High Resolution 31 P NMR Field Cycling Probes Phospholipid Dynamics and Interactions with Phosphatidylinositol‐Specific Phospholipase C

Abstract

31P relaxation times for phospholipids in multicomponent unilamellar vesicles can be measured from 0.004 to 11.7 T by a novel high resolution field cycling technique (Proc. Natl. Acad. Sci. U.S.A. (2004) 101, 17066). The field dependence of the relaxation rate is modeled by a sum of dipolar and chemical shift anisotropy terms from which we extract correlation times for motion on three distinct time-scales: (i) μ s τ v reflecting overall rotational diffusion of the vesicle and by lateral diffusion of the lipids; (ii) ns τ c reflecting faster motions that effectively lead to rotational diffusion perpendicular to the membrane surface; and (iii) sub-ns τ reflecting fast motions of the ester esterified to the phosphate group. The analysis provides a CSA order parameter, Sc, and an effective P-H distance. The ability to monitor relaxation rates at very low fields also yields a good estimate of the time-average angle between the membrane surface and the vector connecting the phosphorus to the glycerol C3 protons (the primary contributors to dipolar relaxation in most phospholipids). Relative differences in P-H orientations can be obtained for different phospholipid headgroups in the same vesicle if smaller unilamellar vesicles are examined. We have used 31P field cycling to monitor association of Bacillus thuringiensis PI-PLC, a peripheral membrane protein, with both PC and several anionic phospholipid bilayers. Micellar diheptanoyl-PC forms complexes with the enzyme that can also be characterized. Differences in phospholipid dynamics induced by the protein binding shed light on how zwitterionic PC molecules activate the enzyme toward its anionic substrate. Supported by NIH GM 60418.