Solid-state Nmr Methods for Structural Studies of Phosphatidylinositol-specific Phospholipase C

Background: Solid-state NMR spectroscopy is rapidly emerging as an integral technique of structural biology. Magic angle spinning NMR methods are becoming increasingly important for structural studies of biological solids inherently lacking long-range order, including membrane proteins [1-4], macromolecular assemblies [5-8], disordered fibrils [9-11], and biomaterials [12]. With the recent advances in multidimensional methods for resonance assignments and structural characterization of proteins and protein assemblies by magic angle spinning NMR, it is now possible to obtain detailed structural information for uniformly enriched proteins with only 0.1-0.5 mmoles of material, as has been demonstrated by several laboratories, including ours [1,7,10,13-26]. Therefore, high-resolution protein structure determination by magic angle spinning solid-state NMR has become a reality [27,28]. Furthermore, our group has pursued development of novel experiments for studies of differentially enriched protein assemblies employing isotope-edited magnetization transfer protocols. These experiments open new possibilities for high-resolution structural characterization of large protein assemblies [26]. Despite these recent breakthroughs, several important classes of solid-state NMR methods for characterization of structure and dynamics of membrane proteins are still missing. In this pilot project, we propose to fill the existing gap and to develop several classes of novel magic angle spinning NMR experiments that are envisioned to be critical for structural analysis of peripheral membrane proteins. As the first step, these experiments will be applied to gaining insight on the catalytic mechanism of PI-PLC. PI-PLC from Bacillus thuringiensis is a peripheral membrane enzyme that catalyzes the specific cleavage of the sn-3-phosphodiester bond in phosphatidylinosoptol and phosphoinositides. PI-PLC is interfacially activated by a non-substrate phosphatidylcholine for the subsequent PI cleavage and cIP hydrolysis [29]. Multiple studies revealed the salient structural and mechanistic aspects of the PI-PLC function [29-36], and the following key features for the interfacial activation have been postulated [32]: i) the initial binding site of PI-PLC to phospholipid interfaces is lined by residues 43-48 (helix B in solution), and single point and double mutants of these residues exhibit lower enzymatic activities; ii) helix B is disrupted when PI-PLC is anchored to the PC-containing membranes; iii) the membrane-activated form of PI-PLC may be a dimer, an interaction thought to involve a tyrosine rich sequence on the protein surface. Despite multiple compelling pieces of evidence supporting this mechanism of interfacial PI-PLC activation, the dimeric form of the wild type PI-PLC has not been yet directly observed, and the structure of the interface of PI-PLC and its specific interactions with the lipids are unknown. In this pilot project, we propose to develop solid-state NMR experiments that will probe the dimeric structure of PI-PLC and the interactions of the protein with the lipids.