Preparation of Amyloidogenic Aggregates from EF-Hand β-Parvalbumin and S100 Proteins

  • Javier Martínez
  • Joana S. Cristóvão
  • Rosa Sánchez
  • Maria Gasset
  • Cláudio M. Gomes
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1779)

Abstract

Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca2+-bound or Ca2+-free states with distinct aggregation propensities. That is the case among β-parvalbumins and S100 proteins, which under certain conditions undergo Ca2+-dependent self-assembly reactions with the formation of oligomers, amyloid-type aggregates and fibrils. These phenomena may be particularly relevant in human S100A6 protein and in fish Gad m 1 allergenic protein, which are implicated in human disease processes. Here, we describe detailed methods to generate and monitor the formation of amyloidogenic assemblies and aggregates of these two EF-hand proteins in vitro.

Key words

Calcium S100A6 Gad m 1 Fish allergens Amyloids EF-hand 

Abbreviation

Gad m 1

Allergome nomenclature of Atlantic cod β-parvalbumin with UniProtKB sequence A51783

ThT

Thioflavin T

Notes

Acknowledgments

This work was partly supported by Fundação para a Ciência e a Tecnologia (FCT/MCTES, Portugal) through grants UID/Multi/04046/2013 (to BioISI/C.M.G.), PTDC/NEU-NMC/2138/2014 (to C.M.G.), IF/01046/2014 (to C.M.G.). Bial Foundation is acknowledged through grant PT/FB/BL-2014-343 (to C.M.G.). AEI/EU-FEDER (Spain) is acknowledged for grants SAF2014-52661-C3 and BFU2015-72271-EXP (to M.G.). J.S.C. was a recipient of a Ph.D. fellowship (SFRH/BD/101171/2014) from Fundação para a Ciência e a Tecnologia (FCT/MCTES, Portugal). G. Fritz (Freiburg University) is gratefully acknowledged for the S100A6 expression plasmid.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Javier Martínez
    • 1
  • Joana S. Cristóvão
    • 1
  • Rosa Sánchez
    • 2
  • Maria Gasset
    • 2
  • Cláudio M. Gomes
    • 1
  1. 1.Biosystems and Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa and Departamento de Química e BioquímicaUniversidade de LisboaLisboaPortugal
  2. 2.Institute of Physical Chemistry RocasolanoSpanish National Research Council (CSIC)MadridSpain

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