Huang Hao, Vogel Hans J
Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.
Methods Mol Biol. 2013;963:99-113. doi: 10.1007/978-1-62703-230-8_7.
The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca(2+)-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the study of the interaction of CIB1 with the cytoplasmic domain of the human integrin αIIb subunit. In this protocol, we describe the purification and isotope labeling strategies for productive NMR studies of CIB1. The same isotope labeling strategies can be implemented to study numerous related regulatory calcium-binding proteins.
钙整合素结合蛋白1（CIB1）已被确定为一种重要的调节性钙结合蛋白，参与多种细胞功能。核磁共振（NMR）光谱法为研究CIB1及相关蛋白的结构、动力学和相互作用提供了一种强大的方法。多维NMR光谱法与各种选择性同位素标记策略相结合，已被证明在CIB1的结构测定中是成功的。此外，当该蛋白结合不同金属离子时，同样的方法能够检测到构象变化，并且有助于研究CIB1与人整合素αIIb亚基细胞质结构域的相互作用。在本方案中，我们描述了用于CIB1高效NMR研究的纯化和同位素标记策略。同样的同位素标记策略可用于研究众多相关的调节性钙结合蛋白。
