Characterization of the EF-Hand Calcium-Binding Domains of Human Plastins

Abstract

The three human plastins (L-plastin, T-plastin, and I-plastin) are important regulatory Ca²⁺-binding proteins that belong to the family of actin-binding proteins. Plastins are involved in the regulation of the actin cytoskeleton as well as the cross-linking of actin filaments. In addition to four calponin-homology (CH) domains, all three plastins contain two N-terminal EF-hand Ca²⁺-binding motifs which together are homologous to a single lobe of the well-known calcium-regulatory protein calmodulin. This part of the protein allows for the regulation of the actin bundling activity in response to elevated calcium levels. In this protocol, we describe the purification of the EF-hand headpiece domains of all three plastins, as well as SPR studies, ITC studies, and NMR interaction studies with different peptides and calcium. In combination, these three experimental techniques provide detailed insights into a novel regulatory mechanism, involving the linker region between the EF-hand domain and the first CH domain of the plastins.

Keywords: Actin bundling; Calcium-binding domain; EF-hand motifs; Fimbrin; I-plastin; ITC; L-plastin; NMR spectroscopy; SPR; T-plastin.