Phosphoinositide-incorporated lipid–protein nanodiscs: A tool for studying protein–lipid interactions

Abstract

Phosphatidylinositol (PtdIns) is phosphorylated at D-3, D-4, and/or D-5 of the inositol ring to produce seven distinct lipid second messengers known as phosphoinositides (PIs). The PI level is temporally and spatially controlled at the cytosolic face of the cellular membrane. Effectors containing PI-binding domains (e.g., PH, PX, FYVE, ENTH, FERM) associate with specific PIs. This process is crucial for the localization of a variety of cell-signaling proteins, thereby regulating intracellular membrane trafficking, cell growth and survival, cytoskeletal organization, and so on. However, quantitative assessments of protein–PI interactions are generally difficult due to insolubility of PIs in aqueous solution. Here we incorporated PIs into a lipid–protein nanoscale bilayer (nanodisc), which is applied for studying the protein–PI interactions using pull-down binding assay, fluorescence polarization, and nuclear magnetic resonance studies, each facilitating fast, quantitative, and residue-specific evaluation of the protein–PI interactions. Therefore, the PI-incorporated nanodisc could be used as a versatile tool for studying the protein–lipid interactions by various biochemical and biophysical techniques.