Stable and Functional Rhomboid Proteases in Lipid Nanodiscs by Using Diisobutylene/Maleic Acid Copolymers

Barniol‐Xicota, Marta; Verhelst, Steven H. L.

doi:10.1021/jacs.8b08441

Cited by 47 publications

(59 citation statements)

References 34 publications

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“…Dynamic light scattering was used to assess the size and homogeneity of solubilized, IMAC-purified four-TM GlpG RNCs and gave a mean peak size of 10.5 nm, which is close to the value of 12.7 nm previously reported for full-length GlpG in DIBMA. 38 The average particle size z (which is the intensity-weighted mean hydrodynamic size of the ensemble) was determined to be 33.0 nm, suggestive of populations of ribosome-bound and unbound discs causing sample polydispersity ( Figure S3d ).…”

Section: Resultsmentioning

confidence: 99%

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies

et al. 2020

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Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo -generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene–maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

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Section: Resultsmentioning

confidence: 99%

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies

et al. 2020

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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%

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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“…45 The rhomboid protease GlpG solubilizes similarly with DIBMA, SMA(3:1), and SMA(2.3:1) and retains about 50 phospholipid molecules of various types, thus preserving native-like activities and stabilities as well as showing suitability for ligand screening. 12,46 The polydispersity and nonselectivity of SMA copolymers may allow for the retention of a broad range of lipid and protein types but also presents technical issues, including peak broadening. High voltages may be needed to release strongly held components within nanodiscs.…”

Section: Mass Spectrometry Of Memteinsmentioning

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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Stable and Functional Rhomboid Proteases in Lipid Nanodiscs by Using Diisobutylene/Maleic Acid Copolymers

Cited by 47 publications

References 34 publications

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies

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

Structures and Interactions of Transmembrane Targets in Native Nanodiscs

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