Biophysical characterisation of SMALPs.

Membrane proteins such as receptors, ion channels and transport proteins are important drug targets. The structure-based study of membrane proteins is challenging, especially when the target protein contains both soluble and insoluble domains. Most membrane proteins are insoluble in aqueous solvent and embedded in the plasma membrane lipid bilayer, which significantly complicates biophysical studies. Poly(styrene-co-maleic acid) (SMA) and other polymer derivatives are increasingly common solubilisation agents, used to isolate membrane proteins stabilised in their native lipid environment in the total absence of detergent. Since the initial report of SMA-mediated solubilisation, and the formation of SMA lipid particles (SMALPs), this technique can directly isolate therapeutic targets from biological membranes, including G-protein coupled receptors (GPCRs). SMA now allows biophysical and structural analyses of membrane proteins in solution that was not previously possible. Here, we critically review several existing biophysical techniques compatible with SMALPs, with a focus on hydrodynamic analysis, microcalorimetric analysis and optical spectroscopic techniques.

Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N',N'-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a 'toolkit' of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.