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Ligand Induced Changes in Stability or Local Conformational Flexibility and Allosteric Interactions in Glutamate Dehydrogenase
Author(s) -
Scheel Carolyn,
Bell Ellis
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a647
Subject(s) - allosteric regulation , chemistry , guanidine , protein subunit , cooperativity , biophysics , ligand (biochemistry) , conformational change , protein structure , stereochemistry , crystallography , enzyme , biochemistry , receptor , biology , gene
Higher eukaryotic glutamate dehydrogenases exhibit complex homo and hetero tropic regulation involving 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 differential scanning calorimetry. 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, fluorescence polarization measurements using the tryptophan fluorescence of the protein and dynamic light scattering allow the dissociation of the subunits to be followed and indicates that Norvaline also affects subunit dissociation. Initial limited proteolysis studies using immobilized trypsin or chymotrypsin is allowing differential effects of some ligands to be localized to specific regions of the polypeptide chain. GDH is digested with immobilized protease and the time dependence of fragmentation followed using MALDI tof mass spectrometry using Protein Prospector to identify cleavage sites from the masses of the resultant fragments. Overall these studies indicate that both activity and allosteric regulation of this complex enzyme are 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 discrete conformational changes is proposed. This work is funded by NSF Grant MCB 0448905 to EB