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ABSTRACT: 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.
Nature Protocol 05/2013; 8(6):1048-57. · 8.36 Impact Factor
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ABSTRACT: 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 <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.
Biophysical Journal 12/2011; 101(11):2679-83. · 3.65 Impact Factor
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ABSTRACT: 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.
Journal of the American Chemical Society 08/2011; 133(37):14507-9. · 9.91 Impact Factor
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ABSTRACT: 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.
Journal of the American Chemical Society 06/2011; 133(24):9370-5. · 9.91 Impact Factor
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ABSTRACT: 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.
Journal of the American Chemical Society 02/2009; 131(5):1652-3. · 9.91 Impact Factor
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ABSTRACT: 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.
Journal of the American Chemical Society 01/2008; 129(51):16042-7. · 9.91 Impact Factor
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ABSTRACT: 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.
Nano Letters 01/2008; 7(12):3875-8. · 13.20 Impact Factor
