Characterization of memebrane-proximal b-rich fragment of GABA̳A receptor α 1 subunit via alanine scanning method
by Liao Yinglei
THESIS
2000
M.Phil. Biochemistry
xiii, 128 leaves : ill. ; 30 cm
Abstract
GABAA receptors are GABA (γ-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. A...[ Read more ]
GABAA receptors are GABA (γ-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs.
A membrane-proximal β-rich fragment of GABAA receptor containing key ligand-binding residues has been successfully overexpressed in E. coli in our lab (Hong Xue,et al.,1997). Binding assay using the method of fluorescence anisotropy shows that this fragment (C166-L296) bear moderate affinity to benzodiazepine ligand. Alanine scanning combined with CD and fluorescent assay method was carrried out on this fragment to investigate the individual contribution of each residue to both the secondary structure and the binding affinity and to further study the structure-function relationship of this fragment.
Present study manifests that substitution by alanine on residues R191, S213, R214, N216, F244, Y252, F253, F272 and L291 result in considerably reduced binding affinity. Among these, the loss of binding affinity on muants R191A, S213A, R214A, N216A are directly as a result of loss of side chain interaction when binding to the ligand, which means residues R191, S213, R214 and N216 may possibly involved in binding site. Nevertheless, the binding affinity loss of mutants F244A, Y252A, F272A and L291A are due to indirect change of structure rather than direct loss of side chain interaction.
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