Abbas Seher, Koch Karl-Wilhelm
Department of Neuroscience, Division of Biochemistry, University of Oldenburg, Oldenburg D-26129, Germany.
Bio Protoc. 2020 Apr 5;10(7):e3580. doi: 10.21769/BioProtoc.3580.
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 Ca-ions requires in particular knowledge about stoichiometries and affinities of Ca-binding to Ca-sensor proteins or Ca-dependent regulators, which can be obtained by employing ITC. We used ITC to measure these parameters for a set of neuronal Ca-sensor proteins operating in photoreceptor cells. Here, we present a step wise protocol to (a) measure Ca interaction with the Ca-sensor guanylate cyclase-activating protein 1, (b) to design an ITC experiment and prepare samples, (c) to remove Ca nearly completely from Ca binding proteins without using a chelating agent like EGTA.
多样而复杂的分子识别是信号转导级联反应的核心要素。这些相互作用模式的强度和性质可通过不同的实验方法来确定。其中，等温滴定量热法（ITC）具有一定优势，它能从滴定系列中提供结合常数和热力学参数，而无需对一个或多个相互作用伙伴进行标记或固定。此外，涉及钙离子的第二信使稳态尤其需要了解钙离子与钙传感器蛋白或钙依赖性调节剂结合的化学计量和亲和力，这可通过使用ITC来获得。我们使用ITC来测量一组在光感受器细胞中起作用的神经元钙传感器蛋白的这些参数。在此，我们提出一个逐步方案，用于（a）测量钙离子与钙传感器鸟苷酸环化酶激活蛋白1的相互作用，（b）设计ITC实验并制备样品，（c）在不使用像乙二醇双四乙酸（EGTA）这样的螯合剂的情况下，几乎完全从钙结合蛋白中去除钙离子。
