This is a proposal to study the effects of selected mutations in p53 on its DNA-binding properties, on its bending effects on the bound DNA and on protein-protein self-association reactions in the presence of DNA. The proposal is developed around a core hypothesis which explains the binding of p53 to DNA in terms of: a) a binding site (response element) on DNA , the sequence of which confers on it the ability to bend coherently; b) specific contacts between the binding domain of p53 and the DNA response element; and c) interpeptide (inter- protein) binding interactions within the p53 binding domain itself that are strengthened by interaction with DNA and supported by interpeptide (inter-protein) interactions arising from - or modulated by - the tetramerization domain. Binding specificity and free energy are the net result of the free energies arising directly from protein-DNA contacts, and indirectly from protein and DNA conformational change and protein self-association reactions. The effects of protein mutations within or outside of the binding domain are to be investigated using different DNA response elements and interpreted within the framework of this hypothesis. The proposed studies will involve isolated binding domains, the extended binding domain region that also contains the tetramerization domain, and the whole protein. Mutants are chosen that do not completely block DNA binding. The effects of combinations of mutant and wild type proteins on different properties are also to be studied.
Showing the most recent 10 out of 12 publications
