Tryptophan Scanning Mutagenesis of EF-Hand Motifs
Ca2+ regulation in living systems occurs via specific structural alterations, subtle or drastic, in the Ca2+-binding domains of sensor proteins. Sensor proteins perform designated nonredundant roles within the dense network of Ca2+-binding proteins. A detailed understanding of the structural changes in calcium sensor proteins due to Ca2+ spikes that vary spatially, temporally, and in magnitude would provide better insights into the mechanism of Ca2+ sensing. This chapter describes a method to study various stages during apo to the holo transition of Ca2+-binding proteins by Trp-mediated scanning of individual EF-hand motifs. We describe the applicability of this procedure to caldendrin, which is a neuronal Ca2+-binding protein and to integrin-binding protein. Tryptophan mutants of full-length caldendrin were designed to reveal local structural changes in each EF-hand of the protein. This method, referred to as “EF-hand scanning tryptophan mutagenesis,” not only allows the identification of canonical and noncanonical EF-hands using very low concentrations of protein but also enables visualization of the hierarchical filling of Ca2+ into the canonical EF-hands.
Key wordsTryptophan scanning EF-hand motif CaBP Caldendrin Fluorescence
The work was supported by the CSIR fast-track SRA to AC, CSIR, DST, and DBT grants to YS and DFG (Kr1879/3-1) to MRK.