Effects of Ca ²⁺ 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 Ca ²⁺ . We use solution and solid-state ¹ H, ³¹ P, and ¹³ C NMR all at natural abundance combined with MD simulations to characterize the structure and dynamics of PIPs. ¹ H and ³¹ P 1D spectra show good resolution at high temperatures with isolated peaks in the headgroup, interfacial, and bilayer regions. Site specific assignment of these 1D reporters were made and used to measure the effects of Ca ²⁺ and PS. In particular, the resolved ³¹ P signals of the PIP3 headgroup allowed for extremely well localized information about PIP3 phosphate dynamics, which the MD simulations were able to help explain. Cross polarization kinetics provided additional site-specific dynamics measurements for the PIP3 headgroups.