AAA+ ATPases convert ATP hydrolysis into mechanical work. It is clear that both human cells and disease causing pathogens use these protein complexes to physically manipulate orther proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. The structural basis on which these molecular machines convert ATP hydrolysis into mechanical work, however, is not known. Such knowledge is vital, not only to our fundamental understanding of energy coupling in general, but also to providing clues to manipulate these proteins to promote human health. Indeed, many diseases are associated with defects in one or more of the 80 AAA+ ATPases that are encoded in the human genome. A major impediment to delineating the mechanisms has been our inability to probe detailed conformational changes that are related to steps in ATP binding, hydrolysis, and product release. We hypothesize that defects in these ATPases will manifest themselves in the manner by which these molecular machines cycle through different stages of ATP hydrolysis. We propose to use novel ensemble scattering and fluorescence single-molecule methods, which are complementary to each other, to aquire solution-phase structural knowledge both under equilibrium and in a time-dependent way. To this end, we will use the highly tractable NtrC (from Escherichia coli) and NtrC1 (from Aquifex aeolicus) proteins as models. These proteins interact with the bacterial transcriptional factor, sigma-54, to remodel RNA polymerase to initiate transcription.
In Aim I, the conformational changes associated with different stages of catalysis will be identified using small- and wide- angle x-ray scattering (SAXS & WAXS). Defects in structural dynamics that are associated with crucial amino acid substitutions will also be determined using single-molecule spectroscopic approaches.
In Aim II, the nucleotide-dependent conformational changes that are associated with the formation of the activator/sigma-54 complex will be identified using both SAXS/WAXS and small-angle neutron scattering (SANS). This will allow us to define the functional roles of nucleotide-dependent conformational changes in these molecular machines. In the course of performing this research, new tools will be developed that are expected to be broadly applicable to similar studies of other proteins that are vital for human health. ? ? ?
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