Effects of Ca²⁺ on the Structure and Dynamics of PIP₃ in Model Membranes Containing PC and PS

Phosphatidylinositol phosphates (PIPs) are a family of seven different eukaryotic membrane lipids that have a large role in cell viability, despite their minor concentration in eukaryotic cellular membranes. PIPs tightly regulate cellular processes, such as cellular growth, metabolism, immunity, and development through direct interactions with partner proteins. Understanding the biophysical properties of PIPs in the complex membrane environment is important to understand how PIPs selectively regulate a partner protein. Here, we investigate the structure and dynamics of PIP₃ in lipid bilayers that are simplified models of the natural membrane environment. We probe the effects of the anionic lipid phosphatidylserine (PS) and the divalent cation Ca²⁺ by using full-length lipids in well-formed bilayers. We used solution and solid-state NMR on naturally abundant ¹H, ³¹P, and ¹³C atoms combined with molecular dynamics (MD) simulations to characterize the structure and dynamics of PIPs. ¹H and ³¹P 1D spectra show good resolution at temperatures above the phase transition with isolated peaks in the headgroup, interfacial, and bilayer regions. Site-specific assignment of the chemical shifts of these reporters enables the measurement of the effects of Ca²⁺ and PS at the single atom level. In particular, the resolved ³¹P signals of the PIP₃ headgroup allow for extremely well-localized information about PIP₃ phosphate dynamics, which the MD simulations can further explain. A quantitative assessment of cross-polarization kinetics provides additional dynamics measurements for the PIP₃ headgroups.