| With this proposal we want to develop novel ways to characterise all the larger (membrane) protein complexes from a specific type of membrane by transmission electron microscopy (EM) in combination with proteomics. The ultimate goal of this approach is to characterise unknown membrane proteins, to discover novel types of association between known proteins and to determine the structural changes in membranes that occur as a response to changing metabolic or stress conditions. In general, structural studies on proteins are heavily based on isolated, highly purified protein samples. For X-ray diffraction studies this is a necessity and for EM, based on crystals, the same is true. However, we want to use a non-crystallographic approach. Single particle EM is a well-developed technique to average the individual projections of large protein complexes. It makes use of sophisticated classification programs to sort out different projections. We have found that this is also possible in complex mixtures of proteins. In this way we will be able to analyse the projection structures of all larger proteins from disrupted membranes. Novel methods in the field of proteomics have dramatically increased the possibility to assign gel electrophoresis spots to specific proteins. We want to use these proteomics methods to assign the EM projection structures to specific proteins. This will be achieved by comparing the frequencies of EM structures with intensities of electrophoresis spots arising from proteins of complete membranes. As a study object we choose the photosynthetic membrane from cyanobacteria and the peroxisome membrane from yeast as starting projects. We expect that by combining EM and proteomics major new discoveries will be achieved because it is a fully novel approach. |
