Characterization of Phosphatidylinositol Phosphate Binding in Lipid Bilayers by Solid-State NMR Spectroscopy

Abstract

Phosphatidylinositol phosphates (PIPs) are a seven membered class of lipid that regulate diverse cell processes in eukaryotic organisms differentiated by the level of phosphorylation at three sites on the lipid head group. The presence of one or more of these lipids in a membrane serves as a biochemical signal for the localization and activation of hundreds of intrinsic and peripheral membrane proteins. Magic-angle-spinning (MAS) solid-state NMR (ssNMR) can provide atomic resolution information about interactions in lipid environments. Direct observation of the lipids and membrane proteins are possible, as well as the interactions between them. While ssNMR is capable of investigating all types of lipids, the existence and importance of up to three additional phosphate groups in PIPs provide a distinct advantage for their investigation. We demonstrate sample preparation and spectroscopic techniques to maximize the sensitivity of 31P spectra of PIP containing liposomes. The location of the PIP binding site on a protein is important to understand if we seek to detect or modify binding. NMR is uniquely suited for mapping the binding sites of PIPs to atomic resolution. Modern ssNMR utilizes very-fast MAS rates (in excess of 100 kHz) and proton detection to streamline the process of assigning the chemical shifts in moderately sized proteins. PH domains are conserved PIP binding domains found in many different proteins. We present progress towards the solid-state assignments of PH domains, a well characterized class of PIP binding domain. These chemical shifts will be used to track the effects of PIP binding on the domain, confirming the binding site as observed by other techniques.