Abstract
The gamma-aminobutyric acid, type A (GABA(A)), receptor is the target for numerous 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 bovine precursor protein) segments of its alpha(1) subunit were overexpressed in Escherichia coli, along with Cys(166)-Leu(296) produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy. Results showed that the protein segments were rich in beta-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 Cys(166)-Leu(296) than to Gln(28)-Glu(165) 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 Trp(273) was closer to the binding site than Trp(198).
Highlights
GABAA receptors serve as the targets of many important neuroactive drugs (6 – 8), and substantial effort has been made to understand the structure of BZ-binding site [9]
Studies with the single Trp mutants W198Y and W273Y indicated that Trp273 was closer to the binding site than Trp198
Sitedirected mutagenesis was applied to the three residues Tyr187, Thr234, and Tyr237, previously identified to be important for ligand binding [16, 17, 18], and to the two Trp residues Trp198 and Trp273 in order to gain insight into the structure-function relationships of BZ binding
Summary
Materials—The cDNA clone, pCDM8-b␣1, encoding bovine ␣1 subunit was a gift from Dr A.
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