SUPRAMOLECULAR SELF-ASSEMBLY OF ESCHERICHIA-COLI GLUTAMINE-SYNTHETASE- CHARACTERIZATION OF DODECAMER STACKING AND HIGH-ORDER ASSOCIATION

Dodecameric glutamine synthetase (GS) from bacteria is formed from two face-to-face hexameric rings of identical subunits. These highly symm etrical aggregates from some bacteria, including Escherichia coli, ''s tack'' in the presence of Zn2+ and other divalent ions to generate pro tein tubes (phase I) and subsequently associate side-to-side to yield ''cables'' and nonspecific aggregates (phase II), In order to understa nd the molecular mechanisms of recognition leading to this macromolecu lar self-assembly, the effects of solution conditions on the kinetics of these processes have been studied. These reactions have been monito red by changes in light scattering and by electron microscopy. Conditi ons' have been established for isolation of phases I and II. At 0.04 m g of GS/mL, pH 7.0, 100 mM KCl, and 1 mM Mn2+, 25 degrees C, minimal s ide-to-side aggregation occurs, and the stacking reaction follows seco nd-order kinetics, with respect to GS, at low extent of reaction. The second-order rate constants determined for phase I, initiated by Zn2or Co2+, demonstrate a pH optimum at 7.0-7.25, whereas phase II is fav ored at pHs below 6.5. The pH profile for the stacking reaction sugges ts that His residues are involved, and modification of 2-3 histidines/ subunit with diethyl pyrocarbonate (DEPC) is sufficient to completely inhibit metal-dependent dodecamer stacking. The effect of ionic streng th on GS stacking was also studied. Although hydrophobic interactions have previously been assumed to dominate this protein-protein associat ion, both phase I and phase II of the assembly are inhibited by KCl an d NaCl, suggesting that ionic interactions also play an essential role . Lastly, the metal ion specificity for the phase I reaction was deter mined. Although Zn2+- and Co2+-mediated GS stacking has been studied m ost extensively, Cu2+ is a more potent initiator of tubule assembly. T he concentration of Cu2+ required for half-maximal rates of stacking ( C-1/2) is 80 mu M. Of the metal ions examined, the order of the effica cy for supporting GS stacking is Cu2+ > Zn2+, Hg2+ > Cd2+ >> Ni2+, Co2 +. The divalent ions Fe2+ and Ca2+ do not support GS stacking. These s tudies indicate that phases I and II of GS association can be controll ed through manipulation of the solution conditions.