Abstract
Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.
Highlights
Gprotein-coupled receptors (GPCRs) play a central role in cell-cell communication and represent the largest group of membrane proteins with over 800 members in the human genome
In Vitro Folding of the Y type 2 receptor (Y2R) into Bicelles
Using lower sodium dodecyl sulfate (SDS)/Y2R ratios resulted in lower reconstitution yields in the subsequent folding step 2, while higher ratios lowered the proportion of active protein
Summary
Gprotein-coupled receptors (GPCRs) play a central role in cell-cell communication and represent the largest group of membrane proteins with over 800 members in the human genome. These molecules transduce signals across the cell membrane via complex formation with extracellular ligands and intracellular interaction partners, namely G-proteins, kinases, and arrestins (Wu et al, 2017). The dynamic nature of these binding processes has recently been shown in structural detail for the ß2-adrenergic receptor (Manglik et al, 2015) and the A2A adenosine receptor (Ye et al, 2016) Influencing these signal transduction pathways holds great potential for pharmaceutical research. Structure based design of highly specific agonists and antagonists targeting GPCRs with reduced side effects requires comprehensive knowledge about the structure and dynamics of these membrane embedded molecules at different stages in their signaling process
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