used to determine subunit stoichiometry either directly or by specifically tagging subunits with other large molecules. Other techniques capable of providing information on the stoichiometry of protein complexes include analytical ultracentrifugation, gel electrophoresis, Fcrster resonance energy transfer (FRET) and mass spectrometry. These methods vary greatly in both experimental complexity and their utility for subunit determination. For example—although costly and time consuming—cryoelectron microscopy and X-ray crystallography are capable of providing exquisite resolution of large protein complexes. On the other hand, FRET only requires judicious chemical modification of two proteins, but is limited to studying the interactions of two (or possibly three) partners in what could be a much larger complex. Single-molecule fluorescence (SMF) microscopy is a relatively new technique capable of resolving the stoichiometry of a fluorescently labelled protein complex. SMF also has the potential to measure the dynamics of protein oligomerisation both in vitro and in vivo, without the need to immobilise the protein on a surface or within a crystal. SMF is not able to provide a structure of an individual molecule (as with AFM or cryoelectron microscopy), however, SMF can be used to visualise molecular complexes tagged with different fluorescent labels. One SMF method capable of resolving subunit stoichiometry is simply to count the number of photobleaching steps within a single complex. Photobleaching is the irreversible loss of fluorescence in a molecule due to changes in its structure following a light-induced chemical reaction. For an individual fluorophore, photobleaching is observed as a step change in fluorescence intensity. If each subunit within a protein complex is labelled with a fluorescent molecule, the total number of photobleaching steps determines the number of subunits within the complex. Multiple photobleaching steps have been observed in a randomly labelled mRNA–ribosome complex and in fluorescently labelled polymers. Photobleaching steps have also been used to determine the number of fluorescently labelled proteins encapsulated within a lipid vesicle, to infer the stoichiometry of stator components within the bacterial flagellar motor and to determine the number of labels on a green fluorescent protein dimer. Here, we employ the stepwise changes in SMF intensity during photobleaching to determine the number of subunits within a membrane–protein complex. b-Barrel pore-forming toxins (b-PFTs) are a class of multimeric membrane proteins that assemble on lipid membranes to form bilayer-spanning pores. Their known stoichiometries vary from small oligomers to large 30–50 subunit complexes. a-Hemolysin (aHL) is the archetypal b-barrel-forming toxin. High-resolution crystallographic, AFM and biochemical studies suggests that aHL forms a heptameric pore. Other AFM experiments have reported hexameric aHL, thus indicating that some variation in subunit stoichiometry might be possible. Leukocidin is a bicomponent b-PFT with significant sequence homology to aHL. It is formed from two different polypeptide subunits, LukF and LukS, both of which have several different isoforms. The structures of both LukS and LukF monomers have been resolved with X-ray crystallography. 30] However, in contrast to aHL, there is much less consensus regarding the structure of the fully assembled leukocidin pore. No crystal structure of the pore complex is available, and the numbers of each component are not conclusively known. Biochemical cross-linking and chemical modification during single-channel recording have provided evidence that leukocidin is an octameric pore with an alternating arrangement of subunits. Other experiments indicate that leukocidin forms pores with alternative stoichiometries: either hexameric or heptameric. Single-molecule FRET experiments have also been performed with leukocidin. Higuchi and co-workers [a] Dr. S. Cheley, Dr. M. I. Wallace, Prof. Dr. H. Bayley Department of Chemistry, University of Oxford Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+1)44-1865-285002 [b] Dr. S. K. Das, Dr. M. Darshi Department of Molecular & Cellular Medicine, The Texas A&M University System Health Science Center College Station, TX 77843-1114 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author: representative image showing individually resolved aHL complexes on a glass cover-slip surface, and a graph showing how the intensity of each single-molecule spot correlates with the number of photobleaching steps detected.

A radical sense of direction: signalling and mechanism in cryptochrome magnetoreception

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

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.

In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers.

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.

Determining Membrane Capacitance by Dynamic Control of Droplet Interface Bilayer Area

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.

Dynamic label-free imaging of lipid nanodomains

Significance Cell membranes are thought to partition into small (10–100 nm) and transient (<100 ms) lipid platforms or “rafts” to control signaling and trafficking across the membrane. The difficulty in observing such species has made “lipid rafts” a contentious topic. Indirect evidence for their existence has been available for decades, but it has not been possible to reveal their dynamics directly. By exploiting the differences in light scattering from different lipid phases, we achieve dynamic imaging of lipid nanodomains. We observe nanodomain formation, destruction, and coalescence—behaviors previously hypothesized but never observed, yet are critical to their proposed function. 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.

High-Speed Single-Particle Tracking of GM1 in Model Membranes Reveals Anomalous Diffusion due to Interleaflet Coupling and Molecular Pinning

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.