Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity: INSIGHTS INTO PHOSPHATIDYLCHOLINE ACTIVATION AND SURFACE DILUTION INHIBITION Export

@article{citeulike:4775452, 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 ({tau}c) 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 {tau}c. PI-PLC activity and phospholipid {tau}c 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 {tau}c, 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. 10.1074/jbc.M809600200}, author = {Pu, Mingming and Fang, Xiaomin and Redfield, Alfred G. and Gershenson, Anne and Roberts, Mary F.}, citeulike-article-id = {4775452}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M809600200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/284/24/16099}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/19336401}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=19336401}, day = {12}, doi = {10.1074/jbc.M809600200}, journal = {J. Biol. Chem.}, keywords = {activation, binding, c, dilution, phospholipase, pu09pdf, surface, vesicle}, month = {June}, number = {24}, pages = {16099--16107}, posted-at = {2009-06-09 00:11:52}, priority = {2}, title = {Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity: INSIGHTS INTO PHOSPHATIDYLCHOLINE ACTIVATION AND SURFACE DILUTION INHIBITION}, url = {http://dx.doi.org/10.1074/jbc.M809600200}, volume = {284}, year = {2009} }

TY - JOUR ID - citeulike:4775452 L3 - citeulike-article-id:4775452 N2 - 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 ({tau}c) 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 {tau}c. PI-PLC activity and phospholipid {tau}c 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 {tau}c, 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. 10.1074/jbc.M809600200 IS - 24 JF - J. Biol. Chem. EP - 16107 TI - Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity: INSIGHTS INTO PHOSPHATIDYLCHOLINE ACTIVATION AND SURFACE DILUTION INHIBITION VL - 284 SP - 16099 KW - activation KW - binding KW - c KW - dilution KW - phospholipase KW - pu09pdf KW - surface KW - vesicle AU - Pu, Mingming AU - Fang, Xiaomin AU - Redfield, Alfred AU - Gershenson, Anne AU - Roberts, Mary PY - 2009/06/12/ UR - http://dx.doi.org/10.1074/jbc.M809600200 ER -