The long-term goal of this work is to understand the mechanism of coupling in F1F0 ATP synthase. Most organisms synthesize ATP via F1F0 ATP synthases so these enzymes are largely responsible for the central function of energy metabolism. In humans, failures in energy metabolism are associated with aging and a variety of inherited disorders. For example, the disease in certain individuals with Leigh's Syndrome is directly attributed to mutations affecting F1F0 ATP synthase. Prokaryotic and eucaryotic F1F0 ATP synthases share common structures and mechanisms. Therefore, the Escherichia coli F1F0 ATP synthase has been chosen as a model. The E. coli system offers a well-characterized enzyme and the use of the broadest range of molecular genetic techniques. Recently, much attention has been focussed on the two stalk structures linking the F1 and F0 sectors of the enzyme. The central stalk is thought to rotate within the enzyme complex during catalysis, while the peripheral stalk serves as a stator holding the bulk of F1 in place. This has led to the widely held view that the peripheral stalk is a rigid static structural feature, but this interpretation is not consistent with all experimental observations. It is hypothesized that the peripheral stalk plays a dynamic role in coupling. The following Specific Aims are proposed to investigate this concept: 1) determining the length of the peripheral stalk in enzymes with deletions in the b subunits; 2) studying the site of interaction between the a and b subunits; 3) investigating the dynamics of the b subunit interaction with F1 by looking for evidence of transient dissociation of the peripheral stalk; 4) studying the preferred intermediates in the assembly of F1F0 ATP synthase.
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