Jaworski, Samantha R.2012-12-042012-12-042012-09https://hdl.handle.net/11299/140034University of Minnesota M.S. thesis. September 2012. Major: Chemistry. Advisor: Dr. Anne Hinderliter. 1 computer file (PDF); viii, 60 pages.The annexins make up to 2% of all intracellular proteins and have been implicated in a multitude of biological processes; thus, they are a model system for peripheral membrane binding proteins. One member, annexin a5, binds membranes enriched in the acidic phospholipid, phosphatidylserine, and calcium ion. We find by Isothermal Titration Calorimetry (ITC) that annexin a5 binds calcium ions with much higher affinity in the presence of non-cholesterol containing neutral and acidic phospholipid mixtures ((60:40) (POPC:POPS)) relative to solution state (without membrane) calcium ion binding. We hypothesize that the shift to higher affinity calcium ion binding is modulated by the composition of the membrane. We tested this hypothesis by introducing cholesterol, a small, but physiologically significant lipid, to form ternary mixtures of (60:40):5, (60:40):10 and (60:40):20 (POPC:POPS):Chol. Cholesterol enhances the demixing behavior of our binary lipid mixture that is a model of the inner leaflet of the plasma membrane of the eukaryotic cell. Cholesterol and POPS have the weak tendency to form complexes with one another in the background of POPC, which enhances the likelihood of domain formation on the membrane surface. We have shown annexin a5 binds calcium ions with similar affinity in the presence of the ternary mixture containing cholesterol compared to the binary mixture without cholesterol. In contrast, when saturated with calcium ions, annexin a5 binds the ternary membrane system with an enhanced affinity as compared to the binary lipid mixture. This suggests that membrane composition alters the responsiveness of annexin a5 to calcium ion. Moreover, this body of work builds upon, and directly tests our new allosteric transition model of how membrane composition poises membrane associated proteins to respond.en-USannexin a5BindingIsothermal titration calorimetryMembrane compositionChemistryThesis or Dissertation