Stability and oligomerization of recombinant GadX, a transcriptional activator of the Escherichia coli glutamate decarboxylase system.
One of the most important strategies that enteric bacteria adopt for maintaining the cytoplasmic pH neutral under acid stress involves the glutamate decarboxylase (Gad) system. The system works by the concerted action of a cytoplasmic, pyridoxal 5'-phosphate-dependent glutamate decarboxylase and a transmembrane antiporter, which imports glutamate and exports gamma-aminobutyrate (GABA), the decarboxylation product, thereby providing local buffering of the extracellular environment. Herein, we provide a preliminary biochemical characterization of GadX, an activator of the Gad system belonging to the AraC/XylS family of bacterial transcriptional regulators. The GadX protein has been purified as a chimeric MalE-GadX with a yield of 15-20 mg/l of bacterial culture. The fusion protein is fairly stable, although a conformational change occurs upon storage, which reduces the binding affinity by a factor of 2, without affecting the binding pattern. Partial removal of the MalE moiety from the fusion protein triggers the formation of a species which is likely to be a heterodimer, or a higher oligomer, of the type GadX/MalE-GadX. This experimental evidence is in line with the well-known tendency of AraC/XylS-like proteins to dimerize via their N-terminal domain.
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Dipartimento di Scienze Biochimiche A. Rossi Fanelli and Instituto di Biologia e Patologia Moleculari del CNR, Università degli Studi di Roma La Sapienza, PiazzaleAldo Moro 5, 00185 Rome, Italy.
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Bossa
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De Biase
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D051777:AraC Transcription Factor / Q000737:chemistry*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005968:Glutamate Decarboxylase / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D033902:Periplasmic Binding Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015534:Trans-Activators / Q000737:chemistry*
D051777:AraC Transcription Factor; D029968:Escherichia coli Proteins; C446316:GadX protein, E coli; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D005968:Glutamate Decarboxylase