Detergent-free solubilisation &amp; purification of a G protein coupled receptor using a polymethacrylate polymer

Lavington, Steven; Watts, Anthony

doi:10.1016/j.bbamem.2020.183441

Cited by 14 publications

(16 citation statements)

References 37 publications

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“…Despite the anticipated advantages over SMA, only recently have the MP solubilisation attributes of the polymer begun to be assessed. Lavington and Watts [ 74 ] successfully performed detergent-free PMA solubilisation of the GPCR neurotensin receptor 1 (NTR1) [ 74 ]. PMA displayed solubilisation efficacy comparable to that of conventional detergents in relatively mild solubilisation conditions (26°C pH 7.4–7.6) with no precipitation in the presence of divalent cations thus showing clear advantage over SMA [ 74 ].…”

Section: Nanodiscsmentioning

confidence: 99%

“…Lavington and Watts [ 74 ] successfully performed detergent-free PMA solubilisation of the GPCR neurotensin receptor 1 (NTR1) [ 74 ]. PMA displayed solubilisation efficacy comparable to that of conventional detergents in relatively mild solubilisation conditions (26°C pH 7.4–7.6) with no precipitation in the presence of divalent cations thus showing clear advantage over SMA [ 74 ]. Such characteristics imply PMA may yet provide a legitimate membrane mimetic for use in biophysical and high-resolution structural studies.…”

Section: Nanodiscsmentioning

confidence: 99%

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Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

Ratkeviciute

Cooper

Knowles

2021

Biochemical Society Transactions

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The solubilisation of membrane proteins (MPs) necessitates the overlap of two contradictory events; the extraction of MPs from their native lipid membranes and their subsequent stabilisation in aqueous environments. Whilst the current myriad of membrane mimetic systems provide a range of modus operandi, there are no golden rules for selecting the optimal pipeline for solubilisation of a specific MP hence a miscellaneous approach must be employed balancing both solubilisation efficiency and protein stability. In recent years, numerous diverse lipid membrane mimetic systems have been developed, expanding the pool of available solubilisation strategies. This review provides an overview of recent developments in the membrane mimetic field, with particular emphasis placed upon detergents, polymer-based nanodiscs and amphipols, highlighting the latest reagents to enter the toolbox of MP research.

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Section: Nanodiscsmentioning

confidence: 99%

Section: Nanodiscsmentioning

confidence: 99%

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

Ratkeviciute

Cooper

Knowles

2021

Biochemical Society Transactions

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“…The interactions of lipids including gangliosides in the outer leaflet and PIs in the inner leaflet can be modelled and reveal multiple potential binding sites in GPCRs of either class A [ 48 ] or class B [ 49 ], illustrating such multivalent interactions ( Figure 3 ). NTSR1 complexes have been expressed in insect cells and solubilized using polymethacrylate (PMA) co-polymer, yielding nanodiscs with higher levels of stimulation of both G q and G i1 heterotrimers than detergent micelle preparations [ 50 ]. PMA offers different sidechains and greater polycation compatibility than SMA, although the yields and purities are lower than those achieved with detergents.…”

Section: Native Gpcr–lipid Complexesmentioning

confidence: 99%

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

et al. 2021

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Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

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“…PMA co-polymers are a third example of alternative polymers and are notable in that their structures are not based on the SMA scaffold (Yasuhara et al 2017). In all cases, these polymers are capable of producing discoidal bilayers with diameters in the 10-20-nm range and have been used to solubilise and purify specific membrane proteins into nanoparticles from biological membranes (Oluwole et al 2017a;Barniol-Xicota and Verhelst 2018;Hall et al 2018;Lavington and Watts 2021).…”

Section: Polymer Lipid Nanoparticlesmentioning

confidence: 99%

“…Whilst the material was not purified, detailed analysis of the stability and activity of the receptor in the resulting nanoparticles showed that receptor ligand binding affinity was similar in DIBMA nanoparticles, membranes and detergent, whilst receptor thermostability was improved by 10°in DIBMA nanoparticles versus detergent (Harwood et al 2020). We have shown that a polymethacrylate (PMA) co-polymer can be used to solubilise NTSR1 from Sf9 membranes (Lavington and Watts 2021). NTSR1 solubilised with PMA can be purified in the presence of divalent cations, with the resulting material showing stimulation of both G q and G s heterotrimers to a degree that is comparable to higher concentrations of detergent-purified NTSR1.…”

Section: Polymer Lipid Nanoparticlesmentioning

confidence: 99%

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

Lavington

Watts

2020

Biophys Rev

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G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.

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Detergent-free solubilisation & purification of a G protein coupled receptor using a polymethacrylate polymer

Cited by 14 publications

References 37 publications

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

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