Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²⁺) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²⁺ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²⁺ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²⁺ affinity to a membrane-associated, higher Ca²⁺ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²⁺ influx is the basis for the cooperative response of annexin a5 toward Ca²⁺, and the role of membrane organization in this response.

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Using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) as a model amphitropic protein, we are investigating how membrane structure and composition affect protein-membrane interactions. Previous work showed that BtPI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles with high curvature. In this work, we monitored single fluorescently labeled BtPI-PLC proteins as they cycled on and off surface-tethered phosphatidylglycerol (PG)/PC small unilamellar vesicles (SUVs) using total internal reflection fluorescence (TIRF) microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of protein-membrane interactions as well as the curvature dependence. BtPI-PLC residence times on SUVs average 300 ms, similar to published residence times (300-400 ms) for other amphitropic proteins that transiently interact with cell surfaces. The kinetics of PI-PLC/membrane interactions is well explained by a simple two state binding model with dissociation and association rate constants averaging 3 s À1 and 0.6 mM À1 s À1 respectively. In addition fluorescence correlation spectroscopy (FCS) measurements indicate that introducing lipid packing defects PG/PC SUVs by incorporating low mole percentages of dioleoylglycerol (DOG) enhances BtPI-PLC binding to SUVs. By combining these single molecule fluorescence results with previous biophysical measurements and molecular dynamics simulations, we have developed a quantitative model showing how the bacterial virulence factor Bt-PI-PLC interacts with cell membranes in molecular detail.

cells. Using surface plasmon resonance and centrifugation assays, we have found that the Smurf1 C2 domain binds to phosphoinositides and phosphatidylserine in an synergistic fashion. Confocal images of Smurf1 C2-GFP demonstrate that the domain localizes to the plasma membrane as well as intracellular vesicles in cells. Site-directed mutagenesis has shown the specific residues in the loop region of the protein involved in its cellular membrane localization. In addition, we have used a rapamycin-inducible phosphoinositide phosphatase system to demonstrate that this domain binds phosphoinositides at the plasma membrane. We conclude that the unique properties of the Smurf1 C2 domain to sense specific lipids in addition to anionic charge enable it to target multiple subcellular locations.

b-like conformations. The antimicrobial activity typically is higher when four arginines are present, yet the activity does not appear to correlate directly with folding or secondary structure.