Advertisement

Tryptophan Scanning Mutagenesis of EF-Hand Motifs

  • Uday Kiran
  • Michael R. Kreutz
  • Yogendra SharmaEmail author
  • Asima ChakrabortyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

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.

Key words

Tryptophan scanning EF-hand motif CaBP Caldendrin Fluorescence 

Notes

Acknowledgments

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.

References

  1. 1.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium: calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529CrossRefGoogle Scholar
  2. 2.
    Zhou Y, Xue S, Yang JJ (2013) Calciomics: integrative studies of Ca2+-binding proteins and their interactomes in biological systems. Metallomics 5:29–42CrossRefGoogle Scholar
  3. 3.
    Raghuram V, Sharma Y, Kreutz MR (2012) Ca2+ sensor proteins in dendritic spines: a race for Ca2+. Front Mol Neurosci 5:61CrossRefGoogle Scholar
  4. 4.
    Mäler L, Blankenship J, Rance M, Chazin WJ (2000) Site–site communication in the EF-hand Ca2+-binding protein calbindin D9k. Nat Struct Mol Biol 7:245–250CrossRefGoogle Scholar
  5. 5.
    Wojciechowski D, Fischer M, Fahlke C (2015) Tryptophan scanning mutagenesis identifies the molecular determinants of distinct barttin functions. J Biol Chem 290:18732–18743CrossRefGoogle Scholar
  6. 6.
    Deacon LJ, Billones H, Galyean AA, Donaldson T, Pennacchio A, Iozzino L, Dattelbaum JD (2014) Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein. Biochimie 99:208–214CrossRefGoogle Scholar
  7. 7.
    De Feo CJ, Mootien S, Unger VM (2010) Tryptophan scanning analysis of the membrane domain of CTR-copper transporters. J Membr Biol 234:113–123CrossRefGoogle Scholar
  8. 8.
    Yamniuk AP, Silver DM, Anderson KL, Martin SR, Vogel HJ (2007) Domain stability and metal-induced folding of calcium-and integrin-binding protein 1. Biochemistry 46:7088–7098CrossRefGoogle Scholar
  9. 9.
    Kiran U, Regur P, Kreutz MR, Sharma Y, Chakraborty A (2017) Intermotif communication induces hierarchical Ca2+ filling of Caldendrin. Biochemistry 56:2467–2476CrossRefGoogle Scholar
  10. 10.
    Seidenbecher CI, Langnaese K, Sanmartí-Vila L, Boeckers TM, Smalla KH, Sabel BA, Garner CC, Gundelfinger ED, Kreutz MR (1998) Caldendrin, novel neuronal calcium-binding protein confined to the somatodendritic compartment. J Biol Chem 273:21324–21331CrossRefGoogle Scholar
  11. 11.
    Reddy PP, Raghuram V, Hradsky J, Spilker C, Chakraborty A, Sharma Y, Kreutz MR (2014) Molecular dynamics of the neuronal EF-hand Ca2+-sensor Caldendrin. PLoS One 9:e103186CrossRefGoogle Scholar
  12. 12.
    Théret I, Baladi S, Cox JA, Sakamoto H, Craescu CT (2000) Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement. Biochemistry 39:7920–7926CrossRefGoogle Scholar
  13. 13.
    Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201CrossRefGoogle Scholar
  14. 14.
    Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66:12–21CrossRefGoogle Scholar
  15. 15.
    Tina KG, Bhadra R, Srinivasan N (2007) PIC: protein interactions calculator. Nucleic Acids Res 35:473–476CrossRefGoogle Scholar
  16. 16.
    Hung HC, Chen YH, Liu GY, Lee HJ, Chang GG (2003) Equilibrium protein folding-unfolding process involving multiple intermediates. Bull Math Biol 65:553–570CrossRefGoogle Scholar
  17. 17.
    Muralidhar D, Jobby MK, Kannan K, Annapurna V, Chary KV, Jeromin A, Sharma Y (2005) Equilibrium unfolding of neuronal calcium sensor-1: N-terminal myristoylation influences unfolding and reduces the protein stiffening in the presence of calcium. J Biol Chem 280:15569–15578CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.CSIR-Centre for Cellular and Molecular Biology (CCMB)HyderabadIndia
  2. 2.RG Neuroplasticity, Leibniz Institute for NeurobiologyMagdeburgGermany
  3. 3.Leibniz Group ‘Dendritic Organelles and Synaptic Function’University Medical Center Hamburg-Eppendorf, Center for Molecular Neurobiology, ZMNHHamburgGermany
  4. 4.Academy of Scientific and Innovative Research (AcSIR)GhaziabadIndia

Personalised recommendations