Ligand binding and structural properties of segments of GABA(A) receptor α1 subunit overexpressed in Escherichia coli

Abstract

The γ-aminobutyric acid, type A (GABA(A)), receptor is the target for numerous therapeutic compounds. In the present study, the Gln28-Leu296, Gln28-Arg276, Gln28-Arg248, and Gln28-Glu165 (numbering of bovine precursor protein) segments of its α1 subunit were overexpressed in Escherichia coli, along with Cys166-Leu296 produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy. Results showed that the protein segments were rich in β-sheet structures. Binding of the fluorescent benzodiazepine Bodipy-FL Ro-1986 was evident from fluorescence resonance energy transfer and fluorescence anisotropy measurements. The binding affinity was in the micromolar range. The binding was attributable more to Cys166-Leu296 than to Gln28-Glu165 and was inhibited by known central benzodiazepine site ligands. Three point mutations, Y187A, T234A, and Y237A, were found to perturb protein secondary structures. Studies with the single Trp mutants W198Y and W273Y indicated that Trp273 was closer to the binding site than Trp198.