Detergent-free solubilization of human Kv channels expressed in mammalian cells

Karľová, Mária; Voskoboynikova, Natalia; Gluhov, Grigory; Abramochkin, Denis V.; Malak, Olfat A.; Mulkidzhanyan, A.; Loussouarn, Gildas; Steinhoff, Heinz‐Jürgen; Шайтан, К. В.; Sokolova, Olga S.

doi:10.1016/j.chemphyslip.2019.01.013

Cited by 27 publications

(20 citation statements)

References 60 publications

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“…approach, the native nanodisc method is still in its early stages of development but has shown great promise in cryo-EM and functional characterization on a selection of membrane proteins expressed in E. coli [5][6][7] and mammalian cells. [8][9][10] However, it is evident from the limited number of studies of membrane proteins embedded in native nanodiscs that the currently available copolymers have several shortcomings.…”

Section: Introductionmentioning

confidence: 99%

Lipid Nanodiscs via Ordered Copolymers

Smith

Autzen

Faust

et al. 2020

Chem

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A highly alternating copolymer composed of acrylic acid and styrene (AASTY) is synthesized with controlled radical polymerization by exploiting the reactivity ratios of the monomers to control the monomer sequence. The AASTY copolymers are effective solubilizers of cellular membranes and their embedded proteins, which improves structural characterization by single-particle cryo-electron microscopy (cryo-EM). These copolymers are promising tools for exploring detergent-free membrane protein solubilization and direct formation of native nanodiscs, facilitating structural and functional analysis of the mammalian proteome.

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Section: Introductionmentioning

confidence: 99%

Lipid Nanodiscs via Ordered Copolymers

Smith

Autzen

Faust

et al. 2020

Chem

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“…Full-length ion channels solubilize into nanodiscs upon SMA treatment to display symmetric tetramers arranged around central pores. 50 The hKCNH5 and hKCNQ1 α-subunits of the neuronal and cardiac voltage-gated potassium (Kv) channels as well as the regulatory KCNE1 β-subunit can be transferred from mammalian cells into SMALPs and exhibit improved behavior over detergent solubilized forms, including greater stability and ease of concentration. The channels appear to arrange themselves randomly on electron microscopy grids to expose diameters of 12-15 nm.…”

Section: Future Directions For Membrane Proteinsmentioning

confidence: 99%

Structures and Interactions of Transmembrane Targets in Native Nanodiscs

Overduin

Esmaili

2019

SLAS Discovery

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Transmembrane proteins function within a continuous layer of biologically relevant lipid molecules that stabilizes their structures and modulates their activities. Structures and interactions of biological membrane–protein complexes or “memteins” can now be elucidated using native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. These linear polymers contain a series of hydrophobic and polar subunits that gently fragment membranes into water-soluble discs with diameters of 5–50 nm known as styrene maleic acid lipid particles (SMALPs). High-resolution structures of memteins that include endogenous lipid ligands and posttranslational modifications can be resolved without resorting to synthetic detergents or artificial lipids. The resulting ex situ structures better recapitulate the in vivo situation and can be visualized by methods including cryo-electron microscopy (cryoEM), electron paramagnetic resonance (EPR), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, small angle x-ray scattering (SAXS), and x-ray diffraction (XRD). Recent progress including 3D structures of biological bilayers illustrates how polymers and native nanodiscs expose previously inaccessible membrane assemblies at atomic resolution and suggest ways in which the SMALP system could be exploited for drug discovery.

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“…The non-selective nature of the method was also demonstrated by the extraction of transmembrane protein complexes from the archaeal photoreceptor-transducer complex (NpSRII/NpHtrII) into a 3:1 SMA copolymer, with a photoreceptor: transducer stoichiometry of 2:2, from Escherichia coli polar lipid liposomes [23]. Detergent-free solubilization of MPs by SMA copolymers has been demonstrated for cellular membranes from yeast [24][25][26][27][28] and mammalian cells [29].…”

Section: Introductionmentioning

confidence: 99%

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy

Voskoboynikova

Karľová

Kurre

et al. 2021

JoF

Self Cite

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The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.

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Detergent-free solubilization of human Kv channels expressed in mammalian cells

Cited by 27 publications

References 60 publications

Lipid Nanodiscs via Ordered Copolymers

Lipid Nanodiscs via Ordered Copolymers

Structures and Interactions of Transmembrane Targets in Native Nanodiscs

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy

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