Ligand binding and structural properties of segments of GABA(A) receptor alpha(1) subunit overexpressed in Escherichia coli

The gamma-aminobutyric acid, type A (GABA(A)), receptor is the target for n umerous therapeutic compounds. In the present study, the Gln(28)-Leu(296), Gln(28)-Arg(276), Gln(28)-Arg(248), and Gln(28)-Glu(165) (numbering of bovi ne precursor protein) segments of its alpha(1) subunit were overexpressed i n Escherichia coli, along with Cys(166)-Leu(296) produced previously, for s tructural analysis by circular dichroism and ligand binding studies by fluo rescence spectroscopy, Results showed that the protein segments were rich i n beta-sheet structures. Binding of the fluorescent benzodiazepine Bodipy-F L Ro-1986 was evident from fluorescence resonance energy transfer and fluor escence anisotropy measurements. The binding affinity was in the micromolar range. The binding was attributable more to Cys(166)-Leu(296) than 60 Gln( 28)-Glu(165) and was inhibited by known central benzodiazepine site ligands , Three point mutations, Y187A, T234A, and Y237A, were found to perturb pro tein secondary structures. Studies with the single Trp mutants W198Y and W2 73Y indicated that Trp(273) was closer to the binding site than Trp(198).