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.

Protein organization on biomembranes and their dynamics are essential for cellular function. It is not clear, however, how protein binding may influence the assembly of underlying lipids or how the membrane structure leads to functional protein organization. Toward this goal, we investigated the effects of annexin a5 binding to biomimetic membranes using fluorescence imaging and correlation spectroscopy. Annexin a5 (anx a5), a peripheral intracellular protein that plays a membrane remodeling role in addition to other functions, binds specifically and tightly to anionic (e.g., phosphatidylserine)-containing membranes in the presence of calcium ion. Our fluorescence microscopy reveals that annexin likely forms assemblies, along with a more dispersed population, upon binding to anionic biomembranes in the presence of calcium ion, which is reflected in its two-component Brownian motion. To investigate the effects of annexin binding on the underlying lipids, we used specific acyl chain-labeled phospholipid analogs, NBD-phosphatidylcholine (NBD-PC) and NBD-phosphatidylserine (NBD-PS). We find that both NBD-labeled lipids cluster under anx a5 assemblies, as compared with when they are found under the dispersed annexin population, and NBD-PS exhibits two-component lateral diffusion under the annexin assemblies. In contrast, NBD-PC diffusion is slower by an order of magnitude under the annexin assemblies in contrast to its diffusion when not localized under anx a5 assemblies. Our results indicate that upon binding to membranes, the peripheral protein annexin organizes the underlying lipids into domains, which may have functional implications in vivo.

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the wild-type protein, face the apolar environment of the lipid bilayer. We then measured the thermodynamic stability of the wild-type protein and of each of the sequence variants by chemical denaturation. We only made these measurements when the proteins appeared to be at reversible equilibrium between folded and denatured states. We also characterized the folded states of each protein by fluorescence spectroscopy and by a functional assay. Those characterizations revealed additional information about how the lipid bilayers may accommodate an arginine.