Description

3Phosphoglycerate Dehydrogenase is a tetramer with three different types of subunit interactions, one at the serine binding regulatory domain, one at the cofactor binding domain, and a third between residues that interact across the central cavity of the tetramer. The serine domain interactions have been well documented but interactions involving the other interfaces are not well characterized. A single tryptophan per subunit lies at the cofactor interface and crosses over between subunits. To examine the effects of substrates on subunit interactions we have used a protein fluorescence collision quenching approach with both charged and neutral quenchers to investigate the effects of ligands on both the exposure and asymmetry of the cofactor subunit interfaces of the native enzyme and a mutant, D264N, shown to have altered cofactor binding and is not regulated by serine binding. Studies with guanidine hydrochloride unfolding have been used to assess the impact of ligands on overall stability and the cooperativity of unfolding. To develop an alternative approach to investigate the strength of subunit interactions and the stability of the structure, the protein has been histidine tagged at the C terminal and preliminary work is underway to attempt to utilize atomic force microscopy to further probe the effects of ligands in both global and interface stability.

This work is supported by NSF Grant MCB 0448905 to EB

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