Structural-functional relationships in muscle Glycogen Synthase

Glycogen Synthase (GS) and glycogenin catalyze the biosynthesis of mammalian glycogen. GS itself plays the pivotal role and is highly regulated. The great relevance of GS in overall glycogen metabolism, homeostasis of glycaemia, the pathogenesis of diabetic complications emphasizes the necessity for a better understanding of the regulatory mechanisms. This goal is presently prevented by the lack of structural information, which are essentially limited to the primary structure, the oligomeric state and the location of the phosphorylation regulatory sequences. Our present efforts are directed to define the domain structure of the active and the inactive state, taking into account the effects of the ligands. Studies were perfume on purified GS by means of i) thermal inactivation kinetics; ii) spectrofluorimetric analysis in the tryptophan emission spectrum and iii) Differential Scanning Calorimetry (DSC). Experiments were carried out on phosphorylated and dephosphorylated GS in the absence and in the presence of ligands. In this preparation, glycogenin is regularly present at tight-binding complex with GS. Experiments of thermal inactivation on both GS a and b, revealed marked differences in their inactivation kinetics. Spectra of tryptophan fluorescence emission were characterized by a biphasic profile with a maximum at 332 and 356 nm. UDO-glucose and glucose 6-Pbring about a moderate quenching of tryptophan fluorescence, with parallel contribution by buried and exposed tryptophan, suggesting that ligand induced conformational changes do not grossly modify the protein ternary structure. DSC Thermograms of unfolding purified GS a demonstrate a complex pattern with main transition at 62 C°, consisting with a structure with multiple domains with slightly different melting temperatures. AT variance, unfolding of the phosphorilated b form takes place at lower temp. (Tm 56 C°), indicative of consistent conformational changes following phosphorilation. We are now investigating effects of ligands on the DSC unfolding profile. Finally, limited proteolysis of the GS-glycogenin complex indicated extensive degradation of glycogenin, with only limited nicking of GS, as if glycogenin is located in a more superficial position in the native protein complex. The present experiments confirm that GS is a multidomain protein and that its complex regulation of catalytic activity is linked to conformational changes triggered by allosteric or by covalent modification.