Introduction. G protein-coupled receptors (GPCRs) are the largest class of signalling proteins in humans. They typically bind to an extracellular ligand and following a conformational change transduce the signals intracellularly by coupling to G proteins. Although recent crystallography studies have advanced our understanding of the structure of GPCRs, the structural dynamics underlying the signal transducing process remain poorly understood. In principle, such dynamics could be obtained by NMR. The α1A and α1B adrenergic receptors are essential to hypertrophic responses and neuromodulation. However, like all GPCRs these receptors are unstable in detergent micelles. We have engineered detergent-stable variants of human α1AAR and α1BAR using directed evolution methods (1) which has enabled biophysical characterization by techniques such as NMR spectroscopy.
Aims. To characterize and compare the conformational dynamics of α1A/1BAR in the apo and ligand bound states.
Methods. The methyl groups of the amino acids have favourable relaxation properties. As these residues are well dispersed in proteins they serve as excellent probes for monitoring subtle conformational changes. As an initial experiment we have labelled α1AAR with 13CH3-methionine.
Results and Discussion. We obtained 13C,1H two dimensional spectra of unliganded receptor as well as receptor bound to an inverse agonist and agonist. Through single site mutations, we have assigned all six methionines in α1AAR. Importantly, methionines distant from the orthostatic binding site showed significant changes on binding agonist, including line broadening suggesting an increase in protein motion whereas binding of antagonist showed line narrowing suggesting a reduction in motion. Also, methionines near the ligand binding pocket undergo peak shifting when bind to different ligands. So current results show valuable dynamics information of α1-adrenoceptor.