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 symmetrical aggregates from some bacteria, including Escherichia coli, "stack" in the presence of Zn2+ and other divalent ions to generate protein tubes (phase I) and subsequently associate side-to-side to yield "cables" and nonspecific aggregates (phase II). In order to understand the molecular mechanisms of recognition leading to this macromolecular self-assembly, the effects of solution conditions on the kinetics of these processes have been studied. These reactions have been monitored by changes in light scattering and by electron microscopy. Conditions have been established for isolation of phases I and II. At 0.04 mg of GS/mL, pH 7.0, 100 mM KCl, and 1 mM Mn2+, 25 degrees C, minimal side-to-side aggregation occurs, and the stacking reaction follows second-order kinetics, with respect to GS, at low extent of reaction. The second-order rate constants determined for phase I, initiated by Zn2+ or Co2+, demonstrate a pH optimum at 7.0-7.25, whereas phase II is favored at pHs below 6.5. The pH profile for the stacking reaction suggests 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 strength on GS stacking was also studied. Although hydrophobic interactions have previously been assumed to dominate this protein-protein association, both phase I and phase II of the assembly are inhibited by KCl and NaCl, suggesting that ionic interactions also play an essential role.(ABSTRACT TRUNCATED AT 250 WORDS)
Yanchunas
J
J
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle 98195.
Dabrowski
M J
MJ
Schurke
P
P
Atkins
W M
WM
D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D006207:Half-Life; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008027:Light; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012542:Scattering, Radiation; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D046911:Macromolecular Substances; D003035:Cobalt; D006639:Histidine; D011189:Potassium Chloride; D003300:Copper; D005974:Glutamate-Ammonia Ligase; D015032:Zinc