ENGINEERING THE AGGREGATION PROPERTIES OF DODECAMERIC GLUTAMINE-SYNTHETASE - A SINGLE AMINO-ACID SUBSTITUTION CONTROLS SALTING-OUT

Escherichia coli glutamine synthetase (GS) is a dodecamer of identical subunits which are arranged as two face-to-face hexameric rings, In t he presence of 10% ammonium sulfate, wild type GS exhibits a pH-depend ent 'salting out' with a pK(a) of 4.51, Electron micrographs indicate that the pH-dependent aggregation corresponds to a highly specific sel f-assembly of GS tubules, which result from stacking of individual dod ecamers. This stacking of dodecamers is similar to the metal ion-induc ed GS tubule formation previously described, Site-directed mutagenesis experiments indicate that the N-terminal helix of each subunit is inv olved in the salting out reaction, as it is in the metal-induced stack ing, A single substitution of alanine for His4 completely abolishes th e (NH4)(2)SO4-induced aggregation. However, the H4C mutant protein doe s nearly completely precipitate under the same salting out conditions. Mutations at other residues within the helix have no effect on the st acking reaction, Differential catalytic activity of unadenylylated GS versus adenylylated GS has been used to determine whether wild type do decamers 'complement' the H4A mutant in the stacking reaction, The com plementation experiments indicate that His4 residues on both sides of the dodecamer-dodecamer interfaces are not absolutely required for sal ting out, although the wild type dodecamers clearly stack preferential ly with other wild type dodecamers, Approximately 20% of the protein p recipitated from the mixtures containing the wild type GS and the H4A mutant is the mutant, The implications of these results for protein en gineering are discussed.