Background

Isothermal titration calorimetry (ITC) is a label free method to investigate bio-molecular interaction processes by measuring heat release or heat consumption during a binding process. These processes could occur between different forms of molecules including macromolecules and/or small ligands. The principle design of the measuring device consists of two cells (a reference and sample cell) in an adiabatic jacket. One binding partner is present in the sample cell, while the other interacting molecule is injected into the sample cell. The reference cell contains a buffer solution of water. Temperature differences between sample cell and reference cell are sensed by detectors and an equal temperature between both cells is maintained by a combination of a heater system and a feedback circuit. Interaction of two molecule species either releases or consumes heat, which is detected based on the heater/feedback system. ITC can be applied for example to study a wide range of interaction processes including protein-protein (Pierce et al., 1999), protein-carbohydrate, protein-DNA, enzyme-substrate and cation-protein (Wiseman et al., 1989; García-Hernández et al., 2003; Kozlov and Lohmann, 2012; Mazzei et al., 2014). Alternative methods like surface plasmon resonance (SPR), back scattering interferometry or fluorescence-based techniques utilize different principles and each technique has its own advantages and disadvantages. SPR requires immobilization of one binding partner and fluorescence would in most cases need site-specific labelling. ITC is a method of choice to measure direct ion binding to proteins for deriving binding constants. Furthermore, ligand binding or protein-protein interactions can be studied without the need to label or immobilize one binding partner and has also shown promises in drug discovery science (Ward and Holdgate, 2001; Weber and Salemme, 2003; Lim et al., 2009; Sulmann et al., 2015). The values extracted from ITC measurements are the binding constants (K_a), binding stoichiometry (n), free energy (ΔG), enthalpy (ΔH) and entropy (ΔS). In another words, it provides thermodynamic parameters in addition to binding constants. Since Ca²⁺ ions play important roles in intracellular signaling, it’s important to understand the intracellular detection of Ca²⁺ by Ca²⁺-sensor proteins and to investigate the interaction processes in quantitative terms. Among Ca²⁺-sensors, one subgroup of neuronal Ca²⁺-sensor (NCS) proteins is important for the primary processes of vision in photoreceptor cells, where they are involved in Ca²⁺-feedback mechanisms to control deactivation processes and sensitivity of photoreceptor cells to light. Guanylate cyclase-activating proteins (GCAPs) are NCS proteins that regulate the activity of membrane bound guanylate cyclases in a Ca²⁺-dependent manner, thereby controlling the cytoplasmic concentration of cyclic GMP, the second messenger of photoexcitation in photorecepotor cells. Several point mutations in GCAP1 lead to severely impaired vision or blindness and amino acid substitutions in different positions appear to cause some common features (Marino et al., 2015; Vocke et al., 2017; Marino et al., 2018; Peshenko et al., 2019). For example, mutant GCAP1 proteins undergo different conformational changes due to impaired Ca²⁺-binding, and it often results in constitutive activity of the GCAP1/guanylate cyclase complex. Eventually it causes an imbalance of the Ca²⁺/cyclic GMP homeostasis. ITC has been applied for measuring the binding of Ca²⁺ and Mg²⁺ to GCAPs (Lim et al., 2009; Sulmann et al., 2015; Peshenko et al., 2019), but also to recoverin (Dell'Orco et al., 2010; Abbas et al., 2019), calmodulin (Beccia et al., 2015) visinin-like protein 3 (Li et al., 2016) neuronal calcium sensor 1 (NCS-1) (Tsvetkov et al., 2018) and other Ca²⁺ sensor proteins. An alternative method specifically developed for measuring Ca²⁺-protein bindings is the Chelator assay, which utilizes the presence of a detectable and titrable Ca²⁺-chelator (e.g., BAPTA). It measures the binding between the protein of interest and free Ca²⁺ (Linse, 2002). Both techniques are to some degree complementary and have been used in combination in a recent publication (Elbers et al., 2018).

In this protocol, we provide a step-by-step description for using ITC in Ca²⁺- and Mg²⁺-binding studies. Since Ca²⁺-sensor proteins can contain sometimes binding sites of very high affinity, it is a challenging task to remove bound Ca²⁺-ions nearly completely from the protein. Therefore, we also provide a protocol to decalcify Ca²⁺-sensor proteins without using a chelating substance like EGTA. This protein decalcification protocol can be used for preparing Ca²⁺-binding proteins used in other techniques (surface plasmon resonance, backscattering interferometry, and Chelator assay). In general, the protocol defines the essentials to design an ITC experiment and analysis of ITC data.