Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology

Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug target, yet significant functional and structural questions remain unanswered for this superfanrily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta(2)-adrenetgic receptor (beta(2)AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta(2)AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allow., unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta(2)AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.