A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs

Long, Ashley R.; O'Brien, Catherine C.; Malhotra, Ketan; Schwall, Christine T.; Albert, Arlene D.; Watts, Anthony; Alder, Nathan N.

doi:10.1186/1472-6750-13-41

Cited by 124 publications

(142 citation statements)

References 52 publications

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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

129

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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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

129

170

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“…A recently developed alternative is the use of an amphipathic styrene maleic acid (SMA) copolymer (Figure 1 A) that is able to remove the protein from a membrane with its associated lipids intact in the form of a protein/lipid nanodisc bound by the polymer. [2][3][4][5] This approach has been used to successfully solubilize membrane proteins from artificial liposomes [2,6] and native membranes, [7,8] and is one of a number of alternatives being developed for housing integral membrane proteins outside the native membrane. [9] Figure 1.…”

Section: Biophysical Characterization Of Integral Membrane Proteinsmentioning

confidence: 99%

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability

et al. 2014

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Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications.

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“…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%

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

Gulati

Jamshad

Knowles

et al. 2014

Biochemical Journal

177

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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A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs

Cited by 124 publications

References 52 publications

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

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

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability

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

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