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

  • Oleg Khassan
  • Katharine V. Jensen
  • Andrew G. Woodman
  • Hans J. VogelEmail author
  • Hiroaki Ishida
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

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.

Key words

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

Notes

Acknowledgments

This research is currently supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Oleg Khassan
    • 1
  • Katharine V. Jensen
    • 1
  • Andrew G. Woodman
    • 1
  • Hans J. Vogel
    • 1
    Email author
  • Hiroaki Ishida
    • 1
  1. 1.Biochemistry Research Group, Department of Biological SciencesUniversity of CalgaryCalgaryCanada

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