An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins

Chien, Chih-Ta Henry; Helfinger, Lukas R; Bostock, Mark J.; Solt, Andras; Tan, Yi; Nietlispach, Daniel

doi:10.1021/jacs.7b06730

Cited by 35 publications

(67 citation statements)

References 30 publications

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“…Here, the size-adaptable nature of the SapA scaffold allows for incorporation of both smaller and larger IMPs into Salipro nanoparticles by using the very same scaffold protein and workflow. The flexibility of the Salipro technology has been demonstrated by the reconstitution of a multitude of IMP sizes and shapes, ranging from small monomeric proteins, such as the 16.5-kDa bacterial outer membrane protein OmpX (Chien et al, 2017), to large protein oligomers, illustrated by the tetrameric peptide transporter PepT So2 with a total of 56 transmembrane helices (Frauenfeld et al, 2016). The Salipro nanoparticles have been shown to be compatible with a wide range of high-resolution structural, biophysical and biochemical methods (Frauenfeld et al, 2016;Chien et al, 2017;Lyons et al, 2017;Flayhan et al, 2018;Kintzer et al, 2018;Nguyen et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, Salipro nanoparticles can be functionalized by a diverse range of tags on the SapA scaffold protein, useful for downstream applications relevant to pharmaceutical research. In addition, IMPs embedded in Salipro nanoparticles have been shown to be functional and can undergo conformational changes, highlighting the flexibility of the SapA scaffold to preserve the conformational changes required for protein function (Chien et al, 2017;Flayhan et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…In both cases, the reconstituted protein is shown to form pure, stable and homogenous Salipro nanoparticles. Previously, detergent-purified IMPs reconstituted into Salipro nanoparticles were reported to remain structurally intact and functionally active (Chien et al, 2017;Flayhan et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019). We envision that the DirectMX methodology will enable studies on IMPs that remained so far inaccessible to other solubilization or reconstitution methods, in particular IMPs representing unstable therapeutic targets from the families of GPCRs, ion channels and transporters.…”

Section: Discussionmentioning

confidence: 99%

“…Homogenized crude cell membranes were prepared and subsequently incubated in the presence of the mild detergent digitonin (1% final concentration), which increases the membrane fluidity at 4 • C, thus rendering lipids and IMPs more accessible for the following reconstitution step. In addition, detergents have been shown to activate SapA molecules from a closed into an open, lipid-binding conformational state at physiological pH (Chien et al, 2017). After removal of insoluble material by low speed centrifugation, the remaining material was incubated with an excess of SapA molecules to allow for the reconstitution of all IMPs, including their associated lipids, into Salipro nanoparticles (Figures 1A,B).…”

Section: Direct Reconstitution Into Monodisperse Salipro-slc Nanopartmentioning

confidence: 99%

“…Salipro nanoparticles are highly homogeneous, displaying both increased thermostability and half-life. In addition, Salipro-reconstituted IMPs have been shown to be functional and structurally intact by a wide range of biochemical and biophysical methods such as cryogenic EM, nuclear magnetic resonance spectroscopy or enzymatic assays (Frauenfeld et al, 2016;Chien et al, 2017;Flayhan et al, 2018;Kintzer et al, 2018;Nguyen et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

Lloris-Garcerá¹,

Klinter²,

Chen

et al. 2020

Front. Bioeng. Biotechnol.

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Integral membrane proteins (IMPs) are central to many physiological processes and represent ∼60% of current drug targets. An intricate interplay with the lipid molecules in the cell membrane is known to influence the stability, structure and function of IMPs. Detergents are commonly used to solubilize and extract IMPs from cell membranes. However, due to the loss of the lipid environment, IMPs usually tend to be unstable and lose function in the continuous presence of detergent. To overcome this problem, various technologies have been developed, including protein engineering by mutagenesis to improve IMP stability, as well as methods to reconstitute IMPs into detergent-free entities, such as nanodiscs based on apolipoprotein A or its membrane scaffold protein (MSP) derivatives, amphipols, and styrene-maleic acid copolymer-lipid particles (SMALPs). Although significant progress has been made in this field, working with inherently unstable human IMP targets (e.g., GPCRs, ion channels and transporters) remains a challenging task. Here, we present a novel methodology, termed DirectMX (for direct membrane extraction), taking advantage of the saposin-lipoprotein (Salipro) nanoparticle technology to reconstitute fragile IMPs directly from human crude cell membranes. We demonstrate the applicability of the DirectMX methodology by the reconstitution of a human solute carrier transporter and a wild-type GPCR belonging to the human chemokine receptor (CKR) family. We envision that DirectMX bears the potential to enable studies of IMPs that so far remained inaccessible to other solubilization, stabilization or reconstitution methods.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Direct Reconstitution Into Monodisperse Salipro-slc Nanopartmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

Lloris-Garcerá¹,

Klinter²,

Chen

et al. 2020

Front. Bioeng. Biotechnol.

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On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

et al. 2018

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NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.

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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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An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins

Cited by 35 publications

References 30 publications

DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

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

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