Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins

Nguyen, Kim‐Anh; Peuchmaur, Marine; Magnard, Sandrine; Haudecoeur, Romain; Boyère, Cédric; Mounien, Saravanan; Benammar, Ikram; Zampieri, Veronica; Igonet, Sébastien; Chaptal, Vincent; Jawhari, Anass; Boumendjel, Ahcène; Falson, Pierre

doi:10.1002/ange.201713395

Cited by 7 publications

(7 citation statements)

References 24 publications

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“…Due to its calixarene platform, calixarene detergent absorbs at 280 nm, which makes protein quantification difficult and limits the use of some biophysical charaterization, such as circular dichroism or tryptophan fluoresence. To adress this limitation, new classes of compounds with similar architecture but without the calixarene platform have been designed and applied successfully to membrane protein stabilization, 34 and this could be a promising future direction for MRP4 studies. The addition of mild groups such as saccharide heads or cholesterol-like groups provides more diversity for such classes of stabilizing detergents.…”

Section: Discussionmentioning

confidence: 99%

Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents

et al. 2019

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Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. However, elucidating the structure and function of native MP is notoriously challenging as their original structure has to be maintained once removed from the lipid bilayer. Conventionally, detergents have been used to solubilize MP with varying degrees of success concerning MP stability. To try to address this, new, more stabilizing agents have been developed, such as calixarene-based detergents and styrene–maleic acid (SMA) copolymer. Calixarene-based detergents exhibit enhanced solubilizing and stabilizing properties compared with conventional detergents, whereas SMA is able to extract MPs with their surrounding lipids, forming a nanodisc structure. Here we report a comparative study using classical detergents, calixarene-based detergents, and SMA to assess the solubilization and stabilization of the human ABC transporter MRP4 (multidrug resistance protein 4/ABCC4). We show that both SMA and calixarene-based detergents have a higher solubility efficiency (at least 80%) than conventional detergents, and show striking overstabilization features of MRP4 (up to 70 °C) with at least 30 °C stability improvement in comparison with the best conventional detergents. These solubilizing agents were successfully used to purify aggregate-free, homogenous and stable MRP4, with sevenfold higher yield for C4C7 calixarene detergent in comparison with SMA. This work paves the way to MRP4 structural and functional investigations and illustrates once more the high value of using calixarene-based detergent or SMA as versatile and efficient tools to study MP, and eventually enable drug discovery of challenging and highly druggable targets.

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Section: Discussionmentioning

confidence: 99%

Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents

et al. 2019

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“…The elution peak was then pooled and stored at 4 °C before use. BmrA was particularly stable when not concentrated as previously reported (30). Thermostabilisation assays were carried out as previously reported (30).…”

Section: Biochemistrymentioning

confidence: 80%

“…Diffraction patterns of the resulting crystals displayed a lattice-doubling problem that prevented their processing and which we overcame by designing a series of tailored amphiphiles 3a-e (Fig. 1B; Chemistry section in Supplementary Materials) with a scaffold based on glycosyl-substituted dicarboxylates surfactants (30). Of note, these additives increase the thermal stability of BmrA up to ~30 °C for 3d (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

Chaptal

Zampieri

Wiseman

et al. 2021

Preprint

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Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved two ATP-Mg2+-bound outward-facing (OF) conformations of the Bacillus subtilis (homodimeric) BmrA, one by X-ray crystallography without drug, and another by single-particle cryo-EM with rhodamine 6G (R6G). Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5[prime]-6[prime] of the other. R6G induces a rearrangement of TM1-2, highlighting a flexibility that was confirmed by H/D exchange and molecular dynamics simulations. The latter also shows a fast post-release occlusion of the cavity driven by hydrophobicity. Altogether, these data support a new swing mechanism for drug transport.

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“…Small amphipathic agents, in contrast, are widely used because they are not only efficient at extracting membrane proteins, but are also effective at preserving structural integrity during membrane protein purification and crystallisation. Representatives include hemifluorinated surfactants (HFSs), cholate‐ or resorcinarene‐based facial amphiphiles (FAs and RGAs), steroid‐based amphiphiles (e.g., chobimalt and GDN) and neopentyl glycol (NG) amphiphiles (GNGs and MNGs), pentasaccharide‐based amphiphiles (PSEs), mannitol‐based amphiphiles (MNAs), neopentyl glycol‐derived triglucosides (NDTs), dendronic trimaltosides (DTMs) and glycosyl‐substituted dicarboxylate detergents (DCODs) . Among these small amphipathic agents, steroid‐based amphiphiles such as chobimalt and GDN have received attention because cholesterol is an important component of eukaryotic membranes (Figure S1 in the Supporting Information), and using its derivatives [e.g., cholesteryl hemisuccinate (CHS)] as additives can improve protein stability through specific interaction with the membrane proteins .…”

Section: Introductionmentioning

confidence: 99%

Steroid‐Based Amphiphiles for Membrane Protein Study: The Importance of Alkyl Spacers for Protein Stability

et al. 2018

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Membrane proteins allow effective communication between cells and organelles and their external environments. Maintaining membrane protein stability in a non-native environment is the major bottleneck to their structural study. Detergents are widely used to extract membrane proteins from the membrane and to keep the extracted protein in a stable state for downstream characterisation. In this study, three sets of steroid-based amphiphiles-glyco-diosgenin analogues (GDNs) and steroid-based pentasaccharides either lacking a linker (SPSs) or containing a linker (SPS-Ls)-have been developed as new chemical tools for membrane protein research. These detergents were tested with three membrane proteins in order to characterise their ability to extract membrane proteins from the membrane and to stabilise membrane proteins long-term. Some of the detergents, particularly the SPS-Ls, displayed favourable behaviour with the tested membrane proteins. This result indicates the potential utility of these detergents as chemical tools for membrane protein structural study and a critical role of the simple alkyl spacer in determining detergent efficacy.

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Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins

Cited by 7 publications

References 24 publications

Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents

Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents

Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

Steroid‐Based Amphiphiles for Membrane Protein Study: The Importance of Alkyl Spacers for Protein Stability

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