The aim of our research is to determine the structure of enzymes and supercomplexes that make up the mitochondrial and bacterial respiratory chain by cryo-electron microscopy and state of the art image analysis techniques. The importance of understanding the function and relationships between enzymes in the respiratory chain is highlighted by the fact that many mitochondrial diseases can be traced back to defects in the genes encoding complex I. Examples are LHON-disease (Leber's hereditary optic neuropathy), Ligh's disease and Kearns-Sayre syndrome. We will determine the native structures of complex I from the strictly aerobic yeast Yarrowia lipolytica and from the bacterium Aquifex aeolicus. The arrangement of the subunits in complex I will be determined. Knowledge of their architecture within the complex will provide the information needed to test existing functional models and identify the subunits involved. Three-dimensional immuno cryo-electron microscopy with monoclonal and polyclonal antibodies against different subunits will be used to localize subunits and determine their architecture in the complex. We will calculate structures of the complex with and without antibody labeling. The location of the binding epitopes will be determined from the difference between each two structures in three dimensions. We will further analyze supercomplexes of the complete electron transfer chain from the membrane of P. denitrificans to investigate direct coupling of the enzymes. We will determine the structure of this supercomplex by three-dimensional electron microscopy using either tomographic techniques of individual particles or the Random Conical Reconstruction Technique applied to large data sets with extensive classification. Antibody labeled supercomplexes will be analyzed to identify the relative arrangement of the individual electron transfer chain complexes within the supercomplex. ? ?
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