Wageningen University and Research, Laboratory of Biochemistry, Wageningen, The Netherlands.
Methods Mol Biol. 2024;2731:295-310. doi: 10.1007/978-1-0716-3511-7_22.
The field of plant receptor biology has rapidly expanded in the past three decades. However, the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Identifying and quantifying protein-ligand interactions is crucial for understanding how they regulate certain physiological processes. An important aspect is the quantification of different parameters of the interaction, like binding affinity, kinetics, and ligand specificity that drive the formation of signaling complexes. In this chapter, we discuss Isothermal Titration Calorimetry (ITC) as a label-free technique to measure thermodynamic parameters of ligand binding with high accuracy and reproducibility. We provide a detailed guideline how to design, perform, analyze, and interpret ITC measurements using as an example the interaction between the SCHENGEN3/GASSHO1 (SGN3/GSO1) leucine-rich repeat receptor-like kinase and its sulfated peptide ligand CASPARIAN STRIP INTEGRITY FACTOR 2 (CIF2).
在过去的三十年中，植物受体生物学领域迅速发展。然而，受体-配体对之间的直接相互作用的证明仍然是一个挑战。鉴定和量化蛋白-配体相互作用对于理解它们如何调节某些生理过程至关重要。一个重要方面是量化相互作用的不同参数，如结合亲和力、动力学和配体特异性，这些参数驱动信号复合物的形成。在本章中，我们讨论了等温热力学滴定法（ITC）作为一种无标记技术，可高精度和可重复性地测量配体与受体结合的热力学参数。我们提供了一个详细的指南，说明如何设计、进行、分析和解释 ITC 测量，并用 SCHENGEN3/GASSHO1（SGN3/GSO1）亮氨酸丰富重复受体样激酶与其硫酸肽配体 CASPARIAN STRIP INTEGRITY FACTOR 2（CIF2）之间的相互作用作为示例。
