Current research topics in our group include:
1. Studies on the fundamental properties of ion channels and pores
The application of membrane proteins in biotechnology requires a sound understanding of their fundamental properties. We are interested in the stability, folding, assembly and functional properties of membrane proteins. These properties differ considerably from those of their soluble counterparts. In terms of functional properties, we are especially interested in the translocation of polymers through membrane pores such as α-hemolysin (DNA), Wza (oligosaccharides) and autotransporters (polypeptides). We are also investigating the properties of various ion channels, such as the molecular mechanism of voltage-gating in potassium channels.
2. New technologies for ion channels and pores
Pore-forming proteins are being engineered for applications in biotechnology. Our main focus is on α-hemolysin, a bacterial toxin that forms a heptameric transmembrane pore of known three-dimensional structure. By using genetic engineering, unnatural amino acid mutagenesis and targeted chemical modification, pores are being made with diverse functional properties. We are also developing methods for high-throughput screening with membrane proteins. These include the use of proteins made in cell-free systems, techniques for the rapid insertion of proteins into lipid bilayers and the development of chips for the parallel processing of hundreds of membrane proteins samples.
3. Stochastic sensing and ultra-rapid DNA sequencing
We have engineered many variants of the α-hemolysin protein pore for single molecule detection (stochastic sensing), which is effected by monitoring the modulation of the ionic current passing through the pore. The approach allows the analysis of a wide variety of analytes: metal cations, small organic molecules, nucleic acids and proteins. Current directions include the incorporation of ligands such as aptamers and oligosaccharides into the α-hemolysin pore. We are also investigating the use of protein pores for sequencing single molecules of DNA, either by the exonuclease approach in which bases (nucleoside monophosphates) are sequentially released into a pore, or by the strand sequencing approach where single strands of DNA are pulled past a "reading head" located within the pore. Nanopore sequencing might be adapted for the identification of epigenetically modified DNA bases and the direct sequencing of RNA.
4. Single molecule chemistry and catalysis
Protein pores can also be used to study covalent chemistry at the single-molecule level. We have investigated a wide variety of chemistry in this way, including the formation and cleavage of arsenic-sulfur bonds, the photochemistry of 2-nitrobenzyl protecting groups, the photoisomerization of azobenzenes, the observation of polymerization one step at a time and recently a hydrogen-deuterium isotope effect. New directions include the incorporation of unnatural amino acids into the α-hemolysin pore with which to expand the range of chemistry that can be investigated and the examination of catalysis at the single-molecule level.
5. Droplet interface bilayers
Recently, we discovered a process by which aqueous droplets submerged in a hydrocarbon solvent can be connected by means of lipid bilayers to form networks. The incorporation of protein pores into the bilayers allows the droplets to communicate. In the area of synthetic biology, considerable effort has been put in the preparation of artificial cells, known as protocells. By contrast, assemblies of interacting protocells have not been reported. The droplet networks are a first effort in this direction. By using engineered pores in the interface bilayers, we have been able to produce droplet networks that form batteries, detect light and rectify electrical signals. Work continues to build more complex two-dimensional and three-dimensional droplet networks.