Insights Into the Interaction of a Model Membrane Binding Protein, Annexin A5, with Calcium ion and Phospholipids Garnered via Isothermal Titration Calorimetry

Abstract

Coupled equilibrium is a theme that runs throughout biochemical systems. It enables the ability to modulate a protein's response as a function of changing ligand levels. Isothermal titration calorimetry is a powerful tool to capture the thermodynamic signature of interactions as it relies upon the inherent heat of an interaction and not upon non-physiological probes. It is also a technique rift with potential complications due to the need of relatively high concentrations of macromolecule and ligand, but, more critically, only limited and somewhat simple models are readily available for analysis. Both of these issues are amplified in systems that contain acidic membrane and calcium ion, as precipitation is present and well developed binding models for membrane are not. The benefits, however, outweigh the potential for failure, and the resultant global analysis of the thermodynamic cycle of the interaction of a membrane-binding protein with phospholipid and calcium ion is presented. A single model was found to globally fit the interaction of annexin a5 with calcium ion, with calcium ion in the presence of phospholipid, with phospholipid in the presence of calcium ion, and annexin a5 with phospholipid. We find that the protein in the presence of phospholipid conserves its number of calcium-binding sites with near solution state affinity. In addition, binding phospholipid induces the formation of a number of new high affinity calcium sites; overall, this is a type of allosteric transition model. These low and high affinity calcium sites have very different calorimetric profiles. The model suggests that the small population of protein that binds membrane serves to thermodynamically position the protein such that it is able to bind calcium ion with much higher affinity than in solution.