Quantitative Determination of Ca²⁺-binding to Ca²⁺-sensor Proteins by Isothermal Titration Calorimetry

Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from titration series without a need to label or immobilize one or more interaction partners. Furthermore, second messenger homeostasis involving Ca²⁺-ions requires in particular knowledge about stoichiometries and affinities of Ca²⁺-binding to Ca²⁺-sensor proteins or Ca²⁺-dependent regulators, which can be obtained by employing ITC. We used ITC to measure these parameters for a set of neuronal Ca²⁺-sensor proteins operating in photoreceptor cells. Here, we present a step wise protocol to (a) measure Ca²⁺ interaction with the Ca²⁺-sensor guanylate cyclase-activating protein 1, (b) to design an ITC experiment and prepare samples, (c) to remove Ca²⁺ nearly completely from Ca²⁺ binding proteins without using a chelating agent like EGTA.