Publication Date:
2015-02-28
Description:
G-protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. Here we report the first solubilization and purification of a functional GPCR (human adenosine A 2A receptor; A 2A R), in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid co-polymer (SMA) direct from the membrane into a nanoscale SMA-lipid particle (SMALP). Furthermore, the A 2A R-SMALP, generated from yeast ( Pichia pastoris ) or mammalian cells, exhibited increased thermostability (~5 °C) compared to detergent ( n -dodecyl- β -D-maltopyranoside)-solubilized A 2A R controls. The A 2A R-SMALP was also stable when stored for prolonged periods at 4 °C and was resistant to multiple freeze-thaw cycles, in marked contrast to the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast to nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, Circular Dichroism spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([ 3 H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
Print ISSN:
0144-8463
Electronic ISSN:
1573-4935
Topics:
Biology
,
Chemistry and Pharmacology
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