Supported Cell Membranes: The next level in model membrane systems

All living cells are surrounded by a thin membrane that shields and separates the inside of these cells from their surroundings. These thin membranes contain many proteins that transport specific compounds, like nutrients and salt, across the membrane. Some of these proteins actively transport protons across the membrane using the energy that is released from electron transfers. Consequently, the concentration of many compounds is different on the inside of the membrane compared to the outside. These gradients play a crucial role in biology and many reactions in the cell are dependent on it. When we study the structure and function of protein normally located in membranes we often study them in an environment that is different from the membrane. In this proposal we aim to develop a new tool that allows the study of membrane proteins in their natural environment. For this to be achieved, we will place the complete membrane on a specially designed surface that interacts with the membrane. These surfaces can then 'interrogate' the membrane proteins that are involved in the transmembrane transport of charged species (salt) or electrons. By studying these proteins we will learn more about how they function inside their natural membrane. Another important benefit is that the membrane proteins do not need to be purified, but that total membrane extracts can be used. This makes the experimental system relatively easy to prepare and therefore allows intergration in devices such as 'lab-on-chip'.

Model-membrane systems are proven to be very valuable tools in the study of biological membranes. Data from these model systems bridge the scientific gap that exists between the levels of molecular biology and in vivo studies of the cell membranes. A very successful model-membrane system is the so-called supported lipid bilayer in which a phospholipid bilayer is positioned planar to a solid surface. This 2D orientation naturally matches the 2-dimensionality of the cell membrane and allows the use of a range of surface-spectroscopic tools which provide structural and functional information not readily obtainable from phospholipid vesicles or cell membranes. In this project we will raise the supported bilayer system to the next level, namely that of the supported cell membrane. In this system, planar membranes are formed using whole cell membranes instead of purified phospholipids. Supported cell membranes will be a key step forward because, unlike the supported lipid bilayer, they still contain the numerous membrane proteins and lipids of the natural membrane and studies can be performed in a more native like lipid environment. Like the supported lipid bilayers, supported cell membranes will enable the simultaneous characterisation of charge transfer through the membrane and redox activity of membrane proteins under a controllable electrochemical gradient (i.e., electric field). This type of data can potentially solve some long-standing questions on redox proteins that generate a proton-motive force (pmf) as part of their catalytic cycle. Finally, since these systems do not need the protein of interest to be purified, the supported cell membranes will be relatively easy to prepare while it could provide new insides into the importance of the native membrane environment on the activity of membrane proteins. Pilot studies reported in this proposal show that supported cell membrane systems can be prepared from bacterial membranes while retaining the functional activity of a membrane protein, ubiquinol oxidase. In this project we propose to further investigate (i) the structure of the supported cell membrane and how it relates to the previous studied supported lipid bilayer systems, (ii) how these complex structures are accommodated by the surface and (iii) to which extend the supported cell membranes are still ideal barriers for polar molecules. Finally, we will (iv) establish how the activity of ubiquinol oxidase in the supported cell membranes compares to that of supported lipid bilayers (see part 1). The outcome of these experiments could result in an easy-to-prepare system for functional studies of redox-active membrane proteins and/or ion channels. To test the wider applicability of this system we propose to test it with membranes from eukaryotic cells and from bacterial strains that over-express different classes of membrane proteins, like sugar transport proteins.