Investigation of an energetic coupling between ligand binding and protein folding
The cellular environment presents a protein with many small molecules with which it may interact. Many novel interactions between proteins and non-substrate metabolites are being uncovered through proteome-wide screens. The homodimeric Escherichia coli cofactor-dependant phosphoglycerate mutase (dPGM) was identified as an ATP binding protein in a proteome-wide screen, but dPGM does not use ATP for catalysis. This dissertation elucidates the effect of ATP and other non-substrate metabolites on dPGM. Initial investigations revealed a partially unfolded, monomeric intermediate of dPGM that forms during equilibrium unfolding. ATP binding was found to occur at the active site of dPGM and to be energetically coupled with dimerization; ligand binding events reduce the population of intermediate. An investigation into the structure of the dPGM intermediate revealed a cooperative folding unit that couples the active site and dimer interface of dPGM. By coupling the two binding sites, the cooperative unit is responsible for conveying the allosteric effect observed between dimerization and ligand binding. We found that physiological salts reduce but do not prevent non-substrate metabolite binding at physiological concentrations. Further, anions bind specifically to dPGM and chloride was found to bind to both of the energetically coupled sites on dPGM, the active site and dimer interface. Our findings illustrate how a cooperative link between a ligand binding site and oligomer interface can promote higher order oligomers and reduce intermediate populations. The physiological effect of the cooperative link and ligand binding to dPGM is a large enhancement in the stability of the dimer over the monomer intermediate and, possibly, competitive inhibition.
Park, Purdue University.
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