Preparation of Lipid Nanodiscs with Lipid Mixtures

Li, Mavis Jiarong; Atkins, William M.; McClary, Wynton D.

doi:10.1002/cpps.100

Cited by 23 publications

(26 citation statements)

References 33 publications

(42 reference statements)

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“…Although nanodiscs can be formed with natural lipid extracts, most native MS experiments have used nanodiscs made of a single synthetic phospholipid because they produce well-resolved spectra. There is an intrinsic distribution in the number of lipids per nanodisc, so the spectra show a regular series of peaks separated by the mass of the lipid. , Adding a new lipid to the nanodisc adds another layer of complexity that makes resolution difficult.…”

Section: Introductionmentioning

confidence: 99%

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

et al. 2021

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Native mass spectrometry (MS) with nanodiscs is a promising technique for characterizing membrane protein and peptide interactions in lipid bilayers. However, prior studies have used nanodiscs made of only one or two lipids, which lack the complexity of a natural lipid bilayer. To better model specific biological membranes, we developed model mammalian, bacterial, and mitochondrial nanodiscs with up to four different phospholipids. Careful selection of lipids with similar masses that balance the fluidity and curvature enabled these complex nanodiscs to be assembled and resolved with native MS. We then applied this approach to characterize the specificity and incorporation of LL-37, a human antimicrobial peptide, in single-lipid nanodiscs versus model bacterial nanodiscs. Overall, development of these model membrane nanodiscs reveals new insights into the assembly of complex nanodiscs and provides a useful toolkit for studying membrane protein, peptide, and lipid interactions in model biological membranes.

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Section: Introductionmentioning

confidence: 99%

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

et al. 2021

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“…Similar to prior studies, it was best to assemble nanodiscs at the phase transition temperature of the most abundant lipid, which is 41 °C for DPPC. 15 After optimizing the ratios and assembly times, we were able to prepare reproducible DPPC/SOPE nanodiscs at a molar ratio of 75/25 ( Figure 2).…”

Section: Resultsmentioning

confidence: 99%

“…9 Native MS studies with nanodiscs have uniquely enabled the investigation of membrane protein oligomers within intact lipid bilayers. [10][11] Nanodiscs have also proven useful for studying the mechanism of membrane-active peptides with native MS. [12][13][14] Although nanodiscs can be formed with natural lipid extracts, 15 most native MS experiments have used nanodiscs made of a single synthetic phospholipid because they produce well-resolved spectra. There is an intrinsic distribution in the number of lipids per nanodisc, so spectra show a regular series of peaks separated by the mass of the lipid.…”

Section: Introductionmentioning

confidence: 99%

“…Drying multiple lipids together in chloroform allowed them to be resolubilized as a mixed membrane with the addition of detergent solution. 15 For proper nanodisc assembly, we discovered that mixed lipid chloroform solutions needed to be clear and fully solubilized prior to evaporation. For DPPG-and DPPC-based nanodiscs, lipids in chloroform usually required heat (around 60 ⁰C) to fully solubilize.…”

Section: Introductionmentioning

confidence: 99%

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Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

Kostelic¹,

Zak²,

Jayasekera³

et al. 2021

Preprint

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Native mass spectrometry (MS) with nanodiscs is a promising technique for characterizing membrane protein and peptide interactions in lipid bilayers. However, prior studies have used nanodiscs made of only one or two lipids, which lack the complexity of a natural lipid bilayer. To better model specific biological membranes, we developed model mammalian, bacterial, and mitochondrial nanodiscs with up to four different phospholipids. Careful selection of lipids with similar masses that balance the fluidity and curvature enabled these complex nanodiscs to be assembled and resolved with native MS. We then applied this approach to characterize the specificity and incorporation of LL-37, a human antimicrobial peptide, in single lipid nanodiscs versus model bacterial nanodiscs. Overall, development of these model membrane nanodiscs reveals new insights into the assembly of complex nanodiscs and provides a useful toolkit for studying membrane protein, peptide, and lipid interactions in model biological membranes.

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“…The lipidic environment modulates membrane protein functionality [ 12 , 82 , 83 ] by molecular interactions between the protein and lipids of the phospholipid bilayer. To answer key questions regarding the requirement of specific phospholipids for optimal activity or stabilization of particular conformations, nanodisc technology provides a controllable membrane environment by regulating the lipid composition with precision [ 96 ]. However, simulation of the cellular native environment requires the selection of an appropriate lipid composition for nanodisc assembly.…”

Section: Lipid Membrane Mimicsmentioning

confidence: 99%

Membrane Protein Stabilization Strategies for Structural and Functional Studies

2021

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Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

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Preparation of Lipid Nanodiscs with Lipid Mixtures

Cited by 23 publications

References 33 publications

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

Membrane Protein Stabilization Strategies for Structural and Functional Studies

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