G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

Abstract

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. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]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.

Overcoming bottlenecks in the membrane protein structural biology pipeline

“…Proteins within SMALPs have been shown to bind ligands comparably to those in the native membrane [44]. The use of SMALPs has facilitated improved thermostability and homogeneity of membrane proteins such as P-glycoprotein (P-gp) and the adenosine A2a receptor [39,42,44]. To date no crystal structure has been reported using SMALPs but both negative stain and cryo-EM low resolution structural maps have been determined [39,43].…”

“…During the formation of SMALPs from a biological membrane, membrane proteins can become trapped inside these discs (as shown in Figure 1D), solubilising them into small particles (much like nanodiscs when using membrane scaffolding proteins), but without the need for detergent at any stage. The proteins within a SMALP can be effectively purfied using affinity chromatography [39,[42][43][44]. The particles are stable once formed so there is no need to supplement buffers during purification as there is when using detergents; this is a major cost advantage.…”

“…The membrane protein that is encompassed inside the SMALP is now stable in its native lipid environment, allowing access to both sides of the membrane protein and aiding in functional and structural studies. Proteins within SMALPs have been shown to bind ligands comparably to those in the native membrane [44]. The use of SMALPs has facilitated improved thermostability and homogeneity of membrane proteins such as P-glycoprotein (P-gp) and the adenosine A2a receptor [39,42,44].…”

Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers

“…It has been shown that this polymer is able to directly solubilize lipid membranes into nanodisk particles without the help of detergents. In this way, membrane proteins can be solubilized in their native lipid environment (4-7), which was found to help stabilize the protein (5,6,8). This has opened options to purify membrane proteins that are unstable in detergent micelles and to study native protein-lipid interactions by biochemical methods.…”

“…A further advantage of these so-called native nanodisks is that they are small, with sizes in the range of 10-25 nm (4)(5)(6)(9)(10)(11)(12)(13)(14)(15). This makes them suitable to be characterized by a variety of biophysical approaches including UV/Vis-and fluorescence spectroscopy (5,6,8), as well as light scattering techniques (6,14).…”