1. Electrostatics and binding properties of Phosphatidylinositol-4,5-bisphosphate in model membranes
- Author
-
Graber, Zachary T.
- Subjects
- Biochemistry, biomembranes, membrane structure, phosphatidylinositol-4,5-bisphosphate, phosphoinositides, calcium, electrostatics, PTEN, 31P NMR
- Abstract
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is an important signaling lipid in the cell plasma membrane, playing an important role in many diverse signaling processes. It is important to gain an understanding of how PI(4,5)P2’s role in these signaling processes is regulated. The headgroup of PI(4,5)P2 is highly negatively charged and electrostatics play a significant role in interactions between PI(4,5)P2 and proteins. In this thesis we have extensively studied the ionization properties of PI(4,5)P2 and its interactions with common components of the plasma membrane inner leaflet and the cytosol. Using a new fitting procedure we have developed a model for the ionization of the phosphatidylinositol polyphosphates within the membrane system. While this ionization had been described before in a qualitative manner, this new fitting model has allowed us to quantitatively describe the ionization and measure pKa values for each ionization step. The ionization of PI(4,5)P2, was also examined in complex ternary lipid systems. We investigated PI(4,5)P2 ionization with each of the plasma membrane inner leaflet lipids, PE, PI, and PS. We found evidence for a significant interaction between PE and PI(4,5)P2. PE forms a hydrogen-bond with the PI(4,5)P2 headgroup, leading to a shift in the ionization of PI(4,5)P2 to lower pKa values. PI was also found to interact with PI(4,5)P2, resulting in the formation of large bulge shaped PI(4,5)P2 rich domains. It has been proposed that lateral localization of PI(4,5)P2 within the plasma membrane is critical for signaling, with multiple pools of PI(4,5)P2 used for different signaling purposes. In vitro studies have indicated that both Ca2+ and cholesterol have the capacity to promote formation of PI(4,5)P2 clusters in model membranes. To shed light on this we have examined the interaction of PI(4,5)P2 with Ca2+, Mg2+, and cholesterol. Cholesterol was found to have a small but significant effect on the ionization of PI(4,5)P2. This interaction may be part of the mechanism for cholesterol induced PI(4,5)P2 domain formation. Ca2+ was found to have a large effect on PI(4,5)P2 ionization, while Mg2+ had a smaller effect. With 1 mM Ca2+ in solution, we found a 1,000 fold surface enrichment of Ca2+ ions to a total of 1.6 Ca2+ ions / PI(4,5)P2. The binding of Ca2+ leads to condensation of the PI(4,5)P2 molecules as revealed by x-ray reflectivity. Mg2+ was found to compete with Ca2+ and reduce Ca2+ binding, but Ca2+ was able to out-compete Mg2+ and seems to eliminate Mg2+ at a 1:1 molar ratio. The critical interaction between PI(4,5)P2 and the tumor suppressor PTEN was also examined. Using 31P NMR we found that PTEN interacts with the PI(4,5)P2 headgroup. We observed a greater effect on the 5-phosphate, indicating the importance of this phosphomomonoester for the interaction. Within the protein, the R12, K13, and Y16 residues were found to be involved in the interaction, and we suggest that these residues may be essential for this highly specific interaction.
- Published
- 2014