Abstract：
CBL1 is an EF hand Ca2+ binding protein from A. thaliana that is involved in the detection of cellular Ca2+ signals and the downstream signal transmission by interaction with the protein kinase CIPK23. So far, the structure and calcium ion binding affinities of CBL1 remain elusive. In this study it was observed that CBL1 tends to form higher oligomeric states due to an intrinsic hydrophobicity and the presence of the detergent BriJ35 was required for the purification of monomeric and functional protein. Functional insights into the in vitro Ca2+ binding capabilities of CBL1 were obtained by isothermal titration calorimetry (ITC) of the wildtype protein as well as single site EF hand mutants. Based on our results, a binding model of CBL1 for Ca2+in vivo is proposed. Additionally, upon both, ITC measurements and the analysis of an AlphaFold2 model of CBL1, we could gain first insights into the formation of the dimer interface. We could identify an area around EF hand 4 to be relevant for the structural and functional integrity of monomeric CBL1 and likely EF hand 1 to be involved in the dimer interface.
摘要：
CBL1是来自拟南芥的EF手Ca2结合蛋白，通过与蛋白质激酶PK23相互作用，参与细胞Ca2信号的检测和下游信号传递。到目前为止,CBL1的结构和钙离子结合亲和力仍然难以捉摸。在该研究中,观察到CBL1由于固有的疏水性而倾向于形成较高的寡聚状态,并且需要去污剂BriJ35的存在来纯化单体和功能性蛋白质。通过野生型蛋白和单位点EF手突变体的等温滴定量热法（ITC）获得了对CBL1体外Ca2结合能力的功能见解。根据我们的结果,提出了CBL1在体内对Ca2的结合模型。此外,在两者之上,ITC测量和CBL1的AlphaFold2模型的分析，我们可以首先了解二聚体界面的形成。我们可以确定EF手4周围的区域与单体CBL1的结构和功能完整性相关，并且可能EF手1涉及二聚体界面。
