new approaches for fresh perspectives on quinol/quinone oxidoreductases

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All living cells are surrounded by a thin membrane that shields and separates the inside of these cells from their surroundings. In more advanced organisms, organelles are located inside the cell with specific functions. Also these organelles are separated (compartmentalised) from the rest of the cell by membranes. These thin membranes contain many proteins that actively transport compounds, like nutrients and salt, specifically across the membrane. 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 them, like photosynthesis and metabolism. Some of these proteins actively 'pump' protons across the membrane using energy that is released from electrons that are formed when sugars and fats are 'burned' by the cell. These electrons do not flow freely in the cell, but are attached to small molecules which 'float' in the membranes of the cell. These molecules are called quinones or co-enzyme Q. This proposal aims to develop a new tool with which we can study the proteins that are located in the membrane and react with quinones. Why do we want to learn more about these proteins? These proteins are involved in many important reactions. For instance, in bacteria they are responsible for all reactions involving nitrogen and carbon dioxide and therefore control how these elements are recycled in our atmosphere. In humans, similar proteins are involved in the burning of sugars and fat and the production of energy; Any problems with these proteins and we become ill. Finally, quinones themselves are 'anti-oxidants' and known to take away so-called 'radicals' which are thought to play an important role in diseases and aging. When we study the structure and function of proteins and quinones in the lab, they are normally taken out of the membrane and thus the environment of these proteins and quinones is changed a great deal. This is done because membranes do not dissolve in water and most of our experiments are performed in water; we thus need to take the membrane away. However, in this proposal we aim to develop a new tool that allows the study of membrane proteins and the quinone in their natural environment, the membrane. For this to be achieved, we will first place a 'membrane protein' that normally receives or gives electrons to the quinones on a solid surface. This solid is conducting (like metal wires) and we will carefully control the properties of the surface so that it will be possible to give or take electrons to or from the protein. We will then place a membrane on top of the proteins and this membrane will contain quinones. If everything works as we think it will, the protein will give or take electrons to or from the quinones. As the transfer of electrons is nothing more than electrical current, we can measure very accurately how fast these electrons are passed from the surface to the proteins and into the quinones (or the other way around). Once this system is complete, we can use these surfaces to 'interrogate' these membrane proteins in almost the same membrane environment they encounter in the cell. By studying these proteins we will thus learn more about how they function inside their natural membrane.