GPCR–styrene maleic acid lipid particles (GPCR–SMALPs): their nature and potential

Abstract

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Non-detergent isolation of a cyanobacterial photosystem I using styrene maleic acid alternating copolymers

“…In the SMALP eld, it's recently been shown that GPCRs exhibit higher activity in vitro when extracted with SMA system as compared to detergent isolation methods, supporting the hypothesis that preservation of the native membrane environment inuences activity of membrane bound proteins. 48,66,71 The SMA buffer used in this study (pH ¼ 9.5), lack of divalent ions and high monovalent ionic strength (125 mM KCl), make this buffer system non-physiological for most proteins. Beyond that, it makes the direct comparison of DDM and SMA extracted proteins in certain downstream analyses difficult, as seen in our P700 + reduction kinetics (Fig.…”

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

“…The styrene-maleic acid (SMA) copolymer has recently gained much attention as solubilizing agent in the field of membrane protein and lipid bilayer research (for reviews, see Dörr et al (1), Lee et al (2), and Wheatley et al (3)). It has been shown that this polymer is able to directly solubilize lipid membranes into nanodisk particles without the help of detergents.…”

“…1,2-Dimyristoylsn-glycero-3-phosphocholine (di-14:0 PC) is selected as lipid because it has been used in different SMA studies where the mode of action of SMA (3,15) and the structural properties of styrene maleic acid lipid particles (14) have been characterized in molecular detail. In the systematic comparison presented here, we focus on different steps in the solubilization process of lipid membranes by SMA according to our previously proposed model (15).…”

Membrane proteins: is the future disc shaped?

“…Characterizing the function of membrane receptors like GPCRs can be achieved with binding assays, which are possible in SMALPs [18,41]. For channels and transporters it relies on measuring changes in the concentration of the substrate on either side of the protein, which generally requires reconstitution of the protein into proteoliposomes or another bilayer system.…”