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Nanodisc single-molecule pulldown to study lipid-protein interactions.

Adriana Reyes-Ordoñez1, Shweta Shree2, Nilmani Singh3

  • 1Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Journal of Lipid Research
|June 22, 2025
PubMed
Summary
This summary is machine-generated.

We developed a Nanodisc-based single-molecule pulldown (SiMPull) assay to study protein-lipid interactions. This method uses Nanodiscs to achieve true single-molecule resolution, revealing critical interactions for AKT signaling.

Keywords:
AKTNanodiscSiMPulllipidphosphatidylinositol phosphatesingle-molecule

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Phospholipids, including phosphatidylinositol phosphates (PIPs), are crucial signaling molecules regulating cellular processes.
  • Studying specific PIP-protein interactions is vital for understanding cell signaling.
  • Existing methods using small unilamellar vesicles have limitations in membrane modeling.

Purpose of the Study:

  • To develop a robust and convenient method for studying protein-lipid interactions with single-molecule resolution.
  • To improve upon the limitations of previous lipid-SiMPull assays by utilizing Nanodiscs as a membrane model.
  • To investigate the structural basis of AKT signaling pathway interactions.

Main Methods:

  • Development of a Nanodisc-based lipid single-molecule pulldown (Nanodisc SiMPull) assay.
  • Utilizing Nanodiscs containing various PIPs to capture fluorescently tagged proteins.
  • Analysis of protein binding kinetics, including dissociation rates (koff), using dwell time analysis.

Main Results:

  • The Nanodisc SiMPull assay specifically pulls down PIP-binding proteins with a detection threshold of 10-20 μM.
  • The assay achieves true single-molecule resolution, with one protein bound per Nanodisc.
  • Identified an intramolecular interaction critical for stabilizing AKT binding to PI(3,4,5)P3.

Conclusions:

  • The Nanodisc SiMPull assay is a powerful tool for investigating protein-lipid interactions at the single-molecule level.
  • This method overcomes limitations of previous vesicle-based assays, offering a more accurate membrane model.
  • Provides new insights into the structural mechanisms governing AKT signaling pathway regulation.