Interaction of Cibacron Blue F3GA with glutamine synthetase: use of the dye as a conformational probe. 1. Studies using unfractionated dye samples.
Cibacron Blue F3GA dye has been used to probe subtle conformational changes in protein structure associated with the conversion of Escherichia coli glutamine synthetase (GS) between relaxed, taut, oxidized, and dissociated forms. Binding of the dye to each form of the enzyme elicits a different spectral perturbation of the dye which can be detected by difference spectroscopy. By following time-dependent changes in the difference spectrum associated with the binding of dye to the enzyme, it was demonstrated that dissociation of subunits provoked either by urea or by relaxation of the enzyme at pH 8.5 is a multiphasic process. In the presence of 3-4 M urea, dissociation of taut GS is associated with an almost instantaneous, transient increase in absorbancy of the difference spectrum at 638 nm and, after a lag, by a progressive decrease in absorbancy at 585 nm and an increase at 700 nm. The kinetics of these changes vary as a function of temperature, pH, and the concentrations of KCl, MnCl2, and urea, probably reflecting differences in the rates of GS relaxation and in the formation of aggregates of intermediate sizes. Results of direct binding measurements show that the taut and relaxed forms of GS can bind only 1-1.3 equiv of dye per subunit, whereas dissociated subunits bind up to 3.0 equiv per subunit. The Kd of the dye-taut GS complex as calculated from binding data was 0.55 microM. The binding of dye to taut GS was inhibited by its substrate, ADP, and by the allosteric effectors AMP and tryptophan. On the basis of the abilities of ADP, AMP, and tryptophan to inhibit the binding of dye to GS, dissociation constants of the respective GS-ligand complexes were 2.4, 121, and 1170 microM, respectively, in good agreement with previously determined values. From the difference spectra obtained between a given concentration of dye in a 5.0-cm cell and 10 times that concentration in a 0.5-cm cell, it was established that at concentrations greater than 5 microM a significant fraction of the dye is present as stacked aggregates. Because only the dye monomer binds to GS, the difference spectrum between dye and dye bound to GS is due in part to GS-promoted shifts in the equilibrium between stacked and unstacked dye molecules. Consequently, with increasing dye concentrations, the amplitude of the dye vs. dye + GS difference spectrum can continue to increase, even after the GS becomes saturated with dye.(ABSTRACT TRUNCATED AT 400 WORDS)
Federici
M M
MM
Chock
P B
PB
Stadtman
E R
ER
D000227:Adenine Nucleotides / Q000494:pharmacology; D004396:Coloring Agents; D004926:Escherichia coli / Q000201:enzymology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012492:Salts; D013053:Spectrophotometry; D014227:Triazines; D014364:Tryptophan / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D000227:Adenine Nucleotides; D004396:Coloring Agents; D012492:Salts; D014227:Triazines; C012842:Cibacron Blue F 3GA; D014364:Tryptophan; D014508:Urea; D005974:Glutamate-Ammonia Ligase