Abstract
PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is a 40 kDa enzyme that catalyzes the cleavage of GPI-anchored proteins and PI. PI-PLC binds to the membrane via the interfacial binding surface, a special region on the rim of the active site pocket. Hydrophobic and electrostatic interactions (via Trp and Lys residues, respectively) are proposed to play a role in membrane binding. To further examine these interactions, recombinant wild-type (WT) and mutant His6-tagged PI-PLCs (W47A, W242A, W47A/W242A and K44A) were expressed in E. coli and purified. Trp emission of WT and mutant enzymes did not undergo a blue shift upon addition of phosphatidylcholine (PC) liposomes, suggesting that PI-PLC does not deeply penetrate the membrane during membrane association. Trp quenching upon association of WT/mutant PI-PLC with bilayers containing brominated lipids (70% egg PC, 30% 6,7-Br2PC) was equal to that observed for pure egg PC, again suggesting no deep membrane penetration of the enzymes. Stopped-flow fluorescence studies showed first-order quenching of Trp fluorescence upon mixing of WT/mutant PI-PLCs with zwitterionic and negatively charged liposomes, consistent with the notion of a single binding event. The calculated dissociation constants revealed much weaker membrane association of W47A/W242A PI-PLC compared to WT enzyme, and ultimately suggest a role for these Trp residues in membrane association. Introduction of a PI substrate into egg PC liposomes resulted in 5-fold stronger association of WT and mutant PI-PLCs with the membrane surface. Lastly, Langmuir isotherms showed a change in the cross-sectional area per molecule of dimyristoyl-PC and dimyristoylphosphatidylglycerol monolayers upon injection of WT PI-PLC into the subphase, implying that PI-PLC association alters the surface packing of the monolayer. Changes in the area per molecule on PI-PLC binding were more substantial above the Tm of these lipids. (Supported by NSERC).
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