Purification and stable isotope labeling of the calcium- and integrin-binding protein 1 for structural and functional NMR studies.

Hao Huang, Hans J Vogel

Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

Methods in molecular biology (Clifton, N.J.) 2013

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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.

Citation

Hao Huang, Hans J Vogel. Purification and stable isotope labeling of the calcium- and integrin-binding protein 1 for structural and functional NMR studies. Methods in molecular biology (Clifton, N.J.). 2013;963:99-113