Characterization of Ca 2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

Abstract

The interactions between Ca 2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca 2+ or phosphocholine were injected. Binding of Ca 2+ induced a 10-fold higher signal than expected from the molecular weight of Ca 2+. It was interpreted to result from the conformational change that occurs on binding of Ca 2+. Two sites with different characteristics were distinguished: a high-affinity site with K D = 0.03 mM and a low-affinity site with K D = 5.45 mM. The pH dependencies of the two Ca 2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K D = 5 μM at 10 mM Ca 2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca 2+ is an efficient regulator under physiological conditions.