Effects of Ca<sup>2+</sup> on the Structure and Dynamics\nof PIP<sub>3</sub> in Model Membranes Containing PC and PS

Phosphatidylinositol\nphosphates (PIPs) are a family of\nseven different\neukaryotic membrane lipids that have a large role in cell viability,\ndespite their minor concentration in eukaryotic cellular membranes.\nPIPs tightly regulate cellular processes, such as cellular growth,\nmetabolism, immunity, and development through direct interactions\nwith partner proteins. Understanding the biophysical properties of\nPIPs in the complex membrane environment is important to understand\nhow PIPs selectively regulate a partner protein. Here, we investigate\nthe structure and dynamics of PIP<sub>3</sub> in lipid bilayers that\nare simplified models of the natural membrane environment. We probe\nthe effects of the anionic lipid phosphatidylserine (PS) and the divalent\ncation Ca<sup>2+</sup> by using full-length lipids in well-formed\nbilayers. We used solution and solid-state NMR on naturally abundant <sup>1</sup>H, <sup>31</sup>P, and <sup>13</sup>C atoms combined with\nmolecular dynamics (MD) simulations to characterize the structure\nand dynamics of PIPs. <sup>1</sup>H and <sup>31</sup>P 1D spectra\nshow good resolution at temperatures above the phase transition with\nisolated peaks in the headgroup, interfacial, and bilayer regions.\nSite-specific assignment of the chemical shifts of these reporters\nenables the measurement of the effects of Ca<sup>2+</sup> and PS at\nthe single atom level. In particular, the resolved <sup>31</sup>P\nsignals of the PIP<sub>3</sub> headgroup allow for extremely well-localized\ninformation about PIP<sub>3</sub> phosphate dynamics, which the MD\nsimulations can further explain. A quantitative assessment of cross-polarization\nkinetics provides additional dynamics measurements for the PIP<sub>3</sub> headgroups.