Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2

Horsey, Aaron J.; Briggs, Deborah A.; Holliday, Nicholas D.; Briddon, Stephen J.; Kerr, Ian D.

doi:10.1016/j.bbamem.2020.183218

Cited by 21 publications

(20 citation statements)

References 57 publications

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“…Recently, native-mass spectrometry of SMALP-encapsulated proteins has also been demonstrated, enabling investigation of post-translational modifications, association with specific lipids and protein maturation [ 67 ]. Fluorescence correlation spectroscopy of SMALP purified proteins has been utilised to develop a new method for measuring ligand binding to membrane proteins, including both an ABC transporter [ 49 ], and a GPCR [ 68 ]. This approach enables a quantitative, real-time measurement of binding assays with relatively low concentrations of purified protein.…”

Section: Functional Insightsmentioning

confidence: 99%

Biological insights from SMA-extracted proteins

Unger

Ronco-Campaña

Kitchen

et al. 2021

Biochemical Society Transactions

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In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

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Section: Functional Insightsmentioning

confidence: 99%

Biological insights from SMA-extracted proteins

Unger

Ronco-Campaña

Kitchen

et al. 2021

Biochemical Society Transactions

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“…For example, using proteins fluorescently labelled via introduced unnatural amino acids to enable single-molecule fluorescence studies and using FCS to confirm the existence of dimers of the transporter protein ABCG2 and its interaction with substrate. 41,42 This expanding utility of SMALPs has been the driver for developing new amphipathic co-polymers that retain the ability of SMA to solubilise membrane proteins but exhibit different physico-chemical characteristics to SMA. For example, styrene-co-maleimide (SMI) is positively-charged and retains functionality at acidic pH, in contrast to the negative charge and alkaline working range of SMA; 28 styrene maleimide quarternary ammonium (SMA-QA) forms relatively large nanodiscs (∼30 nm diameter) that remain stable between pH 2.5-pH 10; 29 diisobutylene-co-maleic acid (DIBMA) in which the styrene aromatic ring of SMA is replaced by the aliphatic diisobutylene thereby changing how the polymer interacts with the lipid in the nanoparticles 30 and thiolated SMA (SMA-SH) which can be derivatised or immobilised via the introduced thiol.…”

Section: Characterization Of the Slower Diffusing Component (τ D2 )mentioning

confidence: 99%

Single molecule binding of a ligand to a G-protein-coupled receptor in real time using fluorescence correlation spectroscopy, rendered possible by nano-encapsulation in styrene maleic acid lipid particles

et al. 2020

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“…Many proteins within SMALPs have been shown to have much greater stability than when detergent solubilised [10] , [11] , [12] , [13] , [14] , [22] , [23] , [24] . Recently, SMALP-encapsulation has been successful for structural determination of several proteins by cryo-electron microscopy [25] , [26] , [27] , [28] , [29] , [30] , as well as for identifying novel function or developing new assays [31] , [32] , [33] , [34] .…”

Section: Introductionmentioning

confidence: 99%

Membrane protein extraction and purification using partially-esterified SMA polymers

Hawkins

Jahromi

Gulamhussein

et al. 2021

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA 1440 and SMA 17352. SMA 2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation.

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Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2

Cited by 21 publications

References 57 publications

Biological insights from SMA-extracted proteins

Biological insights from SMA-extracted proteins

Single molecule binding of a ligand to a G-protein-coupled receptor in real time using fluorescence correlation spectroscopy, rendered possible by nano-encapsulation in styrene maleic acid lipid particles

Membrane protein extraction and purification using partially-esterified SMA polymers

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