Abstract
The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (tauc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased tauc. PI-PLC activity and phospholipid tauc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC tauc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.
Highlights
Bacterial phosphatidylinositol-specific phospholipase C (PIPLC)2 enzymes aid in organism infectivity [1], whereas the Grants GM60418
To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids and high resolution field cycling 31P NMR methods to estimate internal correlation times (c) of phospholipid headgroup motions
Phospholipid Dynamics and phosphatidylinositol-specific phospholipase C (PI-PLC) Activity clude obtaining information on lipid structure and dynamics from relaxation rates. This limitation is avoided by fc-P-NMR spin lattice relaxation techniques that resolve the dynamics of each phospholipid component in mixed component vesicles by using the high field to separate resonances but allowing nuclei to relax at defined lower fields
Summary
Bacterial phosphatidylinositol-specific phospholipase C (PIPLC)2 enzymes aid in organism infectivity [1], whereas the Grants GM60418 PI-PLC Activity—The specific activity (mol minϪ1 mgϪ1) of PI-PLC toward PI-containing SUVs with a fixed enzyme and total lipid concentration (10 mM) and increasing mole fractions of the activator PC, XPC, was determined.
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