Thesis advisor: Jianmin Gao / Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves the substrate phosphatidylinositol through two steps: the first step occurs in the interface between lipid and solution, while the second step only takes place in water soluble environment. For interfacial catalysis, the enzyme should bind to the lipid surface first before engaging its substrate, therefore interfacial kinetics include both interfacial binding and an interfacial catalytic step. The Bacillus thuringiensis PI-PLC is activated by binding to zwitterionic surfaces; phosphatidylcholine (PC) and two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure, in particular, are critical for this interaction. The helix B region in PI-PLC orients the side chains of Ile43 and Trp47 so that they form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. An earlier crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, showed that the helix B region was reorganized into an extended loop. Whether this conformational change occurred in the wild type PI-PLC was tested with a series of mutations targeting helix B residues and surrounding regions. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is likely to play a role in the activated PI-PLC conformation. Besides the helix B residues, a number of hydrophobic residues along the rim of the <em>f</em>Ñ<em>f</em>Ò-barrel and close to both helix B and the active site were also altered to assess their contribution to membrane binding and kinetic activation. Results showed that Tyr86 and Tyr88, but not Tyr118, contribute to the protein binding to PC vesicles. These residues are capable of cation-<em>f</em>à interactions with the choline headgroup of the phospholipid PC. Although mammalian PLC<em>f</em>Ô1 is a complex multidomain protein, the catalytic domain resembles the bacterial PI-PLC enzymes. Little work has been done to characterize the extent to which this domain contributes to membrane binding. A mutated protein that removes the very anionic X/Y linker region that covers the active site was constructed. The interfacial binding and the corresponding enzyme activity of this mutant against WT were measured in both micelles and large unilamellar vesicles. The results showed at <em>f</em>ÝM protein concentration there was no large difference between the PLC<em>f</em>Ô1 and the deletion mutant in terms of vesicle binding. However, the deletion mutant showed much higher membrane binding affinity at nM concentrations. These results shed some light on the activation or inhibition role of the catalytic domain and pointed to a possible direction of future studies, for example examining specific mutant enzymes in the interfacial loop region. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_101433 |
| Date | January 2011 |
| Creators | Guo, Su |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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