Research

Tethered Membrane

Our aim is to develop novel electrode materials, including nanoparticles, that interact directly with redox enzymes, cell membranes or whole bacteria. This technology either serves as a platform for the development of diagnostics/biosensors and fuel cells or to study the catalytic mechanism of redox-active membrane proteins. The electrode materials, which include ''membrane-modified electrodes" (see Figure), are characterised with a broad spectrum of tools, including Quartz-crystal microballance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). Using electrochemical tools, the electron transport activity of redox enzymes in the membranes is directly converted into a measurable current enabling the detailed study of the enzymes. By combining our electrochemical methods with fluorescent techniques, we aim to obtain information about processes normally not detected by electrochemistry, such as proton pumping or the redox state of single redox site in a multisite enzyme.


Projects

Redox-active membrane enzymes

We have modified electrodes with lipid membranes in which the membranes either adopt a planar geometry (see Figure) or liposomes are immobilised intact on the surface. This allows us to study redox-active membrane proteins in their native environment. We use these systems to study the catalytic activity and proton pumping activity of an ubiquinol oxidase from E. coli, cytochrome bo3. Currently, we have three projects in this area.

Single-enzyme studies of a proton pump

Single Vesicle

By modifying the electrode surfaces, membrane vesicles can be adsorbed on the surface that stay intact. By loading these vesicles with fluorescent probes we can simultaneously control the activity of the ubiquinol oxidase, cytochrome bo3 (using electrochemical methods) and measure its proton pumping activity (see also the ERC project 'MEME'). Currently, this experimental setup is used to study the proton pumping activity of a heme-copper oxidase at the single-enzyme level.

Respiration in Shewanella oneidensis MR-1

Membrane modified electrode of CymA and FccA

An essential functional module of living cells is an active membrane-encapsulated cytoskeleton. The major constituents of this cytoskeleton are a membrane-associated actin cortex and microtubules interacting with the actin cortex. This module is essential for the mechanical properties of cells and for their dynamic 3D internal organization. Our aim is to develop the first steps of a modular approach towards the rational design of cellular mimics. Starting from autonomous parts, we will assemble a synthetic functional "cytoskeleton/membrane" module from purified components. This module will have the most important basic features of a natural cytoskeleton. Its composition will be precisely controlled and its constituents will be designed, emphasizing the truly synthetic nature of this approach. Not only will this lead to a deeper understanding of the biological principles governing the role of membrane proteins and the actin cytoskeleton in the construction of cell architectures but it will also permit us to design and create "de novo" organelles/cells.

Membrane-bound hydrogenase (MBH)

Membrane modified electrode with a membrane-bound hydrogenase

MBHs are hydrogen/proton converting enzymes that donate to or extract electrons from the quinol pool in the lipid membrane. These enzymes are extensively studied using electrochemical methods, but invariably only the two hydrophilic subunits are used, omitting a third membrane subunit that interacts with the quinone pool. In this project, the whole heterotrimeric MBH is placed in the planar lipid membrane on the electrode surface (see image above) and the activity and other properties (such is deactivation) are characterised using electrochemical methods.


Bionanotechnology: Exploiting interactions between nanoparticles and cell membranes

Exploiting Transmembrane Cytochromes for Solar Energy Conversion

MtrF coupled to a TiO2 nanoparticle

There is vast potential for renewable fuel and electricity generation through the photochemical conversion of solar to chemical energy. We wish to tap into what is a largely unused power supply and develop the basic principles for a novel biotechnology that will harness solar energy using a biological synthetic approach, in which a bacterial respiratory machinery is coupled to inorganic photosensitisers. Our biosynthetic approach is inspired by plant photosynthesis where solar energy is absorbed by the special pair of chlorophylls (the photosensitisers) and, ultimately, converted to chemical energy. Our ambition is to overcome the major drawbacks of most artificial homogenous photosynthetic systems: a short-lived charge separated state, which is due to the failure to spatially decouple the reductive and the oxidative sites required to sustain charge separation. In plant photosynthesis, the latter is achieved by rapid transmembrane electron transfer. In this project, which is a collaboration with the Universities of East Anglia and Cambridge, transmembrane charge separation is achieved by exploiting the membrane-spanning electron-transfer conduits of the microbe Shewanella oneidensis MR-1, where the cytochrome conduits play an integral part of their ability to respire on inorganic minerals. As a proof of principle, we aim to photocatalyse the reduction of protons to hydrogen, using both inorganic (platinum) and biological (hydrogenase) catalysts. The resulting photocatalyst adopts the principles of natural photosynthesis (light harvesting, charge separation and catalysis) in a device that spatially separates the sites of photoexcitation and oxidation to that from reduction (hydrogen evolution). The image illustrates a cytochrome condiut protein (MtrF) coupled to a inorganic photosensitiser (TiO2). The assembly is adorbed on a gold electrode and electrochemistry is used to characterise the electron transfer rates between (photoexcited) TiO2 and MtrF.

Microbial fuel cells

A shewanella bacterium coupled to a quantum dot

Recently, an intense interest has emerged in microorganisms that generate electrical current from organic matter in microbial fuel cells (MFCs). MFCs have the ability to generate a clean and renewable energy and to generate continuous power in specialised environments like the bottom of the sea bed or the human body. However, one of the greatest bottlenecks in MFC development is their limited power output, which is still insufficient to drive most electronic devices in society. The limitation in power output is mostly due to the slow rate of electron transfer from the microbes to the anode of the MFC. Using our technology, we aim to 'electrically' connect bacteria in biofilms to the electrode (anode) via nanowires and thereby enhancing the electron transfer rate between microbe and the electrode, and thus the power output of the MFC. To analyse the interactions between bacteria such as Shewanella oneidensis MR-1 and chemically-modified nanoparticles, we make use of quantum dots, which are semi-conducting nanoparticulates that are strongly fluorescent and thus easy to image (see Figure).


Novel supported lipid bilayer platform in biotechnology

Spherical-supported bilayer lipid membranes (ssBLM)

ssBLM

Nanoparticles can be relatively easily extracted from (aqueous) solution via (low-speed) centrifugation or, in the case of (para)magnetic particles, with a magnet. In our lab we coat silica nanoparticles with lipid bilayers. By including membrane proteins in the membrane-coated nanoparticles, we create a technological platform in which the membrane proteins can be easily extracted. Sponsored by MedImmune, we are currently exploring this platform for applications in phage display, raising antibody or antibody mimetic proteins against membrane proteins.

New supported membrane platforms for the study of transporters and membrane metallo-enzymes

double bilayer

For a long time it has been realised that the solid support unerneath supported bilayer will interact with membrane proteins incorported and possibly disrupt the structure or otherwise impair the function the membrane proteins. Several solutions to this problem have been proposed in literature, including a so-called double supported membrane. However, few have been actually tested, let alone used in application with medically relevant membrane proteins. In this project, we are testing the double and multi layer platform in the study of transporters and membrane metallo-enzymes. In one project, we are using the solid-supported membrane platform from the Surfe2r technology that can be used for the biophysical characterisation of ion pumps and transporters. In a second project, we are expanding multi-membrane platforms for the electrochemical study of quinone dehydrogenases


Nanotoxicology and nanomedicine

Nanotox

The toxic effects of nanomaterials are poorly understood and their effects on aquatic wildlife are largely unknown. In the absence of such basic toxicological information, it is difficult to set environmental quality standards or perform risk assessments for these materials. In a project funded by the EU,ENNSATOX, this crucial uncertainty was addressed by seeking to relate the structure and functionality of well-known nanoparticles (silica or ZnO) of varying morphology to its biological activity at the membrane level. Our lab focuses particularly on whether nanomaterials are able to induce damage, either mechanically or via the production of radiacal oxygen species, and whether nanoparticles are able to passively pass a cell membrane, thereby potentially inducing toxic effect in the cytoplasm. Alterantively, by creating nanomaterials that can passively pass the membrane without too many toxic concequences, a drug delivery system can be designed.