Application of polymethacrylate based nanodiscs for the characterization of phosphoinositide/peptide interactions.

Abstract

Phosphoinositides (PIPs) have been shown to mediate many cellular functions by attracting proteins to specific cellular sites. The complexity of biological membranes has prompted the development of a wide variety of simple model systems to study PIP/protein interactions. Among these model systems, lipid nanodiscs are unique due to their small size which generally enables the use of techniques like isothermal titration calorimetry and solution NMR to study PIP/protein interactions. To date, the use of lipid nanodiscs for studying PIP/protein interactions has been limited to membrane scaffold protein based nanodiscs. In this study, we utilize polymethacrylate based nanodiscs to study the interaction of PIPs with bivalent cations and/or peptides. We describe the conditions to obtain PIP containing nanodiscs for different lipid acyl chain compositions (saturated vs. unsaturated) as well as different lipid mixtures. We characterize the nanodiscs using dynamic light scattering and transmission electron microscopy to optimize the size distribution based upon the lipid/polymer ratio. We characterize the binding of cationic species to the PIP containing nanodiscs using isothermal titration calorimetry.