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 Ca(2+)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.