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The Use of Metal Fluoride Compounds as Phosphate Analogs for Understanding the Structural Mechanism in P-type ATPases

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P-Type ATPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1377))

Abstract

The membrane-bound protein family, P-type ATPases, couples ATP hydrolysis with substrate transport across the membrane and forms an obligatory auto-phosphorylated intermediate in the transport cycle. The metal fluoride compounds, BeF x , AlF x , and MgF x , as phosphate analogs stabilize different enzyme structural states in the phosphoryl transfer/hydrolysis reactions, thereby fixing otherwise short-lived intermediate and transient structural states and enabling their biochemical and atomic-level crystallographic studies. The compounds thus make an essential contribution for understanding of the ATP-driven transport mechanism. Here, with a representative member of P-type ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), we describe the method for their binding and for structural and functional characterization of the bound states, and their assignments to states occurring in the transport cycle.

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Acknowledgement

This work was supported by JSPS KAKENHI Grant Number 23370058.

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Authors and Affiliations

  1. Asahikawa Medical University, 2-1-1-1, Midorigaokahigashi, Asahikawa, Hokkaido, 078-8510, Japan

    Stefania J. Danko & Hiroshi Suzuki

Authors
  1. Stefania J. Danko
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  2. Hiroshi Suzuki
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Corresponding author

Correspondence to Hiroshi Suzuki .

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Editors and Affiliations

  1. Department of Biochemistry, Biocenter, University of Oxford, Oxford, Oxfordshire, United Kingdom

    Maike Bublitz

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Danko, S.J., Suzuki, H. (2016). The Use of Metal Fluoride Compounds as Phosphate Analogs for Understanding the Structural Mechanism in P-type ATPases. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_19

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  • DOI: https://doi.org/10.1007/978-1-4939-3179-8_19

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3178-1

  • Online ISBN: 978-1-4939-3179-8

  • eBook Packages: Springer Protocols

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