Reversible stacking of lipid nanodiscs for structural studies of clotting factors

Kirill Grushin, Mark Andrew White, Svetla Stoilova-Mcphie

Research output: Contribution to journalArticle

Abstract

Nanodiscs (ND) are discoidal phospholipid bilayers stabilized by a pair of membrane-scaffolding proteins (MSP). The macromolecular composition and size of ND are ideal for structural and functional studies of membrane and membrane-associated proteins. In this work, we investigate the assembly of ND from a galactosylceramide and dioleoyl phosphatidylserine (PS) lipid mixture with two different MSP and at four MSP-to-lipid ratios. This lipid composition has been optimized for structural and biophysical studies of membrane-bound blood clotting factors that require Ca2+ ions for function. We have demonstrated that CaCl2 induces reversible stacking of the ND that depends on the ND size and Ca2+ concentrations. Our biophysical and electron microscopy (EM) studies show a predominant ND population of ∼12 nm in diameter for both the ND assembled from MSP1D1 to lipids ratio of 1:40 and from MSP1E3D1-to-lipids ratio of 1:80. Approximately half of the ND population assembled at MSP1E3D1-to-lipids ratio of 1:150 has a diameter of ∼16 nm. These larger ND form ordered stacks at 5-mm Ca2+ concentrations, as shown by cryo-EM. The number and length of the ND stacks increase with the increasing in Ca2+ concentration. Adding millimolar concentrations of EDTA reverses the stacking of the ND.

Original languageEnglish (US)
Pages (from-to)139-148
Number of pages10
JournalNanotechnology Reviews
Volume6
Issue number1
DOIs
StatePublished - Feb 1 2017

Keywords

  • cryo-electron microscopy
  • lipid nanodiscs
  • phospholipids
  • stacking of nanodiscs

ASJC Scopus subject areas

  • Biotechnology
  • Medicine (miscellaneous)
  • Materials Science (miscellaneous)
  • Energy Engineering and Power Technology
  • Engineering (miscellaneous)
  • Process Chemistry and Technology

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