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Determination of the Rab27–Effector Binding Affinity Using a High-Throughput FRET-Based Assay

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Rab GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2293))

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Abstract

Thus far, two Rab27 isoforms (Rab27a and Rab27b) have been identified that interact with their eleven downstream effectors proteins, preferentially in their GTP-bound state. In recent years, a number of studies has suggested roles for Rab27–effector protein interactions in the development of cancer cell invasion and metastasis, and immune and inflammatory responses. Here we develop an in vitro fluorescence resonance energy transfer (FRET)-based protein–protein interaction assay to report Rab27 protein interactions with their effectors. We particularly focus on determining the interaction of mouse (m) Synaptotagmin-like protein (Slp)1 and mSlp2 effector proteins with human (h)Rab27. Green fluorescent protein (GFP)-N-terminus Rab27 binding domains (m-Slp1 and m-Slp2) recombinant proteins were used as donor fluorophores, whereas mCherry-hRab27a/b recombinant proteins were used as acceptor fluorophores. The conditions of this assay were validated and optimized, and the specificity of the assay was confirmed. Accordingly, this assay can be used to assess and identify key determinants and/or candidate inhibitors of Rab27–effector interactions.

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Acknowledgments

This work was supported by the Iraqi Ministry of Higher Education and Scientific Research/the Iraqi Cultural Attaché in the UK funded PhD studentship.

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Authors and Affiliations

  1. Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK

    Raghdan Z. Al-Saad, Ian Kerr & Alistair N. Hume

  2. Department of Pharmacology and Toxicology, College of Pharmacy, University of Babylon, Babylon, Iraq

    Raghdan Z. Al-Saad

Authors
  1. Raghdan Z. Al-Saad
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  2. Ian Kerr
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  3. Alistair N. Hume
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Corresponding author

Correspondence to Raghdan Z. Al-Saad .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Guangpu Li

  2. Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

    Nava Segev

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Al-Saad, R.Z., Kerr, I., Hume, A.N. (2021). Determination of the Rab27–Effector Binding Affinity Using a High-Throughput FRET-Based Assay. In: Li, G., Segev, N. (eds) Rab GTPases. Methods in Molecular Biology, vol 2293. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1346-7_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1346-7_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1345-0

  • Online ISBN: 978-1-0716-1346-7

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