Membrane Proteins Solubilized Intact in Lipid Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer

Knowles, Timothy J.; Finka, Rachael; Smith, Corinne J.; Lin, Yu‐Liang; Dafforn, Timothy R.; Overduin, Michael

doi:10.1021/ja810046q

Cited by 473 publications

(537 citation statements)

References 13 publications

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“…SMA preparation SMA polymer was prepared as described previously [17]. Briefly, a 10% (w/v) solution of SMA copolymer in 1M NaOH was refluxed for 2 hours, allowed to cool to room temperature, then dialysed against 20mM Tris buffer pH8 before use.…”

Section: Methodsmentioning

confidence: 99%

“…Here we report the application of a recently developed system for detergent-free extraction of ABC transporters from their native membranes using a readily available, biocompatible and chemically stable polymer, polystyrene-co-maleic acid (SMA) [17][18][19]. The polymer inserts into the membrane isolating small discs of lipid bilayer that are encircled by the polymer, termed SMA lipid particles (SMALPs) [17].…”

Section: Introductionmentioning

confidence: 99%

“…The polymer inserts into the membrane isolating small discs of lipid bilayer that are encircled by the polymer, termed SMA lipid particles (SMALPs) [17]. We demonstrate that the polymer is able to effectively solubilise several different eukaryotic ABC transporters from a range of different cell types, including mammalian cells, insect cells and yeast.…”

Section: Introductionmentioning

confidence: 99%

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Detergent-free purification of ABC (ATP-binding-cassette) transporters

Gulati

Jamshad

Knowles

et al. 2014

Biochemical Journal

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176

204

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Abstract:ABC (ATP Binding Cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilises detergents to solubilise the protein from the membrane, effectively removing it from its native lipid environment. Subsequently lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. We present here the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead using a styrene maleic acid co-polymer (SMA). SMA inserts in a bilayer and assembles into discrete particles, essentially solubilising the membrane into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). We show that this polymer can extract several eukaryotic ABC transporters; P-glycoprotein (ABCB1), MRP1 (ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (ABCC7), from a range of different expression systems. The SMALP encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably to those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared to detergent solubilisation. This study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function. Summary statement:A styrene maleic acid copolymer can be effectively used to extract and purify large eukaryotic transmembrane proteins in the absence of detergents, forming small bilayer discs encapsulating the protein, which have great potential for future structure & function studies.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Detergent-free purification of ABC (ATP-binding-cassette) transporters

Gulati

Jamshad

Knowles

et al. 2014

Biochemical Journal

Self Cite

176

204

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“…This copolymer has the ability to solubilise lipid membranes into nanodisc-like structures termed SMA lipid particles (SMALPs; also known as native nanodiscs or lipodisqs), where a small disc of lipid bilayer is encircled by the SMA co-polymer [39][40][41]. 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.…”

Section: Smalpsmentioning

confidence: 99%

Overcoming bottlenecks in the membrane protein structural biology pipeline

Hardy

Bill

Jawhari³

et al. 2016

Biochemical Society Transactions

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Correspondence to: Alice Rothnie (+44 121 204 4013 a.rothnie@aston.ac.uk) or Anass Jawhari (+33 649 555 606 ajawhari@calixar.com) Key words:Membrane proteins Structural biology SMALP Calixarenes MNG Solubilisation Abbreviations:EM-Electron Microscopy GPCR-G protein-coupled receptors GNG-Glucose Neopentyl Glycol MSP -Membrane Scaffold Protein MNG-Maltose Neopentyl Glycol NMR-Nuclear magnetic resonance SMA-Styrene Maleic Acid SMALPs -SMA Lipid Particles AbstractMembrane proteins account for a third of the eukaryotic proteome, but are greatly underrepresented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and electron microscopy cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as GNG, MNG and calixarene-based detergents can improve protein stability without compromising their solubilising properties. SMALPs focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis.Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Scheidelaar

Koorengevel

Walree

et al. 2016

Biophysical Journal

126

170

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The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as a tool in membrane research, due to its ability to directly solubilize lipid membranes into nanodisk particles without the requirement of conventional detergents. Although many variants of SMA are commercially available, so far only SMA variants with a 2:1 and 3:1 styreneto-maleic acid ratio have been used in lipid membrane studies. It is not known how SMA composition affects the solubilization behavior of SMA. Here, we systematically investigated the effect of varying the styrene/maleic acid on the properties of SMA in solution and on its interaction with membranes. Also the effect of pH was studied, because the proton concentration in the solution will affect the charge density and thereby may modulate the properties of the polymers. Using model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipids at pH > pH agg , we found that membrane solubilization is promoted by a low charge density and by a relatively high fraction of maleic acid units in the polymer. Furthermore, it was found that a collapsed conformation of the polymer is required to ensure efficient insertion into the lipid membrane and that efficient solubilization may be improved by a more homogenous distribution of the maleic acid monomer units along the polymer chain. Altogether, the results show large differences in behavior between the SMA variants tested in the various steps of solubilization. The main conclusion is that the variant with a 2:1 styrene-to-maleic acid ratio is the most efficient membrane solubilizer in a wide pH range.

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Membrane Proteins Solubilized Intact in Lipid Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer

Cited by 473 publications

References 13 publications

Detergent-free purification of ABC (ATP-binding-cassette) transporters

Detergent-free purification of ABC (ATP-binding-cassette) transporters

Overcoming bottlenecks in the membrane protein structural biology pipeline

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

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