Student Research - Biochemistry and Molecular Biology

Sarah Wacker

Ligand Induced Changes in Stability and Allosteric Interactions in Glutamate Dehydrogenase

Abstract: Higher eukaryotic glutamate dehydrogenases exhibit complex homo and hetero tropic regulation involving complex subunit interactions and ligand induced conformational changes. The ability of a wide variety of ligands to support high activity and or cooperativity has been correlated with effects on global conformational stability investigated by heat inactivation, guanidine hydrochloride induced unfolding and DSC. Ligands are shown to either have little effect or significantly increase or decrease the global stability of the protein. Correlation of guanidine hydrochloride induced unfolding and fluorescence polarization measurements using the tryptophan fluorescence of the protein allow the dissociation of the subunits to be followed and indicates that Norvaline also affects subunit dissociation. Dynamic Light Scattering approaches are being developed to give more detail of the effects that some ligands have on the stability of either the trimer-trimer or the trimer-monomer interfaces. Limited proteolysis using immobilized trypsin or chymotrypsin in conjunction with electrophoretic and mass spectrometry analysis has allowed differential effects of some ligands to be localized to specific regions of the polypeptide chain. Overall these studies indicate that both activity and allosteric regulation of this complex enzyme is linked to changes in conformational flexibility rather that discrete ligand induced changes in conformation. A model of allosteric interactions being modulated by ligand induced changes of conformational flexibility as opposed to disctrete conformational changes is proposed.

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Author	Sarah Wacker
Mentor	J. Ellis Bell