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

Pellowe, Grant A.; Findlay, Heather E.; Lee, Karen; Gemeinhardt, Tim M; Blackholly, Laura R.; Reading, Eamonn; Booth, Paula J.

doi:10.1021/acs.biochem.0c00423

Cited by 9 publications

(7 citation statements)

References 67 publications

(173 reference statements)

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“…The gentle isolation of membrane proteins from cellular membranes is a major challenge 1 that is increasingly met by the use of nanodisc-forming amphiphilic polymers such as aliphatic diisobutylene/maleic acid (DIBMA, Figure 1a) [2][3][4][5][6][7][8] or aromatic styrene/maleic acid (SMA) copolymers. 3,[9][10][11][12][13] Unlike traditional head-and-tail detergents, which displace the native lipid environment of membrane proteins, 14 amphiphilic copolymers co-extract a nanoscopic lipid patch from the cellular membrane to form polymer-bounded lipid-bilayer nanodiscs.…”

Section: Introductionmentioning

confidence: 99%

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs

Danielczak

Rasche²,

Lenz

et al. 2022

Nanoscale

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Section: Introductionmentioning

confidence: 99%

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs

Danielczak

Rasche²,

Lenz

et al. 2022

Nanoscale

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“…Polymer extraction of membrane proteins has also enabled the development of new tools to investigate biological processes and functions. Pellowe et al [ 66 ] have utilised DIBMA extraction of E. coli membranes combined with SecM-facilitated nascent chain stalling, to develop a way to isolate ribosome-bound nascent chain complexes for membrane proteins within their native lipid environment, for the first time. This has the potential for future structural and biophysical analysis to gain further insight to the mechanism of co-translational membrane protein folding and membrane insertion.…”

Section: Functional Insightsmentioning

confidence: 99%

“…Thus, it seems more likely that lipid-interactions will be maintained when using SMA than conventional detergents. Several studies have reported analysis of the lipid content of purified protein SMALPs using techniques such as TLC [ 13 , 32 , 37 , 66 , 71 ], HPLC [ 54 ] and/or mass spectrometry [ 22 , 71 , 72 ]. Lipids co-purified from E.coli with SMA-extracted KcsA were found to be enriched in both cardiolipin (CL), and phosphatidylglycerol (PG) [ 13 ], both of which are negatively charged at physiological pH.…”

Section: Protein–lipid Interactionsmentioning

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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“…22,31,32 Therefore, DIBMA lipid particles (DIBMALPs) provide an alternative platform for studying membrane proteins in diverse conditions. For example, DIBMA has permitted the extraction and study of G protein-coupled receptors 31 and ribosomenascent chain complexes 33 in divalent magnesium with a native-like membrane environment. In addition, the intramembrane rhomboid protease, GlpG, was found to be highly functional and stable in DIBMALPs, 24 and α-synuclein was folded into an α-helix in the model membrane inside a DIBMALP.…”

Section: ■ Introductionmentioning

confidence: 99%

Structure of Diisobutylene Maleic Acid Copolymer (DIBMA) and Its Lipid Particle as a “Stealth” Membrane-Mimetic for Membrane Protein Research

Guo

Sumner

Qian

2021

ACS Appl. Bio Mater.

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The study of membrane proteins remains challenging, especially in a native membrane environment. Recently, major progress has been made using maleic acid copolymers, such as styrene maleic acid, to purify membrane proteins and study them directly with native lipids associated with the membrane. Additional maleic acid copolymers, such as diisobutylene maleic acid (DIBMA) membrane-mimetic systems, are being developed and found to have improved spectroscopic properties and pH stability. We studied DIBMA and its lipid particles in solution to better understand its assembly, without and with the lipids, to provide an insight regarding how to use it in solution for better membrane extraction. Using small-angle neutron and X-ray scattering (SANS/SAXS), we show that DIBMA organizes into structures of different size scales at various concentrations and ionic strengths. The polymer performed reasonably well under most solvent conditions except in very low concentrations and high-salt conditions that could result in limited interaction with lipids. To explore DIBMA lipid particles as a suitable membrane-mimetic system for neutron scattering studies of membrane proteins, we measured and determined the contrast-matching point of DIBMA to be ∼12% (v/v) D2O  similar to that of most protiated lipid molecules but distinct from that of regular protiated proteins  providing a natural contrast for separating their neutron scattering signals. Using SANS contrast variation, we demonstrated that the scattering from the whole lipid particle can be annihilated. Further, we determined that a well-defined lipid nanodisc structure with DIBMA was contrast-matched. These results demonstrate that the DIBMA lipid particle is an outstanding “stealth” membrane-mimetic for membrane proteins. The results provide a structural framework for understanding the organization and assembly process of the polymer itself and the lipid molecules. Such an understanding is imperative for structural techniques such as cryo-electron microscopy, nuclear magnetic resonance, small-angle scattering, and other biophysical techniques.

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

Cited by 9 publications

References 67 publications

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs

Biological insights from SMA-extracted proteins

Structure of Diisobutylene Maleic Acid Copolymer (DIBMA) and Its Lipid Particle as a “Stealth” Membrane-Mimetic for Membrane Protein Research

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