Tryptophan Scanning Mutagenesis of EF-Hand Motifs

Abstract

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.