Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli.
The gamma-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A for the protein-detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K(d)) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 microM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.
Li
X D
XD
Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA.
Villa
A
A
Gownley
C
C
Kim
M J
MJ
Song
J
J
Auer
M
M
Wang
D N
DN
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005453:Fluorescence; D018755:GABA Agonists / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009118:Muscimol / Q000378:metabolism; D009557:Nipecotic Acids / Q000378:metabolism; D027361:Organic Anion Transporters; D011485:Protein Binding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D018755:GABA Agonists; D050485:GABA Plasma Membrane Transport Proteins; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; D011993:Recombinant Fusion Proteins; C513601:gabP protein, E coli; C030278:nipecotic acid; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D013917:Thrombin